Gene combination and use thereof

Abstract

Provided are a gene combination used for controlling foreign gene expression in a specific plant tissue, and a method applying the gene combination to cultivate a transgenic plant. The method is used to cultivate, for example, an endosperm zero expression-type transgenic rice, i.e., rice grain endosperm produced by the rice does not contain any transgenic product protein synthesis and accumulation.

Claims

1. A method for controlling the expression of an exogenous target gene in a plant, the method comprising introducing a first expression vector and a second expression vector into a recipient plant, wherein the first expression vector comprises a lock polynucleotide and the exogenous target gene operably linked thereto, wherein the lock polynucleotide comprises SEQ ID NO: 5 and blocks the expression of the exogenous target gene, and wherein the second expression vector comprises a key polynucleotide and a tissue-specific promoter operably linked thereto, wherein the key polynucleotide comprises SEQ ID NO: 3 and switches on the expression of the exogenous target gene blocked by the lock polynucleotide.

2. The method of claim 1, wherein the first expression vector further comprises a constitutive promoter operably linked to the lock polynucleotide, wherein the lock polynucleotide is located between the constitutive promoter and the exogenous target gene, and wherein the constitutive promoter is selected from the group consisting of the cauliflower mosaic virus 35S promoter, the nopaline synthase gene promoter from the T-DNA region of the Agrobacterium tumefaciens Ti plasmid, the rice actin promoter Actin I, and the maize ubiquitin (Ubi) gene promoter.

3. The method of claim 1, wherein the tissue-specific promoter is selected from the group consisting of the rice green tissue-specific promoter ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) promoter, the maize phosphoenolpyruvate carboxylase (PEPC) promoter, the rice green tissue-specific expression DX1 promoter, the rice photosystem II 10 kDa polypeptide D540 promoter, the rice leucine-rich repeat-like receptor protein kinase (LP2) promoter, and the maize chloroplast C4 Pdk promoter; preferably, the tissue-specific promoter is the rbcS promoter.

4. The method of claim 1, wherein the exogenous target gene is an insect resistance gene or a herbicide resistance gene, wherein the insect resistance gene is selected from the group consisting of the cry1Ab, cry1Ac, cry1Ab/1Ac, cry1C, co/2A, and Vip3 like insect resistance genes from Bacillus thuringiensis; the anf and sep genes from Serratia entomophila; the cmb gene of Clostridium bifermentans; the mtx gene from Bacillus sphaericus; the insecticidal protein gene from Xenorhabdus nematophilus; the tca and tcb genes from Photorhabdus luminescens; and the prl gene from Metarhizium anisopliae, and wherein the herbicide resistance gene is selected from the group consisting of the EPSP synthase gene for glyphosate resistance, the Salmonella typhimurium EPSP mutant gene aroA, the bar gene for glufosinate resistance, the ALS mutant gene Ilv G for imidazolone resistance, the AccL-s2 gene for sethoxydim resistance, the bxn gene for bromoxynil resistance, and the csrl gene for chlorsulfuron resistance.

5. The method of claim 1, wherein the plant is selected from the group consisting of rice, wheat, barley, oat, maize, millet, sorghum, pearl barley, sweet potato, potato, lotus seed, soybean, and peanut; preferably, the plant is rice.

6. A method of breeding a transgenic plant, the method comprising: crossing a first parent plant comprising a first expression vector with a second parent plant comprising a second expression vector, thereby obtaining the transgenic plant comprising the first expression vector and the second expression vector; or introducing and integrating the first expression vector and the second expression vector into the genome of the same plant, thereby obtaining the transgenic plant, wherein the first expression vector comprises a lock polynucleotide and an exogenous target gene operably linked thereto, wherein the lock polynucleotide comprises SEQ ID NO: 5 and blocks the expression of the exogenous target gene, and wherein the second expression vector comprises a key polynucleotide and a tissue-specific promoter operably linked thereto, wherein the key polynucleotide comprises SEQ ID NO: 3 and switches on the expression of the exogenous target gene blocked by the lock polynucleotide.

7. The method of claim 6, wherein the method further comprises, prior to the crossing step, introducing and integrating the first expression vector into the genome of the first parent plant, and introducing and integrating the second expression vector into the genome of the second parent plant.

8. The method of claim 6, wherein the first expression vector further comprises a constitutive promoter operably linked to the lock polynucleotide, wherein the lock polynucleotide is located between the constitutive promoter and the exogenous target gene.

9. The method of claim 8, wherein the constitutive promoter is selected from the group consisting of the cauliflower mosaic virus 35S promoter, the nopaline synthase gene promoter from the T-DNA region of the Agrobacterium tumefaciens Ti plasmid, the rice actin promoter Actin I, the maize ubiquitin (Ubi) gene promoter; preferably, the constitutive promoter is the rice actin promoter Actin I.

10. The method of claim 6, wherein the tissue-specific promoter is selected from the group consisting of the rice green tissue-specific promoter ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) promoter, the maize phosphoenolpyruvate carboxylase (PEPC) promoter, the rice green tissue-specific expression DX1 promoter, the rice photosystem II 10 kDa polypeptide D540 promoter, the rice leucine-rich repeat-like receptor protein kinase (LP2) promoter, and the maize chloroplast C4 Pdk promoter; preferably, the tissue-specific promoter is the rbcS promoter.

11. The method of claim 6, wherein the exogenous target gene is an insect resistance gene or a herbicide resistance gene, wherein the insect resistance gene is selected from the group consisting of the cry1Ab, cry1Ac, cry1Ab/1Ac, cry1C, cry2A, and Vip3 like insect resistance genes from Bacillus thuringiensis; the anf and sep genes from Serratia entomophila; the cmb gene of Clostridium bifermentans; the mtx gene from Bacillus sphaericus; the insecticidal protein gene from Xenorhabdus nematophilus; the tca and tcb genes from Photorhabdus luminescens; and the prl gene from Metarhizium anisopliae, and wherein the herbicide resistance gene is selected from the group consisting of the EPSP synthase gene for glyphosate resistance, the Salmonella typhimurium EPSP mutant gene aroA, the bar gene for glufosinate resistance, the ALS mutant gene Ilv G for imidazolone resistance, the AccL-s2 gene for sethoxydim resistance, the bxn gene for bromoxynil resistance, and the csrl gene for chlorsulfuron resistance.

12. The method of claim 6, wherein the plant is selected from the group consisting of rice, wheat, barley, oat, maize, millet, sorghum, pearl barley, sweet potato, potato, lotus seed, soybean, and peanut; preferably, the plant is rice.

13. A first expression vector and a second expression vector for controlling expression of an exogenous target gene in a plant, the first expression vector comprising a lock polynucleotide and the exogenous target gene operably linked thereto, wherein the lock polynucleotide comprises SEQ ID NO: 5 and blocks the expression of the exogenous target gene, and the second expression vector comprising a key polynucleotide and a tissue-specific promoter operably linked thereto, wherein the key polynucleotide comprises SEQ ID NO: 3 and switches on the expression of the exogenous target gene blocked by the lock polynucleotide.

14. The first expression vector and the second expression vector of claim 13, wherein the first expression vector further comprises a constitutive promoter operably linked to the lock polynucleotide, wherein the lock polynucleotide is located between the constitutive promoter and the exogenous target gene.

15. The first expression vector and the second expression vector of claim 14, wherein the constitutive promoter is selected from the group consisting of the cauliflower mosaic virus 35S promoter, the nopaline synthase gene promoter from the T-DNA region of the Agrobacterium tumefaciens Ti plasmid, the rice actin promoter Actin I, and the maize ubiquitin (Ubi) gene promoter.

16. The first expression vector and the second expression vector of claim 14, wherein the constitutive promoter is the rice actin promoter Actin I.

17. The first expression vector and the second expression vector of claim 13, wherein the tissue-specific promoter is selected from the group consisting of the rice green tissue-specific promoter ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) promoter, the maize phosphoenolpyruvate carboxylase (PEPC) promoter, the rice green tissue-specific expression DX1 promoter, the rice photosystem II 10 kDa polypeptide D540 promoter, the rice leucine-rich repeat-like receptor protein kinase (LP2) promoter, and the maize chloroplast C4 Pdk promoter.

18. The first expression vector and the second expression vector of claim 13, wherein the tissue-specific promoter is the rbcS promoter.

19. The first expression vector and the second expression vector of claim 13, wherein the exogenous target gene is an insect resistance gene or a herbicide resistance gene, wherein the insect resistance gene is selected from the group consisting of the cry1Ab, cry1Ac, cry1Ab/1Ac, cry1C, cry2A, and Vip3 like insect resistance genes from Bacillus thuringiensis; the anf and sep genes from Serratia entomophila; the cmb gene of Clostridium bifermentans; the mtx gene from Bacillus sphaericus; the insecticidal protein gene from Xenorhabdus nematophilus; the tca and tcb genes from Photorhabdus luminescens; and the prl gene from Metarhizium anisopliae, and wherein the herbicide resistance gene is selected from the group consisting of the EPSP synthase gene for glyphosate resistance, the Salmonella typhimurium EPSP mutant gene aroA, the bar gene for glufosinate resistance, the ALS mutant gene Ilv G for imidazolone resistance, the AccL-52 gene for sethoxydim resistance, the bxn gene for bromoxynil resistance, and the csrl gene for chlorsulfuron resistance.

20. The first expression vector and the second expression vector of claim 13, wherein the plant is selected from the group consisting of rice, wheat, barley, oat, maize, millet, sorghum, pearl barley, sweet potato, potato, lotus seed, soybean, and peanut.

21. The first expression vector and the second expression vector of claim 13, wherein the plant is rice.

22. A transgenic rice plant comprising a first expression vector and a second expression vector, the first expression vector comprising a lock polynucleotide, a rice actin promoter Actin I, and an exogenous target gene, wherein the lock polynucleotide comprises SEQ ID NO: 5 and is located between the rice actin promoter Actin I and the exogenous target gene, and the second expression vector comprising a key polynucleotide and a rice green tissue-specific promoter ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) promoter operably linked thereto, wherein the key polynucleotide comprises SEQ ID NO: 3, and wherein the exogenous target gene is Bt gene cry1Ab/1Ac.

23. The transgenic rice plant of claim 22, wherein the transgenic rice plant comprises Bt protein expressed in leaves and stems, with less expression of Cry1Ab/1Ac protein in endosperm than in leaves and stems, or no expression of Cry1Ab/1Ac protein in endosperm.

24. The transgenic rice plant of claim 23, wherein the amount of Cry1Ab/1Ac protein is less than 1 ng/g in refined rice endosperm of the transgenic rice plant.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1: Map of pSB130 plasmid vector. The plasmid vector has two T-DNA regions, wherein one carries a hygromycin B-resistant marker gene (hpt) and the other carries a multiple cloning site (MCS) for inserting a key gene or a lock element sequence linked to a target gene.

