CN112812163A

CN112812163A - Application of transcription factor in rice breeding and rice breeding method

Info

Publication number: CN112812163A
Application number: CN202110245121.2A
Authority: CN
Inventors: 方中明; 杭俊楠; 杨修艳; 王荣纳
Original assignee: Guizhou University
Current assignee: Guizhou University
Priority date: 2021-03-05
Filing date: 2021-03-05
Publication date: 2021-05-18
Anticipated expiration: 2041-03-05
Also published as: CN112812163B

Abstract

The invention relates to the technical field of rice molecular breeding, in particular to application of a transcription factor in rice breeding and a rice breeding method. The rice breeding method comprises the steps of improving the expression quantity of OsGAmyb gene and/or OsTCP21 gene protein of rice plants; the amino acid sequence of the OsGAmyb gene is shown as SEQ ID NO.1, and the amino acid sequence of the OsTCP21 gene is shown as SEQ ID NO. 3. After the expression quantity of the protein of the OsGAmyb gene and/or the OsTCP21 gene of the rice plant is improved, the tillering number, the biomass and the rice number of the rice plant are obviously increased, so that the yield of the rice plant is improved. The OsGAmyb and OsTCP21 gene overexpression can be applied to various aspects of genetic improvement of rice plant types and yield, and creates conditions for improving the rice yield and cultivating new plant types of rice.

Description

Application of transcription factor in rice breeding and rice breeding method

Technical Field

The invention relates to the technical field of rice molecular breeding, in particular to application of a transcription factor in rice breeding and a rice breeding method.

Background

"people eat every day and first rice", rice (Oryza sativa L.) is one of the most important grain crops in the world, and the grain safety of the world is determined by the high and low yield of the rice. The rice yield is mainly determined by three agronomic traits, namely the number of ears per plant, the number of grains per ear and the weight of each grain. Wherein the number of ears of a single plant is closely related to the tillering of rice. Rice tillers, as a special branch containing the spike in monocotyledons, can be formed mainly in two stages, namely the initiation of the tillering bud and the elongation of the tillering bud (Leyser O. Regulation of shoot breaking by auxin.2003,8(11): 541) -545.). Normally, rice tillering buds occur mainly in the axilla of each leaf on the main stem of rice, but not all tillering buds can form effective tillering, which affects the yield of rice single plants. Only axillary buds located between ungerminated basal nodes are likely to develop into productive tillers, which in turn produce productive ears affecting rice yield per Plant (Wang Y, Li J. the Plant architecture of rice [ J ]. Plant Molecular Biology,2005,59(1): 75-84.). Thus, the final number of tillers in rice depends on the number of initial tillering buds and the number of tillering buds that can grow. Therefore, in order to increase the yield of individual rice plants, it is most important to increase the number of initial tillering buds and the number of tillering buds that can grow.

Molecular breeding is a breeding means different from traditional crossbreeding, wherein molecular breeding comprises transgenic breeding, namely a breeding method which applies genetic engineering to breeding work and breeds a new variety with certain requirements through gene introduction. The transgenic breeding is carried out on the rice to improve the initial tillering bud number and the number of tillering buds capable of growing, and the method has important significance for improving the rice yield. In the practice of transgenic breeding, the selection of which target gene is very critical for transgenic breeding, and screening and research on genes related to rice development are urgently needed, so that a molecular breeding method capable of remarkably improving the rice yield is obtained.

Disclosure of Invention

The invention aims to provide a rice breeding method to solve the technical problem of increasing the number of initial tillering buds of rice so as to improve the yield of the rice.

In order to achieve the purpose, the invention adopts the following technical scheme:

the rice breeding method improves the expression quantity of OsGAmyb gene and/or OsTCP21 gene protein of rice plants; the amino acid sequence of the OsGAmyb gene is shown as SEQ ID NO.1, and the amino acid sequence of the OsTCP21 gene is shown as SEQ ID NO. 3.

