ROOT-SECRETED PEPTIDE PEP1 IN RICE AND GENE ENCODING THE SAME AND USE THEREOF

Abstract

The disclosure isolates and identifies a peptide PEP1 that regulates plant root development and a gene OsPEP1 encoding the same. Exogenous application of PEP1 could inhibit the plant root development. A recombinant expression vector containing the gene or part of the DNA of the gene is obtained, and a transgenic plant with altered root growth and development is obtained by transforming with the recombinant expression vector. Therefore, the peptide can be used as a plant growth regulator, and the gene encoding the same and precursor protein thereof can be used as a potential molecular breeding target for crop improvement, for example, improving crop yield by regulating the growth and development of crop roots.

Claims

1. A method for shortening or lengthening root length of a plant, comprising administering to the plant a peptide for regulating plant root development, wherein the peptide for regulating plant root development is selected from the group consisting of: a peptide having the amino acid sequence set forth in SEQ ID NO: 1; and a peptide derived from the peptide having the amino acid sequence set forth in SEQ ID NO: 1 with a substitution and/or a deletion and/or an addition of one or more amino acid residues and being related to the plant root development.

2. The method of claim 1, wherein a precursor protein of the peptide is selected from the group consisting of: a precursor protein having the amino acid sequence set forth in SEQ ID NO: 2; and a precursor protein derived from the precursor protein set forth in SEQ ID NO: 2 with a substitution and/or a deletion and/or an addition of one or more amino acid residues and being related to plant development;

3. A method for constructing a transgenic plant, wherein root length of the transgenic plant becomes shortened or lengthened; a gene encoding the peptide for regulating plant root development is selected from the group consisting of: a gene with a coding region having the nucleotide sequence set forth in SEQ ID NO: 3; and a gene derived from the gene with a coding region having the nucleotide sequence set forth in SEQ ID NO: 3 with a substitutions and/or a deletions and/or an addition of one or more nucleotides and being related to plant development.

4. The method of claim 3, wherein the plant is rice.

5. The method of claim 4, wherein the plant is treated with different concentrations of a peptide to obtain a plant with reduced or increased length; the peptide is selected from the group consisting of: a peptide having the amino acid sequence set forth in SEO ID NO: 1; and a peptide derived from the peptide having the amino acid sequence set forth in SEO ID NO: 1 with a substitution and/or a deletion and/or an addition of one or more amino acid residues and being related to the plant root development.

6. The method of claim 3, comprising inserting a DNA molecule having the nucleotide sequence set forth in SEQ ID NO: 3 into a multiple cloning site of plasmid pCAMBIA1300 to obtain a recombinant expression vector I-OsPEP1 overexpression vector; and introducing the recombinant expression vector I-OsPEP1 overexpression vector into a target plant to obtain a transgenic plant with a shortened length.

7. The method of claim 3, comprising inserting a DNA fragment of a gene having the nucleotide sequence set forth in SEQ ID NO: 3 is ligated to a transition vector pBSSK-in in sense and antisense orientations, and then conducting insertion into a plasmid pCAMBIA1300 to obtain a recombinant expression vector II-OsPEP1 RNAi vector; and introducing the recombinant expression vector II-OsPEP1 RNAi vector into a target plant to obtain a transgenic plant with a shortened length.

8. A recombinant expression vector comprising a gene for regulating plant root development or an antisense gene thereof; wherein the gene for regulating plant root development is selected from the group consisting of: a gene with a coding region having the nucleotide sequence set forth in SEQ ID NO: 3; and a gene derived from the gene with a coding region having the nucleotide sequence set forth in SEQ ID NO: 3 with a substitutions and/or a deletions and/or an addition of one or more nucleotides and being related to plant development.

9. The recombinant expression vector of claim 8, wherein the recombinant expression vector is the following (I) or (II): (I) a recombinant expression vector comprising the gene obtained by inserting the DNA molecule set forth in SEQ ID NO: 3 into the multiple cloning site of plasmid pCAMBIA1300; (II) a recombinant expression vector obtained by ligating a DNA fragment of a gene set forth in SEQ ID NO: 3 to a transition vector pBSSK-in in sense and antisense orientations and then conducting insertion into the plasmid pCAMBIA1300.