(2) FIG. 2: a flow diagram of the construction of the expression vector pKey1 of the gene key (Key1; SEQ ID NO: 5) according to one embodiment of the present invention, wherein FIG. 2A shows: the first step of obtaining an NosT fragment by amplifying with the specific primers introduced with KpnI and EcoRI recognition sites and ligating it into the corresponding multiple cloning site of pSB130; and the second step of obtaining a Key1 3′ fragment by amplifying with the specific primers introduced with XbaI/SmaI and KpnI recognition sites and ligating it into the corresponding multiple cloning site of pSB130.

(3) FIG. 2B shows: the third step of obtaining a Key1 5′ fragment by amplifying with the specific primers with SalI and SmaI recognition sites introduced and ligating it into the corresponding multiple cloning site of pSB130; and the last step of obtaining a rbcS fragment by amplifying with the specific primers introduced with HindIII and SalI recognition sites and ligating it into the corresponding multiple cloning site of pSB130 to construct a pKey1 expression vector.

(4) FIG. 3: a flow diagram of the construction of the expression vector pLY1 of the lock element (Lock1; SEQ ID NO: 8) and its linked reporter gene (eYFP) according to one embodiment of the present invention, wherein FIG. 3A shows: the first step of amplifying and cloning the NosT fragment as described for pKey1; and the second step of obtaining the Lock1 fragment by amplifying with the specific primers introduced with PstI/XbaI recognition sites and ligating it into the corresponding multiple cloning site of pSB130. FIG. 3B shows: the third step of obtaining an ActinI fragment by amplifying with the specific primers introduced with HindIII and PstI recognition sites and ligating it into the corresponding multiple cloning site of pSB130; and the last step of obtaining an eYFP fragment by amplifying with the specific primers introduced with XbaI and KpnI recognition sites and ligating it into the corresponding multiple cloning site of pSB30 to construct a pLY1 expression vector.

(5) FIG. 4: a flow diagram of the construction of the expression vector pKey2 of the key gene (Key2; SEQ ID NO: 3) according to another embodiment of the present invention, wherein FIG. 4A shows: the first step of obtaining the NosT fragment by amplifying with the specific primers introduced with SacI and EcoRI recognition sites and ligating it into the corresponding multiple cloning site of pSB130: and the second step of obtaining the rbcS fragment by enzymatic cleavage of the pKey1 expression vector with SalI and HindIII and ligating it into the corresponding multiple cloning site of pSB130. FIG. 4B shows: the third step of obtaining a Key2 3′ fragment by amplifying with the specific primers introduced with SacI and XbaI recognition sites and ligating it into the corresponding multiple cloning site of pSB130; and the last step of obtaining a Key2 5′ fragment by amplifying with the specific primers introduced with SalI and XbaI recognition sites and ligating it into the corresponding multiple cloning site of pSB30 to construct a pKey2 expression vector.

(6) FIG. 5: a flow diagram of the construction of the expression vector pLY2 of the lock element (Lock2; SEQ ID NO: 6) and its linked reporter gene (eYFP) according to another embodiment of the present invention, wherein FIG. 5A shows: the first step of obtaining the Nos fragment by enzymatic cleavage of the pLY1 with KpnI and EcoRI and ligating it into the corresponding multiple cloning site of pSB130; and the second step of obtaining the ActinI fragment by amplifying with the specific primers introduced with PstI and HindIII recognition sites and ligating it into the corresponding multiple cloning site of pSB130. Figure SB shows: the third step of obtaining a Lock2 fragment by amplifying with the specific primers introduced with Sail and PstI recognition sites and ligating it into the corresponding multiple cloning site of pSB130; and the last step of obtaining the eYFP fragment by enzymatic cleavage of the pLY1 with KpnI and Sail and ligating it into the corresponding multiple cloning site of pSB130 to construct a pLY2 expression vector.

(7) FIG. 6: a flow diagram of the construction of the expression vector pLB of the lock element (Lock1; SEQ ID NO: 8) and the target gene (cry1Ab/1Ac) according to one embodiment of the present invention, wherein FIG. 6A shows: the first step of obtaining the Nos fragment by amplifying with the specific primers introduced with KpnI/SalI and EcoRI recognition sites and ligating it into the corresponding multiple cloning site of pSB130; and the second step of obtaining the Lock fragment by amplifying with the specific primers introduced with PsiI and XbaI recognition sites and ligating it into the corresponding multiple cloning site of pSB130. FIG. 6B shows: the third step of obtaining the ActinI fragment by amplifying with the specific primers introduced with HindIII and PsiI recognition sites and ligating it into the corresponding multiple cloning site of pSB130; and the last step of obtaining a cry1Ab/1Ac fragment by amplifying with the specific primers introduced with KpnI and SalI recognition sites and ligating it into the corresponding multiple cloning site of pSB130 to construct a pLB expression vector.

(8) FIG. 7: a graphical representation of the functional verification of a set of lock element and key gene according to one embodiment of the present invention, wherein pLY1-C+pKey1 is the observation results under the confocal microscopy of the undifferentiated pLY1 positive callus without green dots after the re-transformation and transient expression of the key gene pKey1, wherein no fluorescence signal is observed under the dark field condition; and pLY1-S+pKey1 is the observation results under the confocal microscopy of the differentiated pLY1 positive callus with green dots after the re-transformation and transient expression of the key gene pKey1, wherein the fluorescence signal is observed under the dark field condition. In the figure, the line segment=20 μM.

(9) FIG. 8: a graphical representation of the functional verification of a set of lock element and key gene according to another embodiment of the present invention, wherein pLY2C+pKey2 is the observation results under the confocal microscopy of the undifferentiated pLY2 positive callus without green dots after the re-transformation and transient expression of the key gene pKey2, wherein no fluorescence signal is observed under the dark field condition; and pLY2-S+pKey2 is the observation results under the confocal microscopy of the differentiated pLY2 positive callus with green dots after the re-transformation and transient expression of the key gene pKey2, wherein the fluorescence signal is observed under the dark field condition. In the figure, the line segment=20 μM.

(10) FIG. 9: the copy number analysis of the Nipponbare lines with pKey1 and pLB positive transformation, wherein: FIG. 9A is the copy number analysis of 9 samples of the Nipponbare line with pKey1 positive transformation, and the results show that 6 samples are single copied; FIG. 9B is the copy number analysis of 10 samples of Nipponbare lines with pLB positive transformation, and the results show that 6 samples are single copied. According to the important agronomic traits such as seed setting rate, plant height and growth stage and other yield traits, two single-copied transformation lines (shown with the red box in the figure) were selected from pKey1 and pLB independent transformants (abbreviated as KEY and LB, respectively) and used as parents for the subsequent production of the LB/KEY and KEY/LB heterozygous hybrid. The selected material number is directly marked above the rectangular box. In the figure: M is DNA Molecular weight Marker II (DIG-labeled), P is a plasmid positive control, and N is a non-transgenic wild type Nipponbare negative control.

(11) FIG. 10: the homozygosity analysis and the PCR detection results of one of T.sub.2 generation plants of the Nipponbare lines with pLB positive single copy transformation. In the Figure: M-DL2000 represents DL2000 DNA Marker; “+” represents a positive plasmid control, “−” represents a non-transgenic negative control; and 1-24 represents 24 independent individual plants in one of T.sub.2 generation of Nipponbare lines with pLB positive single copy transformation.

(12) FIG. 11: ELISA quantitative analysis results of Bt protein contents in stalk and leaf tissues at the tillering, heading and filling stages of six straight- and back-crossed hybrids of 2 Nipponbare pKey1 single copy transformation lines and 2 Nipponbare pLB single copy transformation lines, wherein: KEY1, KEY2 and LB3, LB7 are single copy homozygous parents screened from pKey1 and pLB transformants; LB3/KEY1, LB7/KEY1, LB3/KEY2, LB7/KEY2, KEY1/LB3, and KEY2/LB3 are the heterozygous hybrids crossed by the above single copy homozygous parents. T51-1 and Zheyou 3 are positive controls.

(13) FIG. 12: the resistance of the lock element and key gene heterozygous hybrid LB3/KEY2 (left panel) and wild-type Nipponbare control (right panel) to the artificially inoculated first instar larvae of Chilo suppressalis (1 egg mass, about 80 worms per egg mass) under field condition. In the figure, it can be seen that a high ratio of white spikes appear on the wild type control, and almost no white spikes appear on the transgenic heterozygous hybrids.

(14) FIG. 13: the resistance of the isolated leaves of the lock element and key gene LB3/KEY2 heterozygous hybrids to the indoor artificially inoculated first instar larvae of Chilo suppressalis. In the figure, A: positive control TT51-1; B: wild type Minghui 63 control; C: LB3/KEY2 heterozygous hybrid; D: wild type Nipponbare negative control; E: Nipponbare KEY2 parent transformation line; and F: Nipponbare LB3 parent transformation line. The picture shows that the resistance of transgenic heterozygous hybrid LB3/KEY2 to Chilo suppressalis is consistent with that of the positive control T51-1.

(15) FIG. 14: the resistance of the isolated stalks of the lock element and key gene LB3/KEY2 heterozygous hybrids and wild-type Nipponbare negative control (Nip-CK) to the indoor artificially inoculated first instar larvae of Chilo suppressalis. The picture shows that no signs of infestation of Chilo suppressalis to the isolated stalks of the transgenic LB3/KEY2 heterozygous hybrids are observed, and almost all of the inoculated Chilo suppressalis are killed; while the wormholes, the chomping marks and the excretes caused by Chilo suppressalis are observed on the control stalks, and there are some alive larvae.

(16) The invention may be better understood by reference to the following non-limiting examples, which are provided as the examples of the invention. The following examples are presented to more fully illustrate the embodiments of the invention and should not be construed as limiting the broad scope of the invention.

EXAMPLES

Example 1: Design of the Gene Combination of the Present Invention and its Feasibility Study and Verification

(17) 1. Design of the Gene Combination

(18) As mentioned above, the complete gene combination is designed to consist of two components, i.e., a lock element (Lock) capable of blocking the expression of the target gene and a key gene (Key) capable of tissue-specifically switching on the expression. For the convenience of use, two sets of such components have been designed, wherein the DNA sequences of the first set of components are SEQ ID NO: 5 (Lock1) and SEQ ID NO: 3 (Key1) respectively, and the DNA sequences of the second set of components are SEQ ID NO: 8 (Lock2) and SEQ ID NO: 6 (Key2) respectively. The lock element is designed to be placed between a constitutive expression promoter such as Actin I and the start codon of the target gene, and its function is to switch off the expression of the target gene; while the key gene is designed to be placed under the control of a green tissue-specific expression promoter such as rbcS, and its function is to switch on the expression of the target gene blocked by the lock element in a specific tissue (such as a green tissue).

(19) In order to verify whether the above-mentioned design of gene combinations has the expected switching function, we first tested each of the components with the enhanced yellow fluorescent protein gene eYFP. Then, upon the positive results obtained, we further performed the application study using the insect-resistant Bt hybrid protein gene cry1Ab/1Ac.

