Use of insecticidal protein

Abstract

Related is a use of an insecticidal protein. The insecticidal protein may be used to control Apolygus lucorum. A method for controlling the Apolygus lucorum includes: allowing the Apolygus lucorum to be at least in contact with an ACh1 protein. In the present application, the ACh1 protein that can kill the Apolygus lucorum is produced in bacteria and/or a plant in vivo to control the Apolygus lucorum.

Claims

1. A method for controlling Apolygus lucorum, comprising allowing the Apolygus lucorum to be at least in contact with an ACh1 protein; preferably, the ACh1 protein is present in a host cell that produces at least the ACh1 protein, and the Apolygus lucorum is in contact with at least the ACh1 protein by ingesting the host cell; and more preferably, the ACh1 protein is present in bacteria or a transgenic plant that produces at least the ACh1 protein, the Apolygus lucorum is in contact with at least the ACh1 protein by ingesting the bacterium or tissue of the transgenic plant, and after contacting, the growth of the Apolygus lucorum is inhibited and/or death is caused, so as to achieve the control of the damage of the Apolygus lucorum to plants.

2. The method for controlling Apolygus lucorum according to claim 1, wherein the transgenic plant is cotton, soybean, or rape.

3. The method for controlling Apolygus lucorum according to claim 1, wherein the tissue of the transgenic plant is a bud, a leaf, a cotton boll, a tassel, an ear, or a filament.

4. The method for controlling Apolygus lucorum according to claim 1, wherein the ACh1 protein is an ACh1_1 protein, an ACh1_2 protein, an ACh1_3 protein, or an ACh1_4 protein.

5. The method for controlling Apolygus lucorum according to claim 4, wherein the ACh1 protein has an amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

6. The method for controlling Apolygus lucorum according to claim 4, wherein the ACh1 protein has a nucleotide sequence in the bacteria that is shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8; and the ACh1 protein has a nucleotide sequence in the transgenic plant that is shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

7. The method for controlling Apolygus lucorum according to claim 1, wherein the transgenic plant further comprises at least one second nucleotide different from the nucleotide encoding the ACh1 protein.

8. The method for controlling Apolygus lucorum according to claim 7, wherein the second nucleotide encodes a Cry-like insecticidal protein, a Vip-like insecticidal protein, a protease inhibitor, lectin, α-amylase, or a peroxidase.

9. The method for controlling Apolygus lucorum according to claim 7, wherein the second nucleotide is a dsRNA that inhibits an important gene in a target insect pest.

10. A method of producing a plant for controlling Apolygus lucorum, comprising introducing a polynucleotide sequence encoding an ACh1 protein into a genome of the plant.

11. The method of producing a plant for controlling Apolygus lucorum according to claim 10, wherein the polynucleotide sequence of the ACh1 protein is shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14.

12. The method of producing a plant for controlling Apolygus lucorum according to claim 10, wherein the ACh1 protein has an amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

13. A method for producing a plant seed for controlling Apolygus lucorum, comprising hybridizing a plant obtained by the method according to claim 10 with a second plant, so as to produce a seed containing a polynucleotide sequence encoding an ACh1 protein.

14. A method for cultivating a plant for controlling Apolygus lucorum, comprising: planting at least one plant seed, wherein a genome of the plant seed comprises a polynucleotide sequence encoding an ACh1 protein; growing the plant seed into a plant; and growing the plant under conditions that the Apolygus lucorum is artificially inoculated and/or the hazard of the Apolygus lucorum naturally occurs, and harvesting a plant that has an attenuated plant damage and/or has an increased plant yield compared with other plants that do not have the polynucleotide sequence encoding the ACh1 protein.

15. The method of cultivating a plant for controlling Apolygus lucorum according to claim 14, wherein the polynucleotide sequence of the ACh1 protein is shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14.

16. The method of cultivating a plant for controlling Apolygus lucorum according to claim 14, wherein the ACh1 protein has an amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] FIG. 1 is a construction flowchart of a recombinant expression vector DBN01-P containing an ACh1 nucleotide sequence for the use of the insecticidal protein according to the present application.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0082] The technical schemes of the use of the insecticidal protein of the present application are further described below by specific embodiments.