10. (canceled)

11. (canceled)

12. (canceled)

13. The method of claim 6, wherein the plant is rice.

14. The method of claim 6, wherein the plant is treated with different concentrations of a peptide to obtain a plant with reduced or increased length; the peptide is selected from the group consisting of: a peptide having the amino acid sequence set forth in SEQ ID NO: 1; and a peptide derived from the peptide having the amino acid sequence set forth in SEQ ID NO: 1 with a substitution and/or a deletion and/or an addition of one or more amino acid residues and being related to the plant root development.

15. The method of claim 6, wherein the plant is rice.

16. The method of claim 6, wherein the plant is treated with different concentrations of a peptide to obtain a plant with reduced or increased length; the peptide is selected from the group consisting of: a peptide having the amino acid sequence set forth in SEQ ID NO: 1; and a peptide derived from the peptide having the amino acid sequence set forth in SEQ ID NO: 1 with a substitution and/or a deletion and/or an addition of one or more amino acid residues and being related to the plant root development.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0081] The specific embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.

[0082] FIG. 1 shows that the gene OsPEP1 (LOC_Os11g09560) in Example 1 encodes a protein containing five amino acids (SDFDR, SEQ ID NO: 1); [0083] A is the amino acid sequence of the protein encoded by the gene OsPEP1 (MGEKERRLRVEGWMGRTEMIDRRRQRLHSGERERRLCVRKRMGSSDFDRGA RFGGVDDGRLGEGTKRCEEMVGAIWDVGFERDNPDRSTRNEDVNISW, SEQ ID NO: 2), the peptide PEP1 with 5 amino acid residues is underlined; [0084] B illustrates LC-MS analysis of the peptide secreted by the roots of wild-type rice plants; [0085] C illustrates LC-MS/MS identification of Ser-Asp-Phe-Asp-Arg (PEP1) (SEQ ID NO: 1), a wild-type rice plant root secreted peptide, with a retention time of 12.25 minutes; [0086] D illustrates LC-MS analysis of root secreted peptide of a transgenic rice plant overexpressing LOC_Os11g09560; [0087] E illustrates LC-MS/MS identification of the root secreted peptide Ser-Asp-Phe-Asp-Arg (PEP1) (SEQ ID NO: 1) of a transgenic rice plant overexpressing LOC_Os11g09560, with a retention time of 12.26 minutes.

[0088] FIG. 2 illustrates the functional analysis of root secreted peptide PEP1 in Example 1; [0089] A shows the phenotype of wild-type plants treated with different concentrations of PEP1 for 7 days, with a scale bar of 5 cm; [0090] B shows statistics of the primary root length of wild-type plants treated with different concentrations of PEP1 for 7 days, Data are shown as Mean±SD (n≥15 independent seedlings, three biological replicates). Different letters indicate significant differences (P<0.01, ANOVA, LSD test); [0091] C shows the longitudinal section phenotype of root apical elongation zone (upper left) and meristem (lower left) of wild-type plants without PEP1 (−PEP1) treatment or that of the elongation zone (top right) and meristem (bottom right) treated with 10.sup.−6 M PEP1 (+PEP1) for 2 days. Bar=100 μm; [0092] D is the statistics of the length of the meristem in panel C, and data are shown as Mean±SD (n≥15 independent seedlings). *** Significant difference from WT (P<0.001, Student's t-test); [0093] E is the statistics of cell length in the elongation zone in C, and and data are shown as Mean±SD (n≥15 independent seedlings). *** Significant difference from WT (P<0.001, Student's t-test);

[0094] FIG. 3 is the expression profile of gene OsPEP1 in Example 2; [0095] A shows expression level of the gene OsPEP1 detected by qRT-PCR in rice roots, stems, leaves, and young ears, which were sampled from 7-day-old rice seedlings: [0096] B shows GUS staining of the Pro.sub.OsPEP1::GUS transgenic plant at the age of 7 days. Bar=1 cm; [0097] C shows GUS staining of the primary root of the 2-day-old Pro.sub.OsPEP1::GUS transgenic plant. Bar=0.1 cm; [0098] D shows the cross-section GUS staining of the primary root elongation zone in C, with a scale bar of 100 μm; [0099] E is the longitudinal section GUS staining of the primary root meristem in C, with a scale bar of 100 μm.