(20) 2. Construction of the Validating Expression Vectors for Verifying the Switching Functions

(21) To construct the first set of expression vectors for the lock element and the key gene, all functional fragments were amplified using specific primers introduced with restriction endonuclease recognition sites (Table 1), and high-fidelity PCR technology, and these functional fragments were then ligated into the pSB130 plasmid vector according to the designed arrangement (FIG. 1), thereby obtaining the corresponding gene key expression vector pSB130::rbcS::Key1::NosT (abbreviated as pKey1) and the lock element expression vector pSB130::ActinI::Lock1::eYFP::NosT (abbreviated as pLY1). The specific construction processes are shown in FIG. 2 and FIG. 3, respectively.

(22) Based on the first set of expression vectors as successfully constructed, a second set of expression vectors were constructed, wherein some functional fragments were obtained from the first set of expression vectors by the enzymatic cleavage, and the other functional fragments were amplified by the specific primers (Table 1) and high-fidelity PCR amplification technology, and then ligated into the pSB130 plasmid vector according to the designed arrangement (FIG. 1), thereby obtaining the corresponding key gene expression vector pSB130::rbcS::Key2::NosT (abbreviated as pKey2) and the lock element expression vector pSB130::ActinI::Lock2::eYFP::NosT (abbreviated as pLY2). The specific construction processes of the two expression vectors are shown in FIG. 4 and FIG. 5, respectively.

(23) 3. Construction of the Applied Expression Vectors for Insect Resistance

(24) Since the validating and applied expression vectors are identical in the portion of key gene, and differ only in the fragment of target gene to which the lock element is attached, the construction of the set of the applied expression vectors is mainly performed for the lock element and the fragment of target gene. At the time of construction, on the one hand, some functional fragments common to the validating expression vectors are obtained by enzymatic cleavage, and on the other hand, the lock element in the first set of expression vectors and two insect-resistant gene functional fragments to be ligated are selected in the examples of the present invention and obtained by the specific primers (Table 1) and high-fidelity PCR amplification technology respectively, then these functional fragments are ligated into the pSB130 plasmid vector (FIG. 1) according to the designed arrangement, thereby obtaining the corresponding applied lock element expression vector pSB130::ActinI::LB1::NosT (abbreviated as pLB1). The specific construction process of the expression vector is shown in FIG. 6.

(25) The pSB130 plasmid vector used in the examples of the present invention comprises two “T-DNA” regions, wherein one “T-DNA” region is used to carry a lock element linking to a target gene, or a key gene expression cassette, and the other is used to carry a hygromycin resistant marker gene Hpt expression cassette. The purpose of constructing the double T-DNA expression vector is to focus on future practical applications, because the transformation of the double T-DNA plasmid vector into rice can enable the possibility of the independent integration of the target gene and the marker gene in the receipt genome in order to facilitate the segregation and knock out of the marker gene by self-crossing in subsequent segregation generations.

(26) TABLE-US-00001 TABLE 1 Primers, amplified functional fragments and their DNA sequences for construction of the lock element and key gene expression vectors Amplified functional Primers fragment DNA sequence pKey1 pKey1-4F HindIII-- GATCAAGCTTCACTTAAATTTTGGTGACAGGAATGTAGTTTTCTG RbcS-SalI (SEQ ID NO: 19) PKey1-4R GTACGTCGACCTCTGCAGCTCACCAAGCTCTCTCCTTCTTTGCTC (SEQ ID NO: 20) pKey1-3F SalI-- TCGAATGGCGGCGGTTAGGAGAAGAGAACGAGATGTGGTTGAAGAGAATGGAGTTAC Key 1  GACGACGACGGTGAAACGAAGGAAG 5′--SmaI (SEQ ID NO: 21) pKey1-3R CTTCCTTCGTTTCACCGTCGTCGTCGTAACTCCATTCTCTTCAACCACATCTCGTTCTCT TCTCCTAACCGCCGCCAT (SEQ ID NO: 22) pKey1-2F XbaI-  GATCTCTAGAGTACCCCGGGATGTCCAATTTACTGACCGTACACCAAAATTTGCCTGCA Sma I-- TT Key1 (SEQ ID NO: 23) PKey1-2R 3′--KpnI GTACGGTACCCTAATCGCCATCTTCCAGCAGGCGCACCATTGCCC (SEQ ID NO: 24) pKey1-1F KpnI-- GATCGGTACCGCTAGCTCGAATTTCCCCGATCGTTCAAACATTTG Nos-- (SEQ ID NO: 25) pKey1-IR EcoRI GTACGAATTCGACACCGCGCGCGATAATTTATCCTAGTTTGCGCG (SEQ ID NO: 26) pLY1 pLY1-3F HindIII-- GATCAAGCTTGTCGAGGTCATTCATATGCTTGAGAAGAGAGTCGG Actin-- (SEQ ID NO: 27) pLY1-3R PstI GTACCTGCAGTCTACCTACAAAAAAGCTCCGCACGAGGCTGCATT (SEQ ID NO: 28) pLY1-2F PstI- GATCCTGCAGATAACTTCGTATAGCATACATTATACGAAGTTATGCTAGCTCGAATTTC Lockl-- CCCGATCGTTCAAACATTTG XbaI (SEQ ID NO: 29) pLY1-2R GTACTCTAGAATAACTTCGTATAATGTATGCTATACGAAGTTATGACACCGCGCGCGAT AATTTATCCTAGTTTGCGCG (SEQ ID NO: 30) pLY1-4F XbaI-- GATCTCTAGAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGT eYFP-- (SEQ ID NO: 31) pLY1-4R KpnI GTACGGTACCTCAAGATCTCTTGTACAGCTCGTCCATGCCGAGAG (SEQ ID NO: 32) pLY1-1F KpnI-- GATCGGTACCGCTAGCTCGAATTTCCCCGATCGTTCAAACATTTG Nos-- (SEQ ID NO: 25) pLY1-1R EcoRI GTACGAATTCGACACCGCGCGCGATAATTTATCCTAGTTTGCGCG (SEQ ID NO: 26) pKey2 pKey2-2F XbaI- CGAGATCTGAAGGAAGCAAAGTGGCAGCA Key2 (SEQ ID NO: 33) pKey2-2R 5′--SalI ACGCGTCGACTACCGCCGCCAATCCTCTT (SEQ ID NO: 34) pKey2-3F SacI- CGAGCTCAATATACGCAGATAAAT Key2 (SEQ ID NO: 35) p.Key2-3R 3′--XbaI CGAGATCTTACCGTAGGTATTTA (SEQ ID NO: 36) pKey2-1F EcoRI-- GGCCTTAAGGGCTAGATCATTGTAT Nos-- (SEQ ID NO: 37) pKey2-1R SacI CGAGCTCCTTAAAGGGGCTAGCAA (SEQ ID NO: 38) pLY2 pLY2-1F Pst- AACTGCAGTCTTCAAGGATATGAAAGATCTCITATCCTTGAAGGGCTAGATCATTGTA Lock2-- TCT SaI (SEQ ID NO: 39) pLY2-1R CGCAGCTGGAAGTTCCTATTCTCTAGAAAGTATAGGAACTTCGAATTTCCCCGATCGTT C (SEQ ID NO: 40) pLY1-2F SalI-- GATCGTCGACATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGT eYFP-- (SEQ ID NO: 31) pLY1-2R KpnI GTACGGTACCTCAAGATCTCTTGTACAGCTCGTCCATGCCGAGAG (SEQ ID NO: 32) pLB pLB-1F HindIII-- GATCAAGCTTGTCGAGGTCATTCATATGCTTGAGAAGAGAGTCGG Actin-- (SEQ ID NO: 27) pLB-1R PstI GTACCTGCAGTCTACCTACAAAAAAGCTCCGCACGAGGCTGCATT (SEQ ID NO: 28) pLB-2F PstI-- GATCCTGCAGATAACTTCGTATAGCATACATTATACGAAGTTATGCTAGCTCGAATTTC Lock1-- CCCGATCGTTCAAACATTTG XbaI (SEQ ID NO: 29) pLB-2R GTACTCTAGAATAACTTCGTATAATGTATGCTATACGAAGTTATGACACCGCGCGCGA TAATTTATCCTAGTTTGCGCG (SEQ ID NO: 30) pLB-3F KpnI-- GATCGGTACCATGGACAACAACTGCAGGCCATACAACTGCTTGAGTAACC crylAb/ (SEQ ID NO: 41) pLB-3R Ac--SalI GTACGTCGACTTATTCAGCCTCGAGTGTTGCAGTAACTGGAATGA (SEQ ID NO: 42) pLB-4F KpnI- GATCGGTACCGTACGTCGACGCTAGCTCGAATTTCCCCGATCGTTCAAACATTTG SalI-- (SEQ ID NO: 43) pLB-4R Nos-- GTACGAATTCGACACCGCGCGCGATAATTTATCCTAGTTTGCGCG EcoRI (SEQ ID NO: 26)

Example 2: Verification of the Functions of Switching Off/on the Expression of the Exogenous Target Gene

(27) A two-step transformation method is used for verifying the functions of switching off/on the expression of the exogenous target gene. In the first step, the two lock elements pLY1 and pLY2 in the validating expression vector prepared in Example 1 were introduced into rice embryogenic callus for persistent expression using Agrobacterium-mediated method (Liu et al., 1998). Thereafter, after several consecutive rounds (3-5 rounds, 12-14 days/round) of antibiotic screening, the resistant callus is subject to pre-differentiation (7-9 days) and differentiation culture until green spots are formed (7-9 days). In the second step, the two key genes pKey1 and pKey2 in the validating expression vectors were bombarded into the differentiated positive callus with green spots and the undifferentiated positive callus without green spots for the transient expression by the gene gun-mediated method (Tu et al., 2000). After 24-36 h, the luminescence of the yellow fluorescent protein was observed under the confocal microscopy. If the designed gene combination has complete switch functions, then the undifferentiated positive callus without green dots transformed by pKey1 and pKey2 does not fluoresce at all, and the differentiated positive callus with green dots transformed by them can fluoresce. The detailed test steps are described as follows:

(28) 1. Receipt Material

(29) The variety used as a receipt for genetic transformation of the above validating lock elements pLY1 and pLY2 is japonica Nipponbare. This variety is the model variety of rice genetic transformation, and its mature embryo callus is easy to be induced and has high genetic transformation efficiency.

(30) 2. Medium and Composition Used in the Rice Transformation and Growth

(31) 2.1. Induction/subculture medium (per liter): 4.1 g/L N6 (Chu et al., 1975) basal salt components+N6 organic components (Table 2)+2 mg/L 2,4-dichlorphenoxy acetic acid (2,4-D)+2.0 g/L hydrolyzed casein+30 g/L sucrose+3 g/L agar, pH 5.9.