Example 1: Acquisition and Synthesis of Gene

[0083] 1. Acquisition of the Nucleotide Sequence

[0084] An amino acid sequence of an ACh1_1 insecticidal protein (309 amino acids) is shown in SEQ ID NO:1 in the sequence listing. An ACh1_1 nucleotide sequence (930 nucleotides) encoding the amino acid sequence corresponding to the ACh1_1 insecticidal protein in bacteria is shown in SEQ ID NO: 5 in the sequence listing. In the transgenic plant, an ACh1_1 plant nucleotide sequence (930 nucleotides) encoding the amino acid sequence corresponding to the ACh1_1 insecticidal protein is shown in SEQ ID NO:11 in the sequence listing.

[0085] An amino acid sequence of an ACh1_2 insecticidal protein (306 amino acids) is shown in SEQ ID NO:2 in the sequence listing. An ACh1_2 nucleotide sequence (921 nucleotides) encoding the amino acid sequence corresponding to the ACh1_2 insecticidal protein is shown in SEQ ID NO: 6 in the sequence listing. In the transgenic plant, an ACh1_2 plant nucleotide sequence (921 nucleotides) encoding the amino acid sequence corresponding to the ACh1_2 insecticidal protein is shown in SEQ ID NO:12 in the sequence listing.

[0086] An amino acid sequence of an ACh1_3 insecticidal protein (309 amino acids) is shown in SEQ ID NO:3 in a sequence listing. An ACh1_3 nucleotide sequence (930 nucleotides) encoding the amino acid sequence corresponding to the ACh1_3 insecticidal protein is shown in SEQ ID NO: 7 in the sequence listing. In the transgenic plant, an ACh1_3 plant nucleotide sequence (930 nucleotides) encoding the amino acid sequence corresponding to the ACh1_3 insecticidal protein is shown in SEQ ID NO:13 in the sequence listing.

[0087] An amino acid sequence of an ACh1_4 insecticidal protein (309 amino acids) is shown in SEQ ID NO:4 in a sequence listing. An ACh1_4 nucleotide sequence (930 nucleotides) encoding the amino acid sequence corresponding to the ACh1_4 insecticidal protein is shown in SEQ ID NO: 8 in the sequence listing. In the transgenic plant, an ACh1_4 plant nucleotide sequence (930 nucleotides) encoding the amino acid sequence corresponding to the ACh1_4 insecticidal protein is shown in SEQ ID NO:14 in the sequence listing.

[0088] 2. Synthesis of Above Nucleotide Sequence

[0089] The ACh1_1 nucleotide sequence (as shown in SEQ ID NO:5 in the sequence listing), the ACh1_2 nucleotide sequence (as shown in SEQ ID NO:6 in the sequence listing), the ACh1_3 nucleotide sequence (as shown in SEQ ID NO:7 in the sequence listing) and the ACh1_4 nucleotide sequence (as shown in SEQ ID NO:8 in the sequence listing) are synthesized by Nanjing Genscript Biotechnology Co., Ltd.

Example 2: Construction of Recombinant Expression Vector and Transformation of Recombinant Expression Vector into Escherichia coli to Obtain ACh1 Protein

[0090] 1. Construction of Recombinant Expression Vector Containing ACh1 Gene

[0091] The synthesized ACh1_1 nucleotide sequence is linked into a protein expression vector pET28a (Novagen, USA, CAT: 69864-3); operation steps are performed according to the specification of the product pET28a vector of Novagen, so as to obtain a recombinant expression vector DBN01-P; and a construction flow is shown in FIG. 1 (where Kan represents a kanamycin resistance gene, f1 ori represents the origin of replication of phage f1, Lac1 is a Lac1 initiation codon, ACh1_1 is an ACh1_1 bacterial nucleotide sequence (SEQ ID NO:5), and MCS represents multiple cloning sites).

[0092] According to the above method for constructing the recombinant expression vector DBN01-P, the synthesized ACh1_2 nucleotide sequence is linked to the protein expression vector pET28a, so as to obtain a recombinant expression vector DBN02-P, and ACh1_2 is the ACh1_2 bacterial nucleotide sequence (SEQ ID NO:6).