The principle and the advantages of the scheme are as follows: after the expression quantity of the protein of the OsGAmyb gene and/or the OsTCP21 gene of the rice plant is improved, the tillering number, the biomass and the rice number of the rice plant are obviously increased, so that the yield of the rice plant is improved. The two genes are knocked out, so that the tillering number, biomass and rice quantity of a single rice plant are obviously reduced, and the single rice plant yield is negatively regulated. Under the condition of not influencing the activity of the OsGAmyb protein (namely not in the active center of the protein), the amino acid sequence shown in SEQ ID NO.1 can be subjected to various substitutions, additions and/or deletions of one or more amino acids by a person skilled in the art to obtain an amino acid sequence with equivalent functions. Therefore, the OsGAmyb protein also comprises a protein with equivalent activity obtained by substituting, replacing and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 1. Under the premise of not influencing the activity of the OsTCP21 protein (namely, not in the active center of the protein), the amino acid sequence shown in SEQ ID NO.3 can be subjected to various substitutions, additions and/or deletions of one or more amino acids by the skilled person to obtain an amino acid sequence with equivalent functions. Therefore, the OsTCP21 protein also comprises a protein with equivalent activity obtained by substituting, replacing and/or adding one or more amino acids in the amino acid sequence shown in SEQ ID NO. 3. In addition, in view of codon degeneracy and codon preference of different species, one skilled in the art can use codons suitable for expression of a particular species as desired.

The inventor clones the cDNA sequence of the OsTCP21 gene from the rice Nipponbare (Ni). The OsTCP21 gene overexpression vector is constructed and is introduced into Nipponbare (Ni) to obtain an OsTCP21 gene overexpression plant, and the length, the number, the biomass and the like of tillering buds of a single rice plant are obviously improved compared with Nipponbare. A gene knockout vector of the OsTCP21 gene is constructed, and the knockout vector is introduced into Nipponbare (Ni) to obtain a gene knockout plant of the OsTCP21 gene, wherein the tillering quantity and the yield of a single rice plant are obviously reduced compared with those of Nipponbare (Ni). The inventor also clones the cDNA sequence of the OsGAmyb gene from the Nipponbare. The OsGAmyb gene overexpression vector is constructed and is introduced into Nipponbare (Ni) to obtain an OsGAmyb gene overexpression plant, the length, the number and the biomass of tillering buds of a single rice plant are obviously improved compared with Nipponbare, but the plant height is relatively reduced. The gene knockout vector of the OsGAmyb gene is constructed and is introduced into Nipponbare (Ni) to obtain a gene knockout plant of the OsGAmyb gene, and the tillering quantity and the yield of a single rice plant are obviously reduced compared with Nipponbare. The research shows that the rice yield can be increased by overexpression of the OsGAmyb gene and/or the OsTCP21 gene in rice plants, and particularly the rice yield is increased by increasing the tiller number, biomass and rice number of a single rice plant.

The OsTCP21 gene and the OsGAmyb gene are both target genes of small-molecule RNA and can be regulated by the small-molecule RNA, so that the growth and development processes of plants are influenced, but the practical operation of rice breeding by using the two transcription factors is not available at present. In the research on the influence of small molecular RNA on the growth and development of rice, the inventor finds that miR319 has a negative regulation effect on tillering and yield improvement of rice. The inventor further researches on downstream molecules regulated by miR319, and finds that the OsTCP21 gene and the OsGAmyb gene are negatively regulated by miR319, namely the OsTCP21 gene and the OsGAmyb gene are target genes. The inventor further tries to construct over-expression rice plants of the OsTCP21 gene and the OsGAmyb gene, finally discovers the positive regulation effect of the two genes due to rice yield increase, and applies the two genes to the practical operation of rice transgenic breeding.

The OsTCP21 gene is a transcriptional regulator of TCPs, which encodes a TCP domain containing a basic helix-loop-helix (bHLH) motif (Shuncihi K, Yuko O.PCF1 and PCF2 specific binding to cis elements in The device stimulating Cell nuclear antigen gene [ J ]. The Plant, 1997,9(9): 1607-. The TCPs family gene is a part of genes that negatively regulate rice tillering, for example, OsTCP19 is used as a transcription factor to negatively regulate rice tillering, the tillering number of an overexpression line is reduced, while the tillering number of an OsTCP19 knockout or knock-down line is significantly increased (Liu Y, Wang H, Jiang Z, et al. genomic diseases of genetic adaptation to soil immunity in rice [ J ]. Nature,2021, doi:10.1038/s 41586-020-. In this scheme, the inventors found that the function of the OsTCP21 gene, which is also a member of the TCPs family, is exactly opposite to that of OsTCP19 through the following experimental studies. No report about improving rice yield of OsTCP21 gene exists in the prior art, and only reports that OsTCP21 is down-regulated to enable rice plants to be easily infected with rice dwarf virus (RRSV) (Zhang C, Ding Z, Wu K, et al.Supposition of Japanese acid-mediated defect by viral-induced microRNA319 viruses in rice Plant [ J ] Molecular Plant,2016,9(9): 1302-1314.). This indicates that the inventors have found new properties and functions of genes and applied the new findings to practical operations of rice breeding to obtain novel rice plants and improve rice yield.