(32) 2.2. Transfection medium (per liter): AA basal medium (Table 3) (Toriyama & Hinata, 1985)+200 μM acetosyringone, pH 5.9.

(33) 2.3. Co-cultivation medium (per liter): CC basal medium (Table 4) (Hiei et al, 1994)+200 μM acetosyringone, pH 5.9.

(34) 2.4. Bacteriostatic medium (per liter): induction/subculture medium+500 mg/L cephalosporin, pH 5.9.

(35) 2.5. Screening medium (per liter): induction/subculture medium+50 mg/L hygromycin+500 mg/L cephalosporin, pH 5.9.

(36) 2.6. Regeneration medium (per liter): 4.1 g/L N6 basal salt components+N6 organic components (Table 2)+2.0 g/L hydrolyzed casein+30 g/L sucrose+6 g/L agar+2 mg/L Kinetin+1 mg/L α-naphthylacetic acid, pH 5.9.

(37) 2.7. Rice rooting medium (per liter): 2.05 g/L N6 basal salt components+½ N6 organic components (Table 2)+1.0 g/L hydrolyzed casein+15 g/L sucrose+3 g/L agar, pH 5.9.

(38) TABLE-US-00002 TABLE 2 N6 basal salt components (Shanghai Shenggong) and organic components Basal salt Component components concentration (mg/L) Major elements KNO.sub.3 2830 (NH4).sub.2SO.sub.4 463 KH.sub.2PO.sub.4 400 MgSO.sub.4 .Math. 7H.sub.2O 185 CaCl.sub.2 .Math. 2H.sub.2O 166 Trace elements FeSO.sub.4 .Math. 7H.sub.2O 27.85 Na.sub.2EDTA 37.25 MnSO.sub.4 .Math. H.sub.2O 4.4 ZnSO.sub.4 .Math. 7H.sub.2O 1.5 H.sub.3BO.sub.3 1.6 KI 0.8 Organic components Inositol 100 Glycine 2.0 Niacin 0.5 Pyridoxine 0.5 Thiamine 1.0

(39) TABLE-US-00003 TABLE 3 Formula of AA medium Basal Concentration of the the amount Stock medium components in the of stock liquor liquor components stock liquor (g/L) added/liter Major element components I KCl 58.8 take 50 mL MgSO.sub.4 .Math. 7H.sub.2O 7.4 to 1 L CaCl.sub.2 .Math. 2H.sub.2O 8.8 KH.sub.2PO.sub.4 3.4 Trace element components II MnSO.sub.4 .Math. H.sub.2O 1.69 takel 10 mL ZnSO.sub.4 .Math. 7H.sub.2O 0.86 to 1 L H.sub.3BO.sub.3 0.62 KI 0.083 CuSO.sub.4 .Math. 5H.sub.2O 0.0025 CoCl.sub.2 .Math. 6H.sub.2O 0.0025 Na.sub.2MoO.sub.4 .Math. 2H.sub.2O 0.025 Organic components III Inositol 10.0 take 10 mL Arginine 22.8 to 1 L VB5 0.05 Aspartic acid 26.6 VB1 0.01 VB6 0.05 Glycine 7.5 Glutamine 87.7 AA basal + sucrose 30 g/L + glucose 10 g/L + hydrolyzed casein 0.5 g/L + 2,4-D 2 mg/L pH 5.9

(40) TABLE-US-00004 TABLE 4 Formula of CC medium Basal Concentration of the the amount of Stock medium components in the stock liquor liquor components stock liquor (g/L) added/liter Major element components I NH.sub.4NO.sub.3 12.80 take 50 mL KNO.sub.3 24.24 to 1 L MgSO.sub.4 .Math. 7H.sub.2O 4.94 KH.sub.2PO.sub.4 2.72 CaCl.sub.2 .Math. 2H.sub.2O 11.76 Trace element components II MnSO.sub.4 .Math. H.sub.2O 1.116 takel 10 mL ZnSO.sub.4 .Math. 7H.sub.2O 0.576 to 1 L H.sub.3BO.sub.3 0.310 KI 0.084 CuSO.sub.4 .Math. 5H.sub.2O 0.0025 CoCl.sub.2 .Math. 6H.sub.2O 0.0028 Na.sub.2MoO.sub.4 .Math. 2H.sub.2O 0.024 Organic components III Inositol 9.00 take 10 mL VB5 0.60 to 1 L VB1 0.85 VB6 0.10 Glycine 0.2 100X Fe-salt IV FeSO.sub.4 .Math. 7H.sub.2O 2.785 take 10 mL Na.sub.2-EDTA 3.725 to 1 L CC basal + sucrose 20 g/L + mannitol 36.43 g/L + hydrolyzed casein 50 mg/L + 2,4-D 2.0 mg/L + glucose 10.0 g/L + agar 3.0 g/L, pH 5.9

(41) 3. Transformation of the Lock Element

(42) As described above, the genetic transformation of the validating lock element expression vectors pLY1 and pLY2 was carried out using the Agrobacterium-mediated method. The Agrobacterium strain used is EHA105 (BioVector NTCC Inc.), and the recipient cell used was Nipponbare mature embryo-induced embryogenic callus. The specific steps of Agrobacterium transformation are as follows.

(43) 3.1 Induction of Rice Embryogenic Callus

(44) Several mature Nipponbare seeds were taken and shelled. The full-grained, good embryo unrefined rice were placed into a pre-autoclaved beaker, soaked in 70% alcohol for 1 min under continuous shaking to remove the surface impurities; then soaked and sterilized with 20% sodium hypochlorite for 20 min (while shaking on a shaker if required); then rinsed with sterile distilled water for 4 to 5 times to dilute and remove the sodium hypochlorite remaining on the surface of the unrefined rice. The aseptically treated unrefined rice were inoculated on MS callus induction and subculture medium containing 2.0 mg/L 2,4-D (Table 2), and cultured at 28° C. in dark for about 2 weeks until a suitable size of nascent callus was produced at the scutellum of mature embryos. Thereafter, the nascent callus was cut and transferred to fresh callus induction and subculture medium, subcultured under the same conditions, subcultured every 2 weeks until the embryogenic callus with the thick cytoplasm, the bright yellow color, the hard texture and the granular cell mass was formed.

(45) 3.2 Genetic Transformation and Shake Culture of Agrobacterium Strain EHA105

(46) 0.5 μL the expression vector plasmid was added into a 1.5 mL centrifuge tube containing 60 μL electrocompetent Agrobacterium EHA105, mixed uniformly with the pipette and transferred into the electrode cuvette. After the electroporation, 1 mL LB liquid medium was quickly added, mixed uniformly with the pipette and then transferred to the previous 1.5 mL centrifuge tube and shaken for 1 h at 28° C. in a shaker. After the recovery of the strain, 100 μL strain solution was pipetted and uniformly plated onto the surface of LB solid screening (containing 50 mg/L kanamycin, 25 mg/L rifampicin) medium (Shanghai Shenggong), and cultured at 28° C. for 2 days. After verification of the positive colonies by PCR, the positive clones were cultured by shaking, and the resulting strain solutions were stored at 50% glycerol concentration and −80° C. for future use.

(47) 3.3 Agrobacterium Transfection and Subscreening of the Rice Callus

(48) The Agrobacterium transfection was carried out according to the procedure reported by Yang et al., (2011). The specific procedure is as follows: the Agrobacterium solution stored at −80° C. was thawed, and 200 μL of the solution was uniformly plated onto the surface of the LB solid medium containing 25 mg/l rifampicin and 50 mg/L kanamycin and cultured at 28° C. overnight. The single colonies were picked and expanded with LB liquid medium. Thereafter, 200-300 μL of the fresh strain solution was removed and inoculated into 20 mL of the LB liquid culture containing 25 mg/L rifampicin and 50 mg/L kanamycin, and cultured under shaking (220 rpm) at 28° C. for 16-18 h. A sufficient amount of the strain solution was centrifuged at 4000 rpm for 15 min, and the supernatant of the LB medium was discarded. The Agrobacterium was resuspended by adding 20 mL 0.1 M MgSO.sub.4 solution (slightly pipetted with a pipette), and centrifuged at 4000 rpm for 10-15 min. The MgSO.sub.4 supernatant containing antibiotics was discarded. The Agrobacterium was resuspended by further adding 5 mL AA transfection medium containing 200 μM Acetosyringone (AS) (Table 3), and an appropriate amount of AA-AS transfection medium was added, so that the OD.sub.600 value of the strain solution was finally adjusted to be between 0.8 and 1.0. After the concentration adjusting, the strain solution was dispensed in sterile 50 mL centrifuge tubes (20-25 mL/tube), until the future use.

(49) Before the Agrobacterium transfection, the embryogenic callus was firstly pre-cultured for about 7 days, and then transferred from the subculture dish to an empty culture dish covered with a sterile filter paper, and air-dried on the ultra-clean workbench for about 10-15 minutes, during which, the callus was fully dried by slowly tumbling it with a sterilized spoon. After drying, it was transferred to a centrifuge tube containing the strain solution, and gently shaken at room temperature for 40 min, and the centrifuge tube was settled on the superclean workbench for 10 min. The strain solution was discarded, and the embryogenic callus was placed on the sterile filter paper and dried for about 15 min, then it was transferred to the CC co-cultivation medium (Table 4) containing AS (200 μM) with the surface covered with a sterile filter paper, and cultured in dark at 28° C. for 50-55 h. The embryogenic callus without the significant growth of the Agrobacterium or contaminated Agrobacterium on the surface was transferred onto the N6 bacteriostatic medium containing 2.0 mg/L 2,4-D, 500 mg/L Cefortaxim, and was bacteriostatic cultured in dark at 28° C. for 3-4 d. After the bacteriostatic culture, the callus was then transferred to the screening medium containing 500 mg/L cephalosporin and 50 mg/L Hygromycin, and cultured in dark at 28° C. The callus in the good growth state was picked for subculture every half month and the concentration of cephalosporin in the culture was adjusted according to the degree of contamination. In general, the concentration can be considered to be halved in the third or fourth round subculture. Such subculture was continued until a resistant callus with the rapid growth, large amount, and vivid color was obtained (4-6 rounds of screening and subculture).

(50) 3.4 the Differentiation Culture of the Resistant Callus

(51) The resistant callus obtained in the above step was transferred to N6 regeneration medium, and was pre-differentiated in the dark chamber at 28° C. for one week, then transferred onto a fresh N6 regeneration medium, and cultured in a light chamber for differentiation at 25° C. until green dots were formed (requiring about two weeks).

(52) 4. Key Gene Transformation

(53) Using the callus with the green dots formed in the above step as a receptor, the validating key gene expression vectors pKey1 and pKey2 prepared according to Example 1 were introduced into the differentiated callus with green dots and the undifferentiated callus without green dots by using the gene gun-mediated method described in Tu et al. (2000) for transient expression. After 24 to 36 h of the recovery culture, the fluorescence signal of the yellow fluorescent protein was observed under a confocal microscope and photographed and stored.