[0093] According to the above method for constructing the recombinant expression vector DBN01-P, the synthesized ACh1_3 nucleotide sequence is linked to the protein expression vector pET28a, so as to obtain a recombinant expression vector DBN03-P, and ACh1_3 is the ACh1_3 bacterial nucleotide sequence (SEQ ID NO:7).

[0094] According to the above method for constructing the recombinant expression vector DBN01-P, the synthesized ACh1_4 nucleotide sequence is linked to the protein expression vector pET28a, so as to obtain a recombinant expression vector DBN04-P, and ACh1_4 is the ACh1_4 bacterial nucleotide sequence (SEQ ID NO:8).

[0095] 2. Transformation of Recombinant Expression Vector into Escherichia coli to Obtain ACh1 Protein

[0096] Then, the recombinant expression vectors DBN01-P, DBN02-P, DBN03-P, and DBN04-P are transformed into Escherichia coli BL21(DE3) competent cells (Transgen, China, CAT: CD501) by a heat shock method; a positive colony is picked and placed in an LB liquid medium (10 g/L of a tryptone, 5 g/L of a yeast extract, 10 g/L of NaCl, 100 mg/L of an ampicillin, and pH is adjusted to 7.5 with NaOH); and culture is performed for 16 h at 37° C. and at 200 r/min. The culture solution is then transferred to an YT culture medium according to the proportion of 1:10; and culture is performed at 37° C. and at 200 r/min. When an OD=600 value of the culture solution reaches 0.6-0.8, IPTG is added until a final concentration is 0.5 mM, so as to perform inducible expression for 6 h, and the culture solution is centrifuged to collect the cells; the supernatant is discarded, resuspending is performed after PBS is added, and then ultrasonic disruption is performed; and the expression protein is detected by SDS-PAGE, the protein concentration is estimated, and preservation is performed at −20° C. for later use.

Example 3, Identification of Inhibitory Activity Against Apolygus lucorum by Feeding ACh1 Protein

[0097] Inhibitory activity against the Apolygus lucorum is detected with the ACh1_1, ACh1_2, ACh1_3, and ACh1_4 proteins obtained in 2 in the Example 2. A total of 4 treatments are designed for each pest, which respectively are ACh1_1, ACh1_2, ACh1_3, and ACh1_4; and 1 negative control treatment is designed, which is GFP. Protein liquid of ACh1_1, ACh1_2, ACh1_3, ACh1_4, and GFP are respectively mixed in feed, and a final concentration is 500 μg/g. Each group of treatments is repeated for 6 times.

TABLE-US-00001 TABLE 1 Anti-insect results of Apolygus lucorum fed with ACh1 protein Serial number Test insect of proteins Apolygus lucorum ACh1_1 + ACh1_2 + ACh1_3 + ACh1_4 + GFP − “+” means that there is inhibitory activity against the pest; and “−” means that there is no inhibitory activity against the pest.

[0098] Results of Table 1 show that, the ACh1_1, ACh1_2, ACh1_3, and ACh1_4 proteins have desirable inhibitory activity against the Apolygus lucorum.

[0099] Therefore, it indicates that the ACh1_1, ACh1_2, ACh1_3, and ACh1_4 proteins show resistance activity against the Apolygus lucorum, and this activity is sufficient to have adverse effects on the growth of the Apolygus lucorum, so that the Apolygus lucorum can be controlled in the fields. In addition, it is also possible to reduce the occurrence of diseases on the transgenic ACh1 plants by controlling the damage of the Apolygus lucorum, thereby greatly improving the yield and quality of the transgenic ACh1 plants.

[0100] In conclusion, through the use of the insecticidal protein of the present application, ACh1 protein that can kill the Apolygus lucorum is produced in bacteria and/or a plant body to control the Apolygus lucorum. Compared with an agricultural control method, a chemical control method and a physical control method used in the prior art, the present application achieves the protection of whole growth period and whole plant on the plants so as to control the infestation of the Apolygus lucorum, and is pollution-free, residue-free, stable in effect, thorough, simple, convenient and economical.

[0101] Finally, it should be noted that the above embodiments are only used to illustrate the technical schemes of the present application and not to limit them. Although the present application is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical schemes of the present application may be modified or equivalently replaced without departing from the spirit and scope of the technical schemes of the present application.