[0100] FIG. 4 is the phenotypic analysis of transgenic plants overexpressing OsPEP1 in Example 2; [0101] A shows the phenotype of 7-day-old seedlings of WT and plant lines OE1, OE2 and OE3 overexpressing OsPEP1. Bar=5 cm; [0102] B is the statistics of the primary root length of the corresponding plants indicated in A. Data are means±SD (n≥15 independent seedlings). *** Significant difference from WT (P<0.001, Student's t-test); [0103] C shows expression level of the gene OsPEP1 detected by qRT-PCR in the corresponding plants indicated in A. Data are means±SD (n=3 biological replicates); [0104] D shows longitudinal phenotype of the root tip elongation zone (top) and meristem (bottom) of the wild-type WT and that of the transgenic plant OE2 overexpressing OsPEP1 treated with 10.sup.−6 M PEP1 for 2 days. Bar=100 μm; [0105] E is the statistics of the apical meristem length indicated in D. Data are means±SD (n≥15 independent seedlings, three biological replicates). Different letters indicate significant differences (P<0.01, ANOVA, LSD test); [0106] F is the statistics of the cell length in the elongation zone indicated in D. Data are means±SD (n≥15 independent seedlings, three biological replicates). Different letters indicate significant differences (P<0.01, ANOVA, LSD test).

[0107] FIG. 5 is the phenotypic analysis of transgenic plants underexpressing OsPEP1 in Example 2; [0108] A shows the phenotype of 7-day-old seedlings of WT and that of plant lines Ri1, Ri2 and Ri3 interfering OsPEP1. Bar=5 cm; [0109] B is the statistics of the primary root length of the corresponding plants indicated in A. Data are means±SD (n≥15 independent seedlings). *** Significant difference from WT (P<0.001, Student's t-test); [0110] C shows the expression level of the gene OsPEP1 detected by qRT-PCR in the corresponding plants indicated in A. Data are means±SD (n=3 biological replicates); [0111] D is the longitudinal phenotype of the root tip elongation zone (top) and meristem (bottom) of the wild-type WT and the transgenic plant Ri2 overexpressing OsPEP1 treated with 10.sup.−6 M PEP1 for 2 days. Bar=100 μm; [0112] E is the statistics of the apical meristem length indicated in D. Data are means±SD (n≥15 independent seedlings, three biological replicates). Different letters indicate significant differences (P<0.01, ANOVA, LSD test); [0113] F is the statistics of cell length in the elongation zone indicated in D. Data are means±SD (n≥15 independent seedlings, three biological replicates). Different letters indicate significant differences (P<0.01, ANOVA, LSD test).

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0114] The present disclosure is further described below in conjunction with specific examples, but the protection scope of the present disclosure is not limited to this.

[0115] The composition of the hydroponic medium is shown in Table 2.

TABLE-US-00002 TABLE 2 Composition of hydroponic culture medium Final Name Components MW Mol g/L 10000× (mM) Stock-1 MgSO4•7H.sub.2O 246.3 0.547 134.800 10 ml 0.5470 (NH.sub.4).sub.2SO.sub.4 132.0 0.365 48.200 0.3650 Stock-2 KH.sub.2PO.sub.4 136.1 0.182 24.800 10 ml 0.1820 Stock-3 KNO.sub.3 101.1 0.183 18.500 10 ml 0.1830 Ca(NO.sub.3).sub.2•4H.sub.2O 236.0 0.366 86.400 0.3660 Stock-4 MnCl.sub.2•4H.sub.2O 197.9 0.005 0.990  1 ml 0.0005 H.sub.3BO.sub.3 61.8 0.03 1.860 0.0030 (NH.sub.4).sub.6Mo.sub.7O.sub.24•4H.sub.2O 1235.9 0.001 1.236 0.0001 ZnSO.sub.4•7H.sub.2O 287.5 0.004 1.150 0.0004 CuSO.sub.4•5H.sub.2O 249.5 0.002 0.518 0.0002 Stock-5 NaFe-EDTA•3H.sub.2O 421.1 0.100 42.100  4 ml 0.0400 MES MES 195.2 0.500 97.620 40 ml 2.0000

[0116] The detection results of gene expression levels in the following examples, unless otherwise specified, are all based on the target gene expression level of the wild-type plant Xiushui 134 as 1, and the target gene expression levels of other plants are compared with those of the wild-type plant.