(54) 5. Experimental Results

(55) The test results are shown in FIGS. 7 and 8. As can be seen from the figures, under the confocal microscope, there is no fluorescence signal observed for the undifferentiated (without green spots) pLY1 and pLY2 positive callus upon retransformation and transient expression of the key gene expression vectors pKey1 and pKey2, while the fluorescence signal can be observed only in the differentiated (having green spots) pLY1 and pLY2 positive callus upon retransformation and transient expression of the key gene expression vectors pKey1 and pKey2. These test results thus confirm that both sets of gene combination as designed can properly and effectively switching off/on the gene expression.

Example 3: Construction of the Applied Lock Element and Key Gene Transformation Lines and Production of the Heterozygous Hybrid

(56) The genetic transformation of the applied lock element and key gene was carried out using the Agrobacterium-mediated method. Excepting using of the applied lock element expression vector pLB1 and the key gene expression vector pKey1 prepared according to Example 1, the selection of the receipt material, the various media used, the genetic transformation of the vector and the pre-differentiation and differentiation cultures of the positive resistant callus were all identical to the corresponding test procedures described in Example 2 until the positive resistant callus was differentiated into green seedlings. After washing away the medium on the root system, the resulting green seedlings were transferred into the Yoshida medium (Table 5) for the intermediate culture, either directly (for the plants having roots and buds differentiated at the same time) or after fully rooting in the N6 rooting medium (for the plants having the buds differentiated firstly). After the growth state was good and stable, it was transplanted to the greenhouse until it was mature.

(57) TABLE-US-00005 TABLE 5 The formula of the Yoshida nutrient solution (Yoshida, 1976) Concentration of the Stock components in the the amount of stock liquor Components stock liquor (g/10 L) liquor added/4 liters A NH.sub.4NO.sub.3 914.0 5 B NaH.sub.2PO.sub.4 .Math. 2H.sub.2O 403.0 5 C K.sub.2SO.sub.4 714.0 5 D CaCl.sub.2 886.0 5 MgSO.sub.4 .Math. 7H.sub.2O 3240.0 MnCl.sub.2 .Math. 4H.sub.2O 15.0 (NH.sub.4).sub.6Mo.sub.7O.sub.24 .Math. 4H.sub.2O 0.74 H.sub.3BO.sub.3 9.34 E ZnSO.sub.4 .Math. 7H.sub.2O 0.35 5 CuSO.sub.4 .Math. 5H.sub.2O 0.31 FeCl.sub.3 .Math. 6H.sub.2O 77.0 Citric acid 119.0 (monohydrate) pH 5.0

(58) After the genetic transformation of the above steps, 42 pKey1 independent transformants and 59 pLB1 independent transformants were obtained. Thereafter, the selections of the homozygous lines were carried out on each of the two groups of the independent transformants, and the production of the heterozygous hybrids between the two groups of the independent transformants and the insect resistance identification were performed. The details are as follows.

Example 4: Molecular Analysis of the Applied Lock Element and Key Gene Transformation Lines

(59) 1. DNA Extraction

(60) 1.1 DNA Mini-Extraction

(61) A fresh leaf (length of 2-3 cm) was removed from the transgenic plants obtained according to Example 3, placed in a mortar, added 500 μL of 1.5×CTAB extraction solution, and ground to homogenate. The homogenate was transferred to a 1.5 mL centrifuge tube, incubated in a 56° C. water bath for 20 min, then add 500 μL chloroform:isopentanol (24:1), and mixed thoroughly (upside down for several times), centrifuged at room temperature for 5 min (14,000 r/min). 300 ul of the supernatant was added to 600 μL anhydrous ethanol (precooled at −20° C.), mixed uniformly and placed at −20° C. for more than 30 min, and centrifuged at room temperature for 5 min (14,000 r/min). The supernatant was discarded and the DNA pellet was washed with 75% ethanol. Thereafter, the ethanol was discarded, the DNA pellet was air-dried at room temperature, and finally dissolved in 200 μL of sterile water for use.

(62) 1.2 DNA Maxi-Extraction

(63) 3-5 adult rice leaves (3-5 g) were removed, added liquid nitrogen and rapidly ground to powder form, transferred to a pre-cooled 50 mL centrifuge tube, immediately added 15-20 mL preheated boiling 1.5×CTAB, shaken uniformly and placed in a 56° C. water bath for 30 min. Then one volume of an extraction solvent (chloroform:isopentanol at 24:1) was added, spun gently for 30 min until the solution is separated into three layers, with the upper, middle and lower lays being yellow, green and black respectively. It was centrifuged at 4000 rpm for 20 minutes at room temperature (temperature>18° C.), the supernatant was removed and added 1/10 volume of 56° C. preheated 10×CTAB, and shaken uniformly. Again, one volume of the extraction solvent (chloroform:isopentanol at 24:1) was added and spun gently for 30 min. It was centrifuged at 4000 rpm for 20 min at room temperature (temperature>18° C.), then the supernatant was discard with a 5 mL pipette tip and added one volume of 1×CTAB. After spin, flocculent DNA formed was picked up with the tip and transferred into the bottom of a 1.5 mL centrifuge tube, which was placed upside down and dried for 10 min on absorbent paper. Then 10 μL RNase and 0.5 ml sterilized 1 M NaCl were added and dissolved at room temperature or in a 56° C. water bath. The dissolved DNA was added to a 2 volumes (−20° C.) of −20° C. pre-cooled 95% ethanol. The flocculent DNA formed was precipitated by spin, and placed into a 1.5 mL centrifuge tube with a pipette tip, added 1.5 mL of 75% ethanol to wash the DNA, then the ethanol was discarded, added ultrapure water and stored at 4° C. for use.

(64) 2. Detection of the Positive Transgenic Plants and Identification of the Homozygous Lines

(65) The detection of the positive transgenic plants and identification of the homozygous lines were performed using PCR techniques. Since the Hpt marker gene and the lock element or key gene were co-transformed by double T-DNA, the identification of the TO generation positive transformation line was based on the target gene only. Therefore, after the detection, only the plants positive for the target gene were cultivated to mature, and the non-positive plants were all discarded to reduce the workload of the subsequent generations.

(66) The primers used for amplifying anti-hygromycin gene are: Hpt-F, 5′-GCTGTTATGCGGCCATTGTC-3′ (SEQ ID NO: 9) and Hpt-R, 5′-GACGTCTGTCGAGAAGTTTC-3′ (SEQ ID NO: 10). The PCR reaction system (25 μL system) is: 1 ul of sample DNA template to be tested, 2.5 μL of 10×PCR buffer, 2 μL of 10 mM dNTP, 0.25 μL of 20 uM primer, 0.5 μL of 2 U/μL Taq polymerase, and ddH.sub.2O, total 25 μL. The PCR reaction procedure is: denaturation at 94° C. for 3 min; followed by 35 cycles of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec, extension at 72° C. for 40 sec; followed by extension at 72° C. for 5 min and then hold at 10° C. The amplified products were identified by 1% agarose gel electrophoresis, photographed and stored.

(67) The primers used for amplifying Bt gene are: BtF, 5′-GGCCATACAACTGCTTGAGT-3′ (SEQ ID NO: 11); BtR, 5′-GCGTTTCCCATAGTTCCATA-3′ (SEQ ID NO:12), and the amplified fragment was 1 Kb in length. The PCR reaction procedure is: denaturation at 94° C. for 3 min; followed by 35 cycles of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec, extension at 72° C. for 1 min; followed by extension at 72° C. for 5 min and then hold at 10° C. The amplified products were identified by 1% agarose gel electrophoresis, photographed and stored. The PCR reaction system is shown in table 6.

(68) The primers used for amplifying the key gene are: KeyF, 5′-AACGAGTGATGAGGTTCGCA-3′ (SEQ ID NO:13); KeyR, 5′-ACCCGGCAAAACAGGTAGTT-3′ (SEQ ID NO:14), and the amplified fragment was 672 bp in length. The PCR reaction procedure is: denaturation at 94° C. for 3 min; followed by 35 cycles of denaturation at 94° C. for 30 sec, annealing at 55° C. for 30 sec, extension at 72° C. for 1 min; followed by extension at 72° C. for 7 min and then hold at 4° C. The amplified products were identified by 1% agarose gel electrophoresis, photographed and stored. The PCR reaction system is shown in table 6.

(69) TABLE-US-00006 TABLE 6 PCR reaction system Ingredients Working solution Stock solution μl/tube PCR buffer (+Mg.sup.2+) 1 X 10 X 2.5 dNTP 0.2 mmol/L 2.5 mmol/L 2 Forward primer 0.2 μmol/L 5 μmol/L 1 (BtF or KeyF or HptF) Reverse primer 0.2 μmol/L 5 μmol/L 1 (BtR or KeyR or HptR) Tag polymerase 0.625U 5U/μL 0.125 Template DNA 50ng 50ng/μL 1 ddH.sub.2O 17.375 Total 25

(70) Thus, 21 pKey1- and 22 pLB-positive transformation lines were identified from 41 pKey1 and 50 pLB independent transformed lines of Nipponbare, respectively.

(71) 3. Detection of Transgenic Copy Number

(72) The detection of transgenic copy number was performed using Southern hybridization techniques. First, the total rice DNA was digested with restriction endonuclease HindIII overnight at 37° C. (8-12 h). The digested DNA was separated by 0.8% (w/v) agarose gel electrophoresis (40V, 12 h). Thereafter, the electrophoretically separated DNA blot was transferred to a nylon membrane (GE Healthcare, UK). The preparation of the hybridization probes and Southern hybridization and hybridization signal detection were carried out according to the instructions provided by Roche DIG-High Prime DNA Labeling and Detection Starter Kit II (Switzerland), wherein the primers for preparing the hybridization probes are respectively:

(73) TABLE-US-00007 pKey1: (SEQ ID NO: 15) Key-1U20, 5′-ATGTCCAATTTACTGACCGT-3′, and (SEQ ID NO: 16) Key-800L20, 5′-GCTTCAAAAATCCCTTCCAG-3′; pLB: (SEQ ID NO: 17) Bt-577U24, 5′-AGGCTGATTGGAAACTACACCGAC-3′, and (SEQ ID NO: 18) Bt-1161L24, 5′-ACAGCGGATGGCAAGTTAGAAGAG-3′.

(74) The copy number analysis results of the pKey1- and pLB-positive transformation lines of Nipponbare are shown in FIG. 9. As can be seen from FIG. 9, among 9 pKey1 positive Nipponbare transformation lines detected (FIG. 9A), 6 lines are single copied, and among 10 pLB positive transformation lines detected (FIG. 9B), 6 lines are single copied.