For the OsGAmyb gene, the OsGAmyb gene is a GAmyb gene. The gene is an R2R3 type MYB transcription factor induced by GAs (Tsuji H, Aya K, Ueguchi-Tanaka M, et al. GAMBB controls differential sets of genes and is differential regulated by microRNA in aleurone cells [ J ]. Plant journal.2006,47(3): 427-) 444.). In rice, OsGAmyb loss of function shows incomplete development of stamens, anthers and pollen (Miyuki K, Yoshiaki I, Miyako UT, et al. Loss-of-function relationships of The rice GAYB gene expression in an alkane and flower reduction [ J ]. The plant.2003, 16(1): 33-44.). OsGAmyb genes are also important for floral organ development and pollen development (Kaneko M, Inukai Y, Ueguchi-Tanaka M, et al, Loss-of-function variants of The rice GAYB gene expression in an ecological and flower development [ J ]. The Plant, 2004,16(1):33-44), and it is found in rice that OsGAmyb gene mutants exhibit a defect in pollen development after The conversion from vegetative to reproductive stages, etc. (Liu Z, Bao W, Liang W, et al, expression of gamy-4 and analysis of The regulation role of The Plant of flower development [ J ]. Journal of expression, Plant, 678 52): 670). However, the research of OsGAmyb in rice focuses on internodes, anther development and the like, and the regulation and control effect on tillering/branching is not reported. In the scheme, the inventor researches and discovers a new function of the gene, and performs quantitative breeding on rice by using the function to obtain unexpected technical effects.

In conclusion, the scheme has the following beneficial effects:

(1) the tillering number and biomass of a single rice plant are obviously increased after the cloned OsTCP21 gene is over-expressed, which shows that the OsTCP21 gene is obvious in improving the rice plant type and increasing the yield of the single rice plant, so that the rice plant type and yield can be genetically improved by improving the expression of the OsTCP21 gene through a genetic engineering technology; after the cloned OsGAmyb gene is over-expressed, the tillering number and biomass of a single rice plant are obviously increased, which shows that the OsGAmyb gene is obvious for improving the rice plant type and increasing the yield of the single rice plant, so that the rice plant type and yield can be genetically improved by improving the expression of the OsGAmyb gene through a gene engineering technology.

(2) Successful cloning of the OsTCP21 gene proves that the transcription factor TCPs not only play a role in plant stress response and stress, but also play an important role in plant tillering and plant growth and development, can enrich the knowledge of the plant TCPs, and has great promotion effect on plant branching and yield genetic improvement. The successful cloning of the OsGAmyb gene proves that the transcription factor GAmyb not only plays a role in the development of plant internodes and anthers, but also plays an important role in plant tillering and plant growth and development, can enrich the understanding of the plant GAmyb, and has great promotion effects on the genetic improvement of plant branches and yield.

Furthermore, the nucleotide sequence of the OsGAmyb gene is shown as SEQ ID NO.2, and the nucleotide sequence of the OsTCP21 gene is shown as SEQ ID NO. 4.

Further, the step of improving the expression level of the OsGAmyb gene of the rice plant is as follows: obtaining cDNA of OsGAmyb gene from wild rice by RT-PCR, and then connecting the cDNA of OsGAmyb gene to pCAMBIA-1306 vector to obtain overexpression vector OsGAmyb-p 1306; an agrobacterium-mediated genetic transformation method is adopted to introduce an overexpression vector OsGAmyb-p1306 into wild rice, and an OsGAmyb gene overexpression plant is obtained through screening and cultivation. Constructing an over-expression vector, and obtaining an over-expression plant through agrobacterium transfection, wherein the operation method is mature and stable, and the OsGAmyb gene over-expression plant with stable hereditary property can be obtained.

Further, primers for obtaining cDNA of OsGAmyb gene from wild type rice by RT-PCR included F1 having a nucleotide sequence shown in SEQ ID NO.5 and R1 having a nucleotide sequence shown in SEQ ID NO. 6. And obtaining cDNA of the OsGAmyb gene through RT-PCR cloning, and further constructing an over-expression vector.