Example 1. Acquisition of Rice Peptide PEP1 and Functional Study Thereof

[0117] 1. Acquisition of Rice Peptide PEP1 and Gene Encoding the Same

[0118] (1). Acquisition of the Root-Secreted Peptide in Rice.

[0119] 30 plump wild-type rice (Xiushui 134) seeds were treated with 0.5% nitric acid for 16-22 hours to break up the dormancy, and washed with tap water for 2-3 times, and then tap water was added to soak the seeds for germination acceleration in an incubator at 37° C. for two days until sprouting. During this period, the water was changed every morning and evening. Finally, the germinated seeds were sown on the nylon mesh floating on the nutrient solution (hydroponics medium), and cultured in an artificial climate chamber. The culture conditions in the artificial climate chamber were as follows: light for 14 hours, the average day and night temperature was 30° C./22° C., the light intensity was maintained at 200 μmol/m.sup.2 s.sup.1, and the humidity was 60%. After 10 days of culture, the medium was collected for subsequent concentration, extraction and precipitation of root exudates. The specific method is as follows: first, a rotary evaporator was used to concentrate the culture solution (500 ml) obtained in the previous step by 20 fold. Then, 20 ml of chlorophenol containing 1% NEM (N-ethylmorpholine, N-ethylmorpholine) was added and a resulting mixture was shaken for 1 minute at room temperature, centrifuged at 10,000 g for 10 minutes. A resulting organic phase was collected, and 20 times the volume of acetone was added to precipitate overnight at room temperature. Then a resulting mixture was centrifuged at 10000 g for 10 minutes, then a resulting precipitate was collected, washed with acetone for 3-4 times, and then vacuum dried to powder. Finally, the powder was sent to the company (Applied Protein Technology, co., Ltd) to identify 234 rice root-secreted peptides using liquid chromatography tandem mass spectrometry (LP-MS/MS).

[0120] (2). Acquisition of Candidate Genes Encoding Rice Root-Secreted Peptides.

[0121] Previous study have shown that the products of genes encoding some small signaling peptides (PSK, PSY1, CLV3/CLE) in Arabidopsis were cysteine-poor proteins with a length of 70-110 amino acids. Therefore, it is speculated in the present disclosure that if proteins encoded by certain genes in rice have these characteristics, the genes may be candidate genes encoding similar peptides. Based on this speculation, a total of 66,343 protein-coding genes from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/index.shtml, March 2017) were downloaded, and then 12,678 protein-coding genes with a length of 50-150 amino acids were retrieved with MICROSOFT WORD 2003 and EXCEL 2003. The secreted peptide had a signal peptide sequence at its N-terminus. In the present disclosure, SignalP 4.1 server in the HMM webpage was used for screening and 704 protein-coding genes with a signal peptide at the N-terminus (P≥0.75) were obtained. Finally, EXCEL 2003 was used to exclude protein-encoding genes containing 6 or more cysteines and 416 candidate rice peptide-encoding genes were obtained.

[0122] (3). Identification of Rice Root-Secreted Peptide PEP1 and Gene Encoding the Same.

[0123] By comparing the 234 root-secreted peptides obtained by method (1) with the 416 putative rice peptide-encoding genes obtained by method (2), a novel peptide (Ser-Asp-Phe-Asp-Arg, SEQ ID NO:1) was identified in the present disclosure and candidate gene encoding the same (LOC_Os11g09560) (A in FIG. 1) was determined. In order to further confirm that LOC_Os11g09560 indeed encodes peptide PEP1, LC-MS/MS was used in the present disclosure to detect the root secretions of the LOC_Os11g09560 overexpressing transgenic plants. The results showed that the peak of this peptide in the root secretions of the LOC_Os011g09560 overexpressing transgenic plants were significantly enhanced (B-E in FIG. 1), indicating that the gene LOC_Os11g09560 indeed encodes peptide PEP1. Thereafter, the peptide (Ser-Asp-Phe-Asp-Arg, SEQ ID NO: 1) was named PEP1, and the gene encoding the LOC_Os11g09560 was named OsPEP1.

[0124] 2. Functional Study of the PEP1

[0125] In the present disclosure, different concentrations of artificially synthetic PEP1 were used to treat rice seedlings (the concentrations were as described in A in FIG. 2, and the treatment started after sowing and lasted for 7 days), and the results showed that exogenous application of PEP1 significantly inhibited rice primary root elongation (A and B in FIG. 2). In order to clarify the cellular basis of the PEP1 inhibitory effect on rice root elongation, we observed the section structure of these rice root tips, and found that after PEP1 treatment, the length of the meristem and cells in the elongation zone of rice root tips were significantly shortened. However, there were no significant changes in the quiescent center (as shown in the meristem of the root section, C-E in FIG. 2).