(75) 4. PCR Amplification Detection and Screening of Single Copy Transgenic Homozygous Lines

(76) The PCR amplification detection and screening of single copy transgenic homozygous lines were performed in T.sub.2 generation lines. T.sub.2 generation seeds and plants were obtained after self-crossing the KEY1 and LB single copy transformation lines for two generations, respectively. Thereafter, the homozygous lines respectively carrying KEY1 and LB were screened out by PCR amplification detection. FIG. 10 shows the PCR detection results of one of T.sub.2 generation homozygous lines of the Nipponbare pLB single copy transformation line. As shown in FIG. 10, 24 individual plants of the T.sub.2 generation line show positive LB gene amplified fragments, indicating that the T.sub.2 generation line is an LB transgenic homozygous line. The PCR amplification detections of other pLB and pKey1 transgenic homozygous lines were also performed in this way (data not shown).

Example 5: Determination of Bt Protein Content in the Stalk and Leaf and Identification of Insect Resistance of the Heterozygous Hybrid Prepared with the Applied Lock Element and Key Gene

(77) 1. Material Planting

(78) The Nipponbare pKey1 single-copy transgenic homozygous lines KEY1 and KEY2, which were screened by the PCR amplification detection described in Example 4 and were excellent in seed setting rate and other agronomic traits, were straight-crossed and back-crossed with the pLB single-copy transgenic homozygous lines LB3 and LB7, respectively. As a result, six hybrids were obtained totally.

(79) In 2014, all tested materials (10 lines total), including wild-type Nipponbare negative control, 1 KEY (KEY1) and 2 LB (LB3 and LB7) transformation parental lines and six hybrids straight- and back-crossed by the above pKey1 single-copy transgenic homozygous line with pLB single-copy transgenic homozygous line were sown on June 25. 15 days later, 6 KEY/LB and LB/KEY straight- and back-crossed hybrid young seedlings were positively detected for positivity using the Bt Cry1Ab/1Ac transgenic rapid test strip (item number: AA0232, Youlong, Shanghai) according to the steps described in the product instruction. The hybrid young seedlings detected positive, together with the wild type negative control and the seedlings of the parent materials, were transplanted to the mud pool with the transgenic special isolation net, with at 1 row/section and 10 lines/row on July 28. The plant spacing was 20 cm and the row spacing was 40 cm. The experiment was repeated 3 times. Two rows of non-transgenic rice plants were planted around the entire test area as protection lines. No pesticides were applied during the whole growth period, and water and fertilizer managements were the same as the local field.

(80) In 2015, the test materials were the same as those in 2014. All the tested materials were sown on May 25, and 15 days later, were detected by Bt Cry1Ab/1Ac transgenic rapid test strips (item number: AA0232, Youlong, Shanghai). The hybrid young seedlings confirmed as positive, together with the wild type control and the seedlings of the parent materials, were transplanted to the mud pool with the transgenic special isolation net, at 2 row/section and 12 lines/row on June 25. The plant spacing was 20 cm, and the row spacing was either 40 cm (wide spacing) or 15 cm (narrow spacing). The experiment was repeated 3 times. The rest of the cultivation managements were the same as those in 2014.

(81) 2. Determination of Bt Protein Content in Stalks and Leaves

(82) (1). ELISA Kit and Rice Samples

(83) The Bt protein contents in the stalks and leaves of Nipponbare KEY/LB and LB/KEY straight- and back-crossed hybrids were detected using an enzyme-linked immunosorbent assay (ELISA) kit from EnviroLogix™ Inc., Portland, USA. Rice stalk and leaf samples were sampled in three stages: tillering stage, heading stage and filling stage.

(84) (2). Preparation of Working Solutions and Samples for Bt Protein Content Determination

(85) The determination working solution includes the extraction solution/dilution solution and the elution solution. The elution solution is prepared by adding the phosphate in the kit to 1 L double distilled water and then stirring to dissolve completely. The extraction solution/dilution solution is prepared by adding 200 μL of the elution solution into 1 mL of Tween-20, and stirring to dissolve completely. Both working solutions are stored at 4° C. and preheated to room temperature before use.

(86) 3 replicates were set for each heterozygous hybrid with 1 plant per replicate. The removed fresh samples were ground into powder with liquid nitrogen, about 20 mg powder was taken for each material, added the extraction/dilution solution in a ratio of 20 mg/500 μL and shaken fully to extract protein sample. The homogenate was placed into a 1.5 mL centrifuge tube and allowed to stand on ice for 30 min, then the supernatant was pipetted, centrifuged at 4000 rpm for 3 min at 4° C., and the supernatant was discarded. After dilution with the extraction/dilution solution at certain dilution factors, the Bt Protein concentration was determined and the dilution of the negative control was unnecessary. The dilution factors for the stalk and leaf samples at various stages are shown in Table 7.

(87) TABLE-US-00008 TABLE 7 Dilution factor of the Bt protein sample Bt protein Stages leaf stalk Cry1Ab/1Ac tillering stage 200 50 heading stage 200 50 filling stage 200 50

(88) 3. ELISA Reaction and Protein Content Calculation

(89) The ELISA reaction process was carried out according to the instructions of the kit. The specific steps were as follows:

(90) (1) 50 μL of enzyme-conjugated Cry1Ab/1Ac antibody was added to each well of the plate (96 wells);

(91) (2) Cry1Ab/1Ac standard samples at different concentration gradients (1, 0.5, 0.25, 0.1 ppb), 50 μL blank sample (extraction/dilution solution) and the sample to be tested were added into the well of the above 96-well plate with 1 sample/well. Two technical replicates were set for one sample, and then the 96-well plate was sealed with sealant and shaken at low speed (200 rpm) for 1-2 h;

(92) (3) The supernatant was discarded, each well of a 96-well plate was added with 300 μL of the elution solution, shaken gently for 1 min. The elution solution was discarded and the plate was placed upside down on the absorbent paper to drain off the residual liquid;

(93) (4) Step 3 was repeated for 4 times;

(94) (5) Each well of a 96-well plate was added 100 μL of the substrate, and the wells corresponding to the samples containing Bt protein would appear blue, and each well was sealed and protected from light, and shaken at low speed (200 rpm) for about 30 min;

(95) (6) Each well of a 96-well plate was added with 100 μL of the reaction stop solution, the blue color became yellow color, and the absorbance value was immediately read.

(96) The absorbance value was determined using a microplate reader (Synergy H1, USA) reading at a wavelength of 450 nm. A standard curve is plotted with the concentration and absorbance of the standard samples. The concentration of the test sample was read from the standard curve, and the protein content in the rice tissue was calculated based on the dilution factor. The protein content was calculated by the following formula: Bt protein content (μg/g fresh weight)=test sample concentration (ng/mL)×dilution factor×sample loading volume (μL)/tissue fresh weight (mg). Differences in protein concentration among the different hybrid combinations, different stages, and tissues were compared using multiple t-tests of the grouping data.

(97) The results of the two-year analysis are shown in FIG. 11. In the three growth stages, the Bt protein contents in the leaves of the six straight- and back-crossed hybrids are consistent with or higher than those in the positive control T51-1 and its two-hybrid rice Zheyou 3 (FIGS. 11A and 11C). The Bt protein contents in the stalks of 3 heterozygous hybrids LB3/KEY2, LB7/KEY2 and KEY2/LB3 are consistent with that in Zheyou 3 positive control. The Bt protein contents in the stalks of the other 3 heterozygous hybrids LB3/KEY1, LB7/KEY1 and KEY1/LB3 are all lower than those in the above two positive controls (FIGS. 11B and 11D).

(98) 4. Evaluation of the Insect Resistance in the Field

(99) The evaluation of the resistance of KEY/LB and LB/KEY straight- and back-crossed hybrids to Chilo suppressalis in the field was carried out using the natural insect inoculation method and the artificial insect inoculation method. The natural insect inoculation method was carried out 5-7 days after the peak of Chilo suppressalis outbreak. The indicators include the number of dead hearts and the number of white spikes. The artificial insect inoculations were carried out for two times at the highest tillering stage and one week before the heading. The eggs were purchased from Jiangxi Shennong Technology Co. Ltd., and the purchased eggs were placed in a glass test tube with a length of 12 cm and a diameter of 2.0 cm with a wet cotton ball at the bottom (one egg mass/tube), the tube was sealed with a cotton plug and tied up with a black cloth on the outside of the tube mount. Egg hatching was carried out at a temperature of 26-28° C. and a humidity of 80% under 16 hours light/8 hours dark condition. The hatched first instar larvae were inoculated together with the test tube to the base of the rice bundle within 6 hours after hatching. The specific procedure is as follows: the black cloth was opened and the cotton plug was removed, the base of the test tube was inserted into the soil of the rice field by making the tube mouth close to the rice straw, so that the larva can climb from the test tube to the rice straw for infestation. The inoculated amount of insects was one egg mass (about 80 first instar larvae)/plant. Two weeks later, the number of dead hearts or the number of white spikes caused by Chilo suppressalis was determined, and meanwhile, the total number of tillers or the total number of effective spikes of the test plants were determined.

(100) The insect resistance of LB/KEY and KEY/LB straight- and back-crossed hybrids to Cnaphalocrocis medinalis in the field was evaluated by the natural insect inoculation method. The number of the damaged leaves and the degree of the damage to leaves per plant caused by Cnaphalocrocis medinalis were determined 5-7 days after the occurrence of the infestation peak.

(101) The statistical analysis of the resistance evaluation indexes of LB/KEY and KEY/LB straight- and back-crossed hybrids to Chilo suppressalis and Cnaphalocrocis medinalis was carried out with SPSS22.0 (SPSS, Chicago, USA) software. The significance of the difference of the Chilo suppressalis harm rate between the hybrids and the wild type Nipponbare and the parental control with the lock element and the Bt gene was done by Dunnett's t test.

(102) The results of the natural insect inoculation identification in the field in 2014 demonstrate that six straight- and back-crossed hybrids are highly resistant to Cnaphalocrocis medinalis. The number of the damaged tillers per plant and the number of the damaged leaves per plant of the wild type Nipponbare and the parental lines carrying the lock element and Bt gene reach 54.81-65.27% and 9.17-14.60%, respectively, while the two indicators of the six straight- and back-crossed hybrids are only 0.00-1.33% and 0.00-0.13% (Table 8). The difference therebetween reaches an extremely significant level of P=0.01. In addition, the four heterozygous hybrids LB3/KEY2, LB7/KEY2, KEY1/LB3 and KEY2/LB3 were also well resistant to the naturally inoculated Chilo suppressalis. Compared with the Chilo suppressalis damage rate of 6.96-8.02% in the wild type Nipponbare and the parental lines with lock element and Bt gene (Table 8), the Chilo suppressalis damage rates in these heterozygous hybrids are only within 0.00-0.63%. The difference therebetween also reaches an extremely significant level of P=0.01.

(103) The results of the natural insect inoculation identification in the field in 2015 are essentially the same as those in 2014. Six straight- and back-crossed hybrids all exhibit the good field resistance to Cnaphalocrocis medinalis and Chilo suppressalis. The difference between the hybrids and the wild type Nipponbare and the parental lines with lock element and Bt gene also reach an extremely significant level of P=0.01 (Table 8).