Further, the steps for improving the expression level of the OsTCP21 gene of the rice plant are as follows: obtaining cDNA of OsTCP21 gene from wild rice by using RT-PCR, and then connecting the cDNA of OsTCP21 gene to pCAMBIA-1306 vector to obtain overexpression vector OsTCP-p 1306; an agrobacterium-mediated genetic transformation method is adopted to introduce the overexpression vector OsTCP-p1306 into wild rice, and an OsTCP21 gene overexpression plant is obtained after screening and cultivation. Constructing an overexpression vector, and obtaining an overexpression plant through agrobacterium transfection, wherein the operation method is mature and stable, and the OsTCP21 gene overexpression plant with stable hereditary property can be obtained.

Further, the cDNA of the OsTCP21 gene obtained from wild type rice by RT-PCR included F3 with a nucleotide sequence shown as SEQ ID NO.9 and R3 with a nucleotide sequence shown as SEQ ID NO. 10. The cDNA of the OsTCP21 gene is obtained by RT-PCR cloning, and then an over-expression vector is constructed.

Further, the application of the transcription factor in rice breeding, the expression level of the transcription factor is increased; the transcription factor comprises OsGAmyb protein and/or OsTCP21 protein; the amino acid sequence of the OsGAmyb protein is shown as SEQ ID NO.1, and the amino acid sequence of the OsTCP21 protein is shown as SEQ ID NO. 3. Based on the functions of OsTCP21 and OsGAmyb genes discovered by the inventor, both can be used for rice breeding. The rice breeding aims to obtain rice plants with higher tillering number of single plants, biomass, rice quantity or single plant yield. The expression of the OsTCP21 gene can be improved by an overexpression technology, or the expression of the OsGAmyb gene can be improved by the overexpression technology, so that rice plants with excellent properties of large tillering number of a single plant, high biomass, large rice quantity, high yield of the single plant and the like can be obtained.

Furthermore, the transcription factor is used for improving the tillering number, the biomass and the grain number of the rice. The expression quantity of OsTCP21 and OsGAmyb genes is increased in rice, so that the quantity of the two transcription factors is increased, the aim of improving the plant type and the yield of the rice can be fulfilled, and the specific expression is to increase the tillering number, the biomass and the grain number of the rice.

Further, constructing an overexpression rice plant of the OsGAmyb gene. In the over-expression rice plant of OsGAmyb gene, the expression level of OsGAmyb protein is increased, and the tillering number, biomass, rice quantity and yield of a single plant are improved.

Further, an overexpression rice plant of the OsTCP21 gene is constructed. In an over-expression rice plant of the OsTCP21 gene, the expression level of the OsTCP21 protein is increased, and the tillering number, the biomass, the rice quantity and the yield of a single plant are improved.

Drawings

FIG. 1 is a histogram of the real-time fluorescent quantitative PCR of example 1 of the present invention, the samples being: control wild type nipponica (Ni), OsTCP21 gene overexpression plants 3 lines OE1, OE2 and OE3(mean ± SD, N ═ 3).

FIG. 2 is a histogram of the real-time fluorescent quantitative PCR of example 2 of the present invention, the samples being: control wild type nipponica (Ni), OsGAmyb gene overexpression plants 3 lines OE1, OE2 and OE3(mean ± SD, N ═ 3).

FIG. 3 is a map showing the alignment of the OsTCP21 gene sequence of the mutant of comparative example 1 of the present invention.

FIG. 4 is a map showing the alignment of the OsGAmyb gene sequence of the mutant of comparative example 2 of the present invention.

FIG. 5 is a rice single seedling tillering bud phenotype map (OsTCP21 gene) in Experimental example 1 of the present invention.

FIG. 6 is a rice single seedling tillering bud phenotype map (OsGAmyb gene) of Experimental example 1 of the present invention.

FIG. 7 is a statistical graph of the number of tillers of individual rice plants (OsTCP21 and OsGAmyb gene) (mean. + -. SD, N ═ 20) in Experimental example 1 of the present invention.

FIG. 8 is a rice biomass statistic map (OsTCP21 and OsGAmyb gene) (mean. + -. SD, N20) of individual rice plants in Experimental example 1 of the present invention.

FIG. 9 is a phenotypic graph of individual rice plants (OsTCP21 gene) planted in a field according to Experimental example 2 of the present invention.