Example 2. Study on Expression Profile of the Rice Peptide PEP1 Encoding Gene OsPEP1

[0126] 1. Expression of OsPEP1 in Different Rice Tissues Determined by qRT-PCR

[0127] Xiushui 134 rice was used as material, and cultured in normal nutrient solution (hydroponic culture medium) for 7 days. The roots, stems, leaves and leaf sheath were collected, and when the Xiushui 134 was in heading age, the young panicles were collected. All samples were frozen in liquid nitrogen and ground rapidly. Then the total RNA was extracted and reverse transcribed to obtain the cDNA. The expression of OsPEP1 in various tissues was detected by qRT-PCR. The results showed that the gene OsPEP1 was expressed in the different tissues tested in the present disclosure, and the expression level was higher in roots (A in FIG. 3). The detection primer sequences were as follows:

TABLE-US-00003 (SEQ ID NO: 9) 5′-GGCGTGGATGACGGGAGACT-3′; (SEQ ID NO: 10) 5′-TACATCCTCATTCCTCGTTG-3′,

[0128] The reaction system and procedures were as follows [0129] 2× Master: 2.5 μl [0130] cDNA template: 0.1 μl [0131] Primer-F (10 μM): 0.1 μl [0132] Primer-R (10 μM): 0.1 μl [0133] H.sub.2O: 2.2 μl [0134] Total: 5 μl;

[0135] The PCR procedures were as follows: [0136] initiation: 95° C. for 1 minute; [0137] amplification: 45 cycles of 95° C. for 10 seconds, [0138] 58° C. for 10 seconds, and [0139] 72° C. for 20 seconds;

[0140] Dissolution curve: 95° C. for 5 seconds, [0141] 65° C. for 1 minute, [0142] 97° C. cooling until 65° C.; [0143] Cooling: 40° C. for 30 seconds;

[0144] 2. The Expression of OsPEP1 in Different Rice Tissues determined by GUS Staining.

[0145] The DNA of Xiushui 134 rice was extracted, and used as a template for PCR amplification to amplify the 2 kb nucleotide sequence of the OsPEP1 promoter. The primers for PCR amplification were as follows:

TABLE-US-00004 (SEQ ID NO: 11) 5′-GCATGCCTGCAGGTCGACGTTTCTCAGCTACGCCCCTG-3′; (SEQ ID NO: 12) 5′-CCATGGTACCGTGGATCCCCGGAGCGCAGCCGTCGTCT-3′,

[0146] The obtained PCR product was inserted between the SalI and BamHI restriction sites of the vector pBI101.3-GUSplus modified in our laboratory by recombinant cloning (Lv et al., 2014) to obtain the Pro.sub.OsPEP1::GUS vector. The vector was verified to be correct by sequencing. The constructed overexpression vector was transferred into Agrobacterium EHA 105 to transform Xiushui 134 plants, with reference to conventional steps, which were as follows: [0147] (1) 500 μl of the cultured bacterial solution was added into a 1.5 ml centrifuge tube, centrifuged at room temperature, 4000 rmp for 2 minutes, and a resulting supernatant was removed. A suspension with 30 ml of AAM-infected bacterial solution containing 200 μmol/L acetosyringone was prepared, and the final concentration of bacterial solution indicated by OD.sub.600 is 0.01; 80 to 120 rice calli grown to a certain size (about 1 cubic centimeter) were collected, put in Agrobacterium suspension, and shaken for 5 minutes on a horizontal shaker; [0148] (2) the calli were taken out and placed on sterile filter paper to drain for 30 to 40 minutes; [0149] (3) the calli were placed on a co-culture medium with a sterile filter paper, and cultured in the dark at 25° C. for 3 days; [0150] (4) the calli were taken out, and then washed with sterile water for 5 to 6 times with constant shaking. The calli were washed twice with sterile water containing 300 mg/L carbenicillin sodium (Carb) and shaken on a horizontal shaker for 30 minutes each time. Finally, the calli were placed on sterile filter paper and drain for 2 hours; [0151] (5) the air-dried calli were transferred to the selection medium containing 300 mg/L carbenicillin sodium with the corresponding selection pressure for a first round of selection, and cultured at 28° C. for 14 days in the light; [0152] (6) the initial calli of the positive calli were transferred to the medium containing 300 mg/L carbenicillin sodium with the corresponding selection pressure for a second round of selection, and cultivated at 28° C. in the light until the granular calli with resistance emerged (about 14 days); [0153] (7) 3 to 5 positive calli with bright yellow color were taken from different calli and then transferred into plastic jars with differentiation medium, sealed with parafilm, and put in a culture room (photoperiod: 16 hours of light) at constant temperature (25° C.) for differentiation into seedlings (about 40 days); and [0154] (8) when the seedlings grew to about 3 cm, the old roots and calli were cut off from the base of the seedlings with scissors, and put into the rooting medium to strengthen the seedlings (about 1 week). The seedlings with well-differentiated roots, stems and leaves were taken from the test tube (if the seedlings grew to the top of the test tube, open the lid in time), the sealing film was removed, an appropriate amount of distilled water or sterile water (to prevent the growth of bacteria in the medium) was added, and the seedlings were trained for 2 to 3 days. The agar was washed off and transplanted to grow in hydroponic conditions in the greenhouse. Primers for the gibberellin-resistant gene were used to detect transgenic plant, and sequences of the primer were as follows:

TABLE-US-00005 (SEQ ID NO: 13) 5′-ATGAAAAAGCCTGAACTCACC-3′; (SEQ ID NO: 14) 5′-CTATTCCTTTGCCCTCGGACG-3′,

[0155] In the obtained transgenic rice of T2 generation, representative lines (transgenic plants with positive GUS staining) were selected for GUS staining to study the expression of OsPEP1 in different tissues of rice. It was showed that the gene OsPEP1 was mainly expressed in rice roots, especially the root cap zone of the root tip, and the cortex of the meristem and mature zones (B-E in FIG. 3).

Example 3. Functional Study of the Gene OsPEP1 Encoding the Rice Peptide PEP1

[0156] 1. Construction of the OsPEP1 Overexpressing Recombinant Vector

[0157] The mRNA of Xiushui 134 was extracted and reverse transcribed into cDNA, and the cDNA was used as a template for PCR amplification to prepare the OsPEP1 sequence (DNA set forth in SEQ ID NO: 3). Primers for PCR amplification were as follows:

TABLE-US-00006 (SEQ ID NO: 15) 5′-ACGGGGGACGAGCTCATGGGAGAGAAGGAGCGGAG-3′; (SEQ ID NO: 16) 5′-GACTCTAGAGGATCCCAACTGATGTTTACATCCTCA-3′,

[0158] The obtained PCR product was inserted between the SacI and PstI restriction sites of the vector pCAMBIA1300 modified in our laboratory (Lv et al., 2014) by recombinant cloning to obtain the OsPEP1 overexpressing vector, and then the vector was verified to be correct by sequencing.

[0159] 2. Acquisition of the OsPEP1 Overexpressing Transgenic Rice Plants

[0160] The OsPEP1 overexpressing vector constructed in step 1 was transferred into Agrobacterium EHA105 for transformation of rice Xiushui 134, and 32 positive transgenic plants were obtained. The specific steps were identical to step 2 of Example 2.

[0161] In the obtained T2 OsPEP1 overexpressing transgenic rice plants, three representative plants (OE1, OE2, OE3) were chosen for phenotypic analysis. It was showed that the primary root length of these transgenic plants were significantly shortened compared with that of Xiushui 134 (A and B in FIG. 4). The relative expression levels of endogenous OsPEP1 in Xiushui 134 and OsPEP1 overexpressing transgenic plants (OE1, OE2, OE3) were analyzed by quantitative real-time PCR (primers: GGCGTGGATGACGGGAGACT (SEQ ID NO: 9); TACATCCTCATTCCTCGTTG (SEQ ID NO: 10)). The results showed that the primary root length of those OsPEP1 overexpressing transgenic plants was positively correlated with the expression of OsPEP1 (see C in FIG. 4). In addition, the results of the observation of the section of these OsPEP1 overexpressing transgenic plants' root tips showed that the length of the meristematic region and the cell in the elongation region were significantly shortened, compared to that of Xiushui 134. Exogenous application of PEP1 could not restore the root tip defected phenotype of OsPEP1 overexpressing transgenic plants to the Xiushui 134 level (D-F in FIG. 4).