(104) As for the artificially insect inoculation identifications, in 2014, two hybrids with low Bt protein content in the stem and leaf tissues, LB3/KEY1 and LB7/KEY1, were selected as the materials to be tested, and the wild type Nipponbare and two parents with the lock element and Bt gene, LB3 and LB7, were used as the insect-sensitive controls. The results show that the Chilo suppressalis damage rates in the four control lines were more than 23%, while those in the two heterozygous hybrids were less than 8% (Table 9), thus showing the good resistance to Chilo suppressalis.

(105) In 2015, the artificial insect inoculation in the field was repeated with one heterozygous hybrid LB3/KEY2 with relatively high Bt protein content in the stalk. The results show that the damage of Chilo suppressalis to the heterozygous hybrid is extremely low and the average damage rate of the tillers or spikes of the rice is only 0.78%, which is significantly lower than 56.26% of the wild-type Nipponbare negative control (Table 9 and FIG. 12).

(106) TABLE-US-00009 TABLE 8 the resistance of six LB/KEY and KEY/LB hybrid combinations, three KEY and LB parents, and wild-type Nipponbare control to Chilo suppressalis and Cnaphalocrocis medinalis under the field conditions.sup.a 2014 2015 Chilo Cnaphalocrocis medinalis Chilo Cnaphalocrocis medinalis suppressalis damage rate (%) suppressalis damage rate (%) damage damaged damage damage damaged damage rate tiller per leaves per rate tiller per leaves per Lines (%) plant plant (%) plant plant WT.sup.b 7.59 ± 0.69.sup.c 63.82 ± 10.70 12.47 ± 1.20 7.22 ± 1.00 59.76 ± 6.14 12.17 ± 0.57 KEY2 6.96 ± 0.79 54.81 ± 0.93*  9.17 ± 0.91** 7.46 ± 1.39 56.71 ± 7.69 10.70 ± 0.61** LB3 8.02 ± 0.77 65.27 ± 8.19 12.47 ± 1.20 6.73 ± 0.56 62.33 ± 2.09 11.67 ± 0.42 LB7 7.39 ± 0.47 58.36 ± 3.59 14.60 ± 0.62** 7.02 ± 0.41 61.49 ± 1.29 13.37 ± 0.67** LB3/KEY1 ND  0.65 ± 1.12**  0.13 ± 0.23** 0.28 ± 0.48**  0.00 ± 0.00**  0.00 ± 0.00** LB7/KEY1 ND  1.33 ± 2.31**  0.13 ± 0.23** 0.00 ± 0.00**  0.25 ± 0.44**  0.03 ± 0.06** LB3/KEY2 0.00 ± 0.00**  0.31 ± 0.53**  0.03 ± 0.06** 0.00 ± 0.00**  0.26 ± 0.45**  0.00 ± 0.00** LB7/KEY2 0.00 ± 0.00**  0.00 ± 0.00**  0.00 ± 0.00** 0.00 ± 0.00**  0.00 ± 0.00**  0.00 ± 0.00** KEY1/LB3 0.63 ± 0.55**  0.63 ± 0.55**  0.07 ± 0.06** 0.00 ± 0.00**  0.26 ± 0.45**  0.07 ± 0.12** KEY2/LB3 0.00 ± 0.00**  0.00 ± 0.00**  0.00 ± 0.00** 0.00 ± 0.00**  0.00 ± 0.00**  0.00 ± 0.00** .sup.aThe data of each line are based on three replicates and 10 individual plants are randomly surveyed per replicate for each line. .sup.bWT is wild-type Nipponbare control, and the identification data of all other 9 lines to be tested are compared with the wild type control. The data are analyzed by Dunnett t test of SPSS 22.0 software package. .sup.cValues represent average ± mean standard deviation. ND. represents no data. **represents a very significant difference P < 0.01.

(107) TABLE-US-00010 TABLE 9 the resistance of LB3/KEY2 hybrid combinations to Chilo suppressalis artificially inoculated at the tillering stage and 7 days before heading under the field conditions The number Chilo Identi- of the The mean suppressalis fication individual tillering damage rate year Plant replicate tested number (%) 2014 Nip-WT 1 10 13.5 36.30 ± 25.75 2 10 14.5 28.97 ± 13.72 3 10 14.3 25.17 ± 10.27 mean 10 14.1 30.15 ± 5.65  KEY1 1 10 12.3 34.96 ± 31.66 2 10 11.4 21.93 ± 18.03 3 10 11.7 32.48 ± 15.03 mean 10 11.8 29.79 ± 6.92  LB3 1 10 11.6 30.17 ± 12.76 2 10 12.9 30.23 ± 23.94 3 10 12.5 28.00 ± 10.99 mean 10 12.3 29.47 ± 1.27  LB7 1 10 14.3 24.48 ± 7.79  2 10 15.0 23.33 ± 7.10  3 10 14.2 23.94 ± 8.79  mean 10 14.5 23.92 ± 0.57* LB3/ 1 10 10.7 11.21 ± 10.79 KEY1 2 10 10.3 4.85 ± 6.10 3 10 10.3 4.85 ± 7.25 mean 10 10.4   6.97 ± 3.67** LB7/ 1 10 10.90 10.09 ± 15.53 KEY1 2 10 10.30 5.83 ± 9.31 3 10 10.00 7.00 ± 9.98 mean 10 10.40   7.64 ± 2.20** 2015 Nip-WT 1 24 12.5 59.47 ± 20.64 2 24 11.9 48.77 ± 23.34 3 24 13.6 60.55 ± 19.14 mean 24 12.7 56.26 ± 6.51  LB3/ 1 24 12.8 0.32 ± 1.46 KEY2 2 24 13.3 0.63 ± 2.55 3 24 11.9 1.40 ± 3.70 mean 24 12.7   0.78 ± 0.56** *represents that the difference from the control reaches P < 0.05 significant level, and **represents that the difference from the control reaches P < 0.01 extremely significant level.

(108) 3. Indoor Insect Resistance Evaluation

(109) The indoor insect resistance evaluation was carried out on Chilo suppressalis. The eggs were also purchased from Jiangxi Shennong Technology Co. Ltd., and the egg hatching method was the same as described the above. The method used for the insect resistance evaluation includes the isolated stalk insect inoculation method and the isolated leaf insect inoculation method.

(110) The isolated stalk insect inoculation method is as follows: the rice stalk after the jointing is removed and cut into 12 cm long stalk segments with the joints and leaf sheaths. Two stalk segments of the same plant and 20 first instar larvae of Chilo suppressalis are placed in each insect tube. The insect tube is sealed with a cotton plug, placed at 25-27° C. and 80% relative humidity, and the larval mortality is counted after 6 days.

(111) The isolated leaf insect inoculation method is as follows: the blade leaves and the penultimate leaves at the booting stage are removed and cut into 8 cm long leaf segments, and placed in a culture dish with filter paper moistened with 2 mL of distilled water. 4 leaf segments and 15 first instar larvae of Chilo suppressalis are placed in each culture dish. The culture dishes are wrapped with PARAFILM four times to prevent larvae from escaping out. Thereafter, they are housed at room temperature (25° C.), and the mortality of the larvae is counted after 6 days.

(112) Each insect tube or culture dish is counted as 1 replicate, with 3 replicates per sample. The resistance of different LB/Key1 and Key1/LB transformed heterologous hybrids to Chilo suppressalis was compared using t test of the grouped stalk and leaf data. The results demonstrate that under the indoor artificial inoculation conditions, the insect resistance performances of the isolated leaves and stalks of LB3/KEY2 heterologous hybrids to Chilo suppressalis are also excellent (FIGS. 13 and 14). The mortality of the Chilo suppressalis larvae fed with the isolated leaves is 100%, and the mortality of the Chilo suppressalis larvae fed with the isolated stalks is also more than 95%, while the mortality of the Chilo suppressalis larvae fed with the isolated control leaves and stalks is no more than 20% (Table 10).

(113) TABLE-US-00011 TABLE 10 Resistance of the leaves and stalks of the LB3/KEY2 heterozygous hybrids to artificially inoculated Chilo suppressalis The mortality of the Chilo suppressalis larvae (%) Leaves Stalks Replicate Replicate Replicate Replicate Lines Replicate I II III Mean Replicate I II III Mean LB3/KEY2 100.0 100.0 100.0 100.0 ± 0.0  100.0 95.0 95.0 96.7 ± 2.9  Nip-WT 6.7 13.3 6.6 8.8 ± 3.8 20.0 10.0 0.0 10.0 ± 10.0

Example 6: Determination of Cry1Ab/1Ac Protein Content in the Endosperm of the Heterologous Hybrids with Applied Lock Element/Key Gene

(114) In order to verify whether the gene combination has the function of preventing the target gene in an endosperm-specific manner, the Cry1Ab/1Ac protein contents in the endosperms of the six lock element/key gene heterozygous hybrids described in Example 5 were further determined.

(115) 1. ELISA Kit and Rice Samples

(116) The Cry1Ab/1Ac protein content in the endosperm of Nipponbare pKey1/pLB heterologous hybrid was also determined using the enzyme-linked immunosorbent assay (ELISA) kit from EnronoLogix™ Inc. (Portland. USA). The rice is detected at the filling period.

(117) 2. Preparation of Working Solutions and Samples for Cry1Ab/1Ac Protein Content Determination

(118) The determination working solution includes the extraction solution/dilution solution and the elution solution. The elution solution is prepared by adding the phosphate in the kit to 1 L double distilled water and then stirring to dissolve completely. The extraction solution/dilution solution is prepared by adding 200 μL of the elution solution into 1 mL of Tween-20, and stirring to dissolve completely. Both working solutions are stored at 4° C. and preheated to room temperature before use.

(119) Each hybrid combination consisted of 3 replicates with 1 plant/replicate and 50 seeds/plant. The harvested seeds were hulled and prepared into unrefined rice for use. Then, some unrefined rice were polished into refined rice using a polishing machine, and 50 unrefined rice and 50 refined rice were selected and ground into flours, and about 20 mg of flours were taken from each material, and the extraction/dilution solutions were added in a ratio of 20 mg/500 μL to extract the protein samples under intense shaking. The resulting homogenate was placed in a 1.5 mL centrifuge tube, allowed to stand on ice for 30 min, and centrifuged at 4000 rpm for 3 min at 4° C., and the supernatant was pipetted for use. Thereafter, the protein samples extracted from the refined rice and unrefined rice of the positive control T51-1 and its two-line hybrid rice Zheyou 3 were diluted 50 times with the extracting/diluting solution, and then the Bt protein concentrations were measured. The dilutions of the samples of the heterozygous hybrid and negative control were unnecessary.

(120) 3. ELISA Reaction and Protein Content Calculation

(121) The ELISA reaction process was carried out according to the instructions of the kit, and the specific steps were the same as described for the Bt protein content determination in the aforementioned stalk and leaf tissues.