FIG. 10 is a rice phenotype map (OsTCP21 gene) of individual rice plants grown in a field according to Experimental example 2 of the present invention.

FIG. 11 is a graph showing the relationship between yield and tiller number of individual rice plants planted in a field (OsTCP21 gene) (mean. + -. SD, N20) in Experimental example 2 of the present invention.

FIG. 12 is a phenotypic profile of individual rice plants (OsGAmyb gene) planted in a field according to Experimental example 2 of the present invention.

FIG. 13 is a rice phenotype map (OsGAmyb gene) of individual rice plants grown in a field according to Experimental example 2 of the present invention.

FIG. 14 is a graph showing the relationship between yield and tiller number of individual rice plants (OsGAmyb gene) (mean. + -. SD, N20) in field planting in Experimental example 2 of the present invention.

FIG. 15 shows the results of real-time fluorescent quantitative PCR detection (OsTCP21 gene, mean. + -. SD, N-3) in Experimental example 3 of the present invention.

FIG. 16 shows the results of real-time fluorescent quantitative PCR detection of the OsGAmyb gene (mean. + -. SD, N-3) in Experimental example 3 of the present invention.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art; the experimental procedures used are conventional and can be carried out according to recombinant techniques already described (see molecular cloning, A laboratory Manual, 2 nd edition, Cold spring harbor laboratory Press, Cold spring harbor, N.Y.; Maxetal, Arobist CRISPR/Cas9 system for meeting, high-efficiency multiplex gene editing in monocot and dicot plants. mol plant.2015,8(8): 1274. sup. 1284.); the materials, reagents and the like used are all commercially available.

Example 1: construction of OsTCP21 gene overexpression plant

RNA of rice Nipponbare (Ni) was extracted and reverse-transcribed into cDNA, and the cDNA of OsTCP21 gene was amplified by PCR using a primer set F1 and R1 (the protein sequence of OsTCP21 gene is shown in SEQ ID NO.1, and the gene sequence of OsTCP21 gene is shown in SEQ ID NO. 2). Primer pair F1 and R1, respectively:

f1: 5'-gagctcggtacccggggatccatggagctcgccggcagcaa-3' (SEQ ID NO.5, containing the enzyme cleavage site KpnI);

r1: 5'-tccaagggcgaattggtcgacgtgcttgcccttctccttgatgt-3' (SEQ ID NO.6, containing the cleavage site BamHI).

Then, the cDNA of OsTCP21 gene was ligated into pCAMBIA-1306 vector (pCAMBIA-1306 vector from Cambia) via two cleavage sites KpnI and BamHI to construct OsTCP-p1306, which is a overexpression vector of OsTCP21 gene. The overexpression vector is introduced into a normal rice variety Nipponbare by adopting an agrobacterium EHA105 mediated genetic transformation method.

Culturing all the obtained transgenic seedlings with a normal nutrient solution added with hygromycin for one week, and if the seedlings can grow normally, indicating that the transgenic plants are positive plants. Transplanting all transgenic positive plants into a basket with soil, watering and fertilizing at regular intervals, planting the transgenic positive plants in a field when seedlings grow to be about 15cm high, harvesting and planting single transgenic rice plants after the seedlings grow until homozygous transgenic plants are identified in the T2 generation, and obtaining OsTCP21 gene overexpression plants. Three kinds of plants with over-expressed genes, OsTCP21-OE1, OsTCP21-OE2 and OsTCP21-OE3, are prepared and obtained in the embodiment.

Taking OsTCP21 gene over-expression plant leaves, extracting RNA and carrying out reverse transcription on the RNA to obtain cDNA, detecting the expression quantity of the OsTCP21 gene in the over-expression plant through real-time fluorescent quantitative PCR, wherein the result shows that the expression quantity of the OsTCP21 gene in the over-expression plant (shown in figure 1) is improved compared with that of a control Nipponbare (the expression quantity of wild type Ni in the figure is determined to be '1'), and experiments show that the transgene is successful, the gene is successfully over-expressed, and the over-expression plant is successfully constructed. The sequences of the primer pairs F2 and R2 used for real-time fluorescent quantitative PCR are respectively as follows:

F2：5'-tctcacctctccagccatca-3'(SEQ ID NO.7)；

R2：5'-gtgcttgcccttctccttga-3'(SEQ ID NO.8)。

the data in fig. 1 were analyzed for variables (ANOVA) using SPSS software, and for significance of differences at the 0.05 level using Duncan's, asterisks indicate significant differences between the different groups compared to the control group (p <0.05, p <0.01, p < 0.001).