[0162] 3. Construction of Recombinant OsPEP1 RNAi Vector

[0163] The mRNA of rice Xiushui 134 was extracted and reverse transcribed into cDNA, and the long cDNA was used as a template for PCR amplification to prepare a partial DNA sequence of OsPEP1:

TABLE-US-00007 (SEQ ID NO: 17) ACTCGGGAGAGAGGGAGCGCAGATTGTGCGTGAGGAAACGGATGGGAAGCA GCGATTTCGATCGAGGGGCGCGATTTGGGGGCGTGGATGACGGGAGACTGG GAGAGGGGACGAAGCGGTGTGAGGAGATGGTGGGAGCGATTTGGG.

[0164] Primers for PCR amplification were as follows:

TABLE-US-00008 (SEQ ID NO: 18) 5′-ACTCGGGAGAGAGGGAGCGC-3′; (SEQ ID NO: 19) 5′-CCCAAATCGCTCCCACCATC-3′,

[0165] The cloned PCR product was ligated with T vector (purchased from TAKARA), and the ligated plasmid was digested with PstI, BamH I and Pst I, Sal I respectively to obtain two fragments; the two fragments were ligated into the pBSSK-in vector (Wang et al. 2019) in two steps, pBSSK-in was first digested with Pst I and BamH I, and then ligated with one fragment, then digested with Nsi I and Sal I, and ligated with another fragment. Finally, the two fragments and intron were excised with Sac I and Sal I. and ligated into the same digested plant binary vector pCAMBIA1300 (Lv et al., 2014) to obtain an RNAi expression vector targeting OsPEP1. The RNAi expression vector targeting OsPEP1 was verified to be correct by sequencing.

[0166] 4. Acquisition of OsPEP1 RNAi Transgenic Plants

[0167] The RNAi vector targeting OsPEP1 constructed in the above step 3 was transferred into Agrobacterium EHA105 for transformation of Xiushui 134, and 53 positive transgenic plants were obtained. The specific steps were identical to the step 2 in Example 2.

[0168] In the obtained T2 OsPEP1 RNAi transgenic plants, three representative plants (Ri1, Ri2, Ri3) were selected for phenotypic analysis. It was showed that the root length of these transgenic plants was significantly shortened compared to the wild type Xiushui 134 (see A and B in FIG. 5). The relative expression levels of endogenous OsPEP1 in Xiushui 134 and those OsPEP1 RNAi transgenic plants (Ri1, Ri2, Ri3) were analyzed by quantitative real-time PCR (primers: GGCGTGGATGACGGGAGACT (SEQ ID NO:9); TACATCCTCATTCCTCGTTG (SEQ ID NO: 10)). The results showed that the length of the primary root of those transgenic plants was positively correlated with the expression of OsPEP1 (C in FIG. 5); and results of section showed that the length of the meristem and the cells in the elongation zone of the those transgenic plants were significantly shortened compared to that of Xiushui 134. Additionally, exogenous application of PEP1 could restore the root tip defected phenotype of OsPEP1 RNAi transgenic plants to the Xiushui 134 level (D-F in FIG. 5).

[0169] Collectively, through comparative study of 234 rice root secreted peptides identified by LC-MS/MS and 416 candidate genes encoding rice peptides, in combination with the genetic analysis, a root-secreted peptide PEP1 (Ser-Asp-Phe-Asp-Arg, SEQ ID NO: 1) associated with rice primary root development was identified and its encoding gene OsPEP1 (LOC_Os11g09560) was determined. The results of physiological experiments of the present disclosure showed that exogenous application of PEP1 inhibited rice root elongation, and the genetic analysis of the present disclosure showed that overexpression or inhibition of OsPEP1 expression inhibited rice root elongation. These results suggest that the rice root secreted peptide may play an important role in rice root development as a signaling molecule, and may control the growth and development of rice roots by regulating the expression of OsPEP1.

[0170] Finally, it should also be noted that the above enumeration is only a few specific embodiments of the present disclosure. Obviously, the present disclosure is not limited to the above embodiments, and many modifications can be made. All modifications by those skilled in the art that can be directly derived or associated from the present disclosure shall be considered to fall within the protection scope of the present disclosure.