(122) 4. Determination of Cry1Ab/1Ac Protein Contents in the Refined Rice Endosperms and the Unrefined Rice Flours of 6 Heterozygous Hybrids

(123) The results of repeated measurements in 2014 and 2015 showed that the Cry1Ab/1Ac protein contents of the refined rice endosperm of 6 heterozygous hybrids were not significant different compared with the detection values of the wild type Nipponbare negative control and the LB3 parental line carrying the lock element and Bt gene. Their measured values were all less than 1 ng/g flour, which was extremely significantly lower than the average measured value of the positive control T51-1 (338.45 ng/g flour) and its two-line hybrid rice Zheyou 3 (184.75 ng/g flour) in the two years (Table 11). This result therefore confirms that the “gene combination” as designed in the present invention does completely switch off the expression of the exogenous target gene in the rice endosperm.

(124) The Cry1Ab/1Ac protein content determined in unrefined rice of some heterozygous hybrid reached 1-3 ng/g flour, significantly higher than the detection value of the unrefined rice of the wild type Nipponbare negative control (less than 1 ng/g flour) (Table 11). This might be related with the fact that the pericarp in the unrefined rice (composed of several parts such as peel, seed coat, perisperm and aleurone layer) contains the chloroplast at the beginning of the development. However, during the milling, the pericarp of the unrefined rice was milled as a bran layer, and therefore, a very small amount of Cry1Ab/1Ac protein accumulated therein was also removed.

(125) TABLE-US-00012 TABLE 11 ELISA quantitative analysis of the Cry1Ab/1Ac protein contents in the unrefined rice and refined rice of the parental KEY and LB lines, hybrid LB/KEY and KEY/LB lines, positive control T51-1 and Zheyou 3 and wild-type negative control Nipponbare Cry1Ab/1Ac protein content (ng/g).sup.a 2014 2015 Lines Refined rice Unrefined rice Refined rice Unrefined rice WT.sup.b .sup. 0.42 ± 0.08.sup.c 0.71 ± 0.11 0.21 ± 0.07 0.24 ± 0.11 LB3 0.67 ± 0.37  1.36 ± 0.59* 0.22 ± 0.08   1.81 ± 0.48** LB3/KEY1 0.44 ± 0.06 1.07 ± 0.13 0.23 ± 0.08 0.33 ± 0.03 LB7/KEY1 0.44 ± 0.04 0.86 ± 0.09 0.17 ± 0.05 0.61 ± 0.25 LB3/KEY2 0.80 ± 0.20   2.09 ± 0.15** 0.25 ± 0.02  1.19 ± 0.49* LB7/KEY2 0.64 ± 0.10   2.52 ± 0.06** 0.28 ± 0.12 1.06 ± 0.27 KEY1/LB3 0.29 ± 0.05 1.04 ± 0.01 0.14 ± 0.08 0.20 ± 0.05 KEY2/LB3 0.60 ± 0.13   1.98 ± 0.17** 0.25 ± 0.08   1.50 ± 0.58** T51-1  371.67 ± 30.85**  879.36 ± 67.71**  305.23 ± 27.44** 1040.33 ± 60.38** Zheyou 3 157.32 ± 27.2**  400.53 ± 34.79**  212.17 ± 47.27** 578.69 ± 1.10** .sup.arepresents that the data in the table are measured from 50 grains/test material/grain sampled randomly (repeated three times in total); .sup.bWT is the wild-type Nipponbare positive control, and the measurement data of all other lines are compared. The data are analyzed by Dunnett t test of SPSS 22.0 software package. .sup.cValues represent average ± mean standard deviation. *represents that the difference from the wild-type Nipponbare negative control reaches P < 0.05 significant level. **represents that the difference from the wild-type Nipponbare negative control reaches P < 0.01 extremely significant level.
Sequence Listing and Annotations

(126) The present invention provides the nucleotide sequence and the putative amino acid sequence thereof of the exogenous target gene Bt, gene lock Lock1/Lock2 and gene key Key1/Key2 for constructing a “gene combination”, wherein the Bt gene cry1Ab/1Ac is artificially synthesized and kindly provided by the Academician Yunliu FAN of the Academy of Agricultural Sciences; the lock element Lock1/Lock2 and the key gene Key1/Key2 were artificially synthesized. The respective sequences listed in the sequence listing are as follows.

(127) TABLE-US-00013 SEQ ID NO: Annotations  1 Bt gene coding sequence; 1833 bp in total, wherein the composition ratio of bases A, T, G and C is 476: 479: 401: 477  2 putative Bt protein sequence; 610 amino acid residues in total, wherein the first 489 amino acid residues are from cry1Ab, and the last 121 amino acid residues are from cry1Ac  3 DNA sequence of key gene Key1; 1116 bp in total, wherein the composition ratio of bases A, C, G and T is 287: 248: 319: 262  4 putative KEY1 protein sequence; 371 amino acid residues in total  5 lock element (Lock1); 333 bp in total, the sequence does not encode protein, and is only the regulatory sequence  6 DNA sequence of key gene Key2: 1428 bp in total, wherein the composition ratio of bases A, C, G and T is 495: 255: 293: 385  7 putative KEY2 protein sequence; 475 amino acid residues in total  8 selectable lock element (Lock2), 333 bp in total  9 hygromycin-resistant gene primer Hpt-F 10 hygromycin-resistant gene primer Hpt-R 11 Bt gene PCR amplification primer BtF 12 Bt gene PCR amplification primer BtR 13 Key gene PCR amplification primer KeyF 14 Key gene PCR amplification primer KeyR 15 pKey1 SOUTHERN hybridization probe preparation primer Key1-1U20 16 pKey1 SOUTHERN hybridization probe preparation primer Key1-800L20 17 pLB1 SOUTHERN hybridization probe preparation primer Bt-577U24 18 pLB1 SOUTHERN hybridization probe preparation primer Bt-1161L24 19-43 Primers used for the expression vector (see Table 1)

REFERENCES

(128) 1. Bock R., 2001, Transgenic plastids in basic research and plant biotechnology. J. Mol. BioL., 312:425-438. 2. Cai M., Wei J., Li X. H., Xu C. G., Wang S. P. 2007, A rice promoter containing both novel positive and negative cis-elements for regulation of green tissue-specific gene expression in transgenic plants. Plant Biotechnol J, 5:664-674. 3. Chu C., Wang C., Sun C., Hsu C., Yin K., Chu C., Bi F., 1975, Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Sci Sin., 18:659-668. 4. Daniell H., 2002, Molecular strategies for gene containment in transgenic crops, Nat. Biotechnol., 20(6):581-586. 5. Hiei Y., Ohta S., Komati T., 1994, Efficient transformation of rice (Oryza saliva L.) mediated by Agrobacterium and sequence analysis of the boundaries of T-DNA. Plant J., 6:271-282. 6. James, Clive, 2014. Global Status of Commercialized Biotech/GM Crops: 2014. ISAAA Brief No. 49. ISAAA: Ithaca, N.Y. 7. Jang I. C., Nahm B. H., Kim J. K., 1999, Subcellular targeting of green fluorescent protein to plastids in transgenic rice plants provides a high-level expression system. Mol Breeding, 5:453-461. 8. Kyozuka J., McElroy D., Hayakawa T., Xie Y. W|u R., Shimamoto K., 1993, Light-regulated and cell-specific expression of tomato rbcS-gusA and rice rbcS-gusA fusion genes in transgenic rice. Plant Physiol, 102:991-1000. 9. Liu Q. Q., Zhang J. L., Wang Z. Y., Hong M. M., Gu M. H., 1998, A highly efficient transformation system mediated by Agrobacterium tumefaciens in rice. Acta Phytophysiol. Sin., 24:259-271. 10. Luo K., Duan H., Zhao D., Zheng X., Deng W., Chen Y., Neal Stewart Jr. C., McAvoy R., Jiang X., Wu Y., He A., Pei Y., Li Y., 2007, “GM-gene-deletor”: fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seeds of tobacco plants. Plant Biotech J., 5(2):263-274. 11. Nomura M., Katayama K., Nishimura A., IshidaY., Ohm S., Komari T., Miyao T. M., Tajima S., Matsuoka M., 2000, The promoter of rbcS in a C3 plant (rice) directs organ-specific, light-dependent expression in a C4 plant (maize), but does not confer bundle sheath cell-specific expression. Plant Mol Biol, 44:99-106. 12. Oliver M., Quisenberry J. E., Trolinder N. L. G., 1998, Control of plant gene expression. United States Patent, US005723765A 13. Paoletti M. G., Pimentel D., 1996, Genetic engineering in agriculture and the environment. Bioscience, 46(9):665-673. 14. Thilmony R., Guttman M., Thomson J. G., Bleeh A. E., 2009, THE LP2 leueine-rich repeat receptor kinase gene promoter directs organ-specific, light-responsive expression in transgenic rice. Plant Biotechnol J., 7:867-882. 15. Toriyama K., Hinata, K., 1985, Cell suspension and protoplast culture in rice. Plant Sci., 41:179-183. 16. Tu J., Zhang G., Datta K., Xu C, He Y., Zhang Q., Khush G. S., Datta S. K., 2000, Field performance of transgenic elite commercial hybrid rice expressing Bacillus thuringiensis S-endotoxin. Nature Biotech, 18:1101-1104. 17. Yanagisawa S., Izui K., 1989, Maize Phosphoenol pyruvate Carboxylase Involved in C4 Photosynthesis: Nucleotide Sequence Analysis of the 5′ Flanking Region of the Gene. J. Biochem., 106:982-987. 18. Yang R., Tang Q., Wang H., Zhang X., Pan G., Wang H., Tu J., 2011, Analyses of two rice (Oryza sativa L.) cyclin-dependent kinase inhibitors and effects of transgenic expression of OsiICK6 on plant growth and development. Annals of Botany. 107:1087-1101. 19. Ye R. J., Zhou F., Lin Y. J., 2012. Two novel positive eis-regulatory elements involved in green tissue-specific promoter activity in rice (Oryza sativa L ssp.). Plant Cell Rep, 31:1159-1172. 20. Yoshida S., Forno D. A., Cock J. H., Gomez K. A., 1976, Routine procedures for growing rice plants in culture solution. In Laboratory Manual for Physiological Studies of Rice pp. 61-66. Philippines, IRRI, Manila. 21. Zhao D., Lv L., He A., Luo K., Duan H., Zheng X., Deng W., Chen Y., An X., He M., 2008. The gene-deletor technology: principle and potential application in genetically engineered agriculture, Molecular Plant Breeding, 2008, 6(3): 413-418.

(129) The invention is not limited to the scope of the specific embodiments described herein. In fact, various modifications of the invention in addition to those described herein will be apparent to those skilled in the art reading the above description. These modifications should fall within the scope of the appended claims.

(130) It should also be understood that all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weight or molecular mass values are approximate and are provided for illustration.