Example 2: construction of OsGAmyb gene overexpression plant

RNA of Nipponbare of rice was extracted and reverse-transcribed into cDNA, and the cDNA of OsTCP21 gene was amplified by PCR using a primer set F3 and R3 (the protein sequence of OsGAmyb gene is shown in SEQ ID NO.3, and the gene sequence of OsGAmyb gene is shown in SEQ ID NO. 4). Primer pair F3 and R3 were:

f3: 5'-gagctcggtacccggggatccatgtatcgggtgaagagcgagag-3' (SEQ ID NO.9, containing the enzyme cleavage site KpnI);

r3: 5'-tccaagggcgaattggtcgactttgaattctgacatttcacaggc-3' (SEQ ID NO.10, containing the cleavage site BamHI).

The overexpression vector OsGAmyb-p1306 for OsGAmyb gene was constructed by ligating into pCAMBIA-1306 vector (pCAMBIA-1306 vector purchased from Cambia Co.) with KpnI and BamHI. The overexpression vector is introduced into a normal rice variety Nipponbare by adopting an agrobacterium EHA105 mediated genetic transformation method.

Culturing all the obtained transgenic seedlings with a normal nutrient solution added with hygromycin for one week, and if the seedlings can grow normally, indicating that the transgenic plants are positive plants. Transplanting all transgenic positive plants into a basket with soil, watering and fertilizing regularly, planting in a field when a young seedling grows to about 15cm, harvesting and planting a single transgenic rice plant after the seedling grows until a homozygous transgenic plant is identified in the T2 generation, and obtaining OsGAmyb gene overexpression plants which are OsGAmyb-OE1, OsGAmyb-OE2 and OsGAmyb-OE3 respectively.

Taking OsGAmyb gene over-expression plant leaves, extracting RNA and carrying out reverse transcription on the RNA to obtain cDNA, detecting the expression quantity of the OsGAmyb gene in the over-expression plant through real-time fluorescent quantitative PCR, wherein the result shows that the expression quantity of the OsGAmyb gene in the over-expression plant (shown in figure 2) is improved compared with that of a control Nipponbare (the expression quantity of wild type Ni in the figure is determined to be '1'), experiments show that the transgene is successful, the gene is successfully over-expressed, and the over-expression plant is successfully constructed. The sequences of the primer pairs F4 and R4 used for real-time fluorescent quantitative PCR are respectively as follows:

F4：5'-cagaagaacaccgggctgt-3'(SEQ ID NO.11)；

R4：5'-caaatgagcggccatccga-3'(SEQ ID NO.12)。

the data in fig. 2 were analyzed for variables (ANOVA) using SPSS software, and for significance of differences at the 0.05 level using Duncan's, asterisks indicate significant differences between the different groups compared to the control group (p <0.05, p <0.01, p < 0.001).

Comparative example 1: construction of OsTCP21 Gene mutant plants (Gene knockout)

A gene knockout carrier OsTCP21-C of an OsTCP21 gene is constructed by utilizing a primer pair F5 and R5, wherein F5 and R5 are respectively as follows:

F5：5'-gctgtggcggtccttcccgcgttttagagctagaaatagcaagtta-3'(SEQ ID NO.13)；

R5：5'-gcgggaaggaccgccacagcaacctgagcctcagcgcagc-3'(SEQ ID NO.14)。

gene knock-out is performed using the CRISPR/Cas9 system, the methods of which are described in prior art documents: maxetal, A robust CRISPR/Cas9 system for meeting, high-efficiency multiplex gene editing in monocot and dicot plants. mol plant.2015,8(8), 1274-. The gene knockout expression vector is introduced into a normal japonica rice variety Nipponbare (Ni) by adopting an agrobacterium EHA105 mediated genetic transformation method. Sequencing the mutant plant in the T0 generation, determining that 3 strains are knocked out of the gene (figure 3), continuously and independently breeding to the T1 generation to obtain the independent mutant plant strains (all homozygous mutants) of the OsTCP21 gene, wherein the independent mutant plant strains are respectively as follows: OsTCP21-C1 (deletion of 2bp), OsTCP21-C2 (deletion of 1bp) and OsTCP21-C3 (deletion of 2 bp).

Comparative example 2: construction of OsGAmyb Gene mutant plants (Gene knockout)

Constructing a gene knockout carrier OsGAmyb-C of OsGAmyb gene by utilizing a primer pair F6 and R6, wherein F6 and R6 are respectively:

F6：5'-ggagcagatggactcgccgggttttagagctagaaatagcaagtta-3'(SEQ ID NO.15)；

R6：5'-ccggcgagtccatctgctccaacctgagcctcagcgcagc-3'(SEQ ID NO.16)。

gene knock-out is performed using the CRISPR/Cas9 system, the methods of which are described in prior art documents: maxetal, A robust CRISPR/Cas9 system for meeting, high-efficiency multiplex gene editing in monocot and dicot plants. mol plant.2015,8(8), 1274-. The gene knockout expression vector is introduced into a normal japonica rice variety Nipponbare (Ni) by adopting an agrobacterium EHA105 mediated genetic transformation method. Sequencing the mutant plant in the T0 generation, determining that 3 strains are knocked out of the gene (figure 4), continuously and independently breeding to the T1 generation to obtain the independent mutant plant strains (all homozygous mutants) of the OsTCP21 gene, wherein the independent mutant plant strains are respectively: OsGAmyb-C1 (deletion of 2bp), OsGAmyb-C2 (deletion of 1bp) and OsGAmyb-C3 (deletion of 1 bp).

Experimental example 1: hydroponic culture test

Seeds of an over-expression plant (example 1), Nipponbare (Ni) and a gene knockout plant (comparative example 1) are soaked in distilled water for 3 days on a culture dish and cultured for 7 days, then the seeds are transferred into a rice nutrient solution for culture, the formula of the nutrient solution refers to the formula of the conventional international rice in the prior art, the rice nutrient solution is cultured for 40 days respectively, the rice phenotype is observed, the number and the biomass (namely the fresh weight of seedlings) of rice tillering buds are counted, and the rice tillering buds are photographed by a fluorescence stereomicroscope. The shooting result of the tillering bud is shown in fig. 5, and the picture shows that compared with a control Nipponbare plant, the tillering number and the length of the tillering bud of the OsTCP21 gene over-expression plant are increased, and the difference is obvious. Compared with a control Nipponbare plant, the OsTCP21 gene knockout mutant plant has the advantages that the tillering number and the tillering bud length are reduced, and the difference is obvious.

Seeds of an over-expression plant (example 2), Nipponbare (Ni) and a gene knockout plant (comparative example 2) are soaked in distilled water for 3 days on a culture dish and cultured for 7 days, then the seeds are transferred into a rice nutrient solution for culture, the formula of the nutrient solution refers to the formula of the conventional international rice in the prior art, the rice nutrient solution is cultured for 40 days respectively, the rice phenotype is observed, the number and the biomass of rice tillering buds are counted, and the rice tillering buds are photographed by a fluorescence stereomicroscope. The shooting result of the tillering bud is shown in fig. 6, and the picture shows that compared with a control Nipponbare plant, the tillering number and the length of the tillering bud of the OsGAmyb gene over-expression plant are increased, and the difference is obvious. Compared with a control Nipponbare plant, the OsGAmyb gene knockout mutant plant has the advantages that the tillering number and the tillering bud length are reduced, and the difference is obvious.

The counting of the number of tillering buds of the hydroponic plants of examples 1 and 2 and comparative examples 1 and 2 is performed, and the statistical result is shown in FIG. 7 (the tillering numbers of repeated experiments are the same and are all 2 for three gene knockout plants). The statistical result of the number of the tillering buds of the rice shows that compared with a control Nipponbare plant, an OsTCP21 gene overexpression plant and an OsGAmyb gene overexpression plant have the advantages that the tillering number and the length of the tillering buds are increased, and the difference is obvious. Compared with a control Nipponbare plant, the OsTCP21 gene knockout mutant plant and the OsGAmyb gene knockout mutant plant have the advantages that the tillering number and the tillering bud length are reduced, and the difference is obvious. The biomass statistics of the hydroponic plants of examples 1 and 2 and comparative examples 1 and 2 shows that the biomass statistics result (figure 8) shows that the biomass of the OsTCP21 gene overexpression plant and the OsGAmyb gene overexpression plant is increased compared with that of the control Nipponbare plant, and the difference is obvious. Compared with a control Nipponbare plant, the OsTCP21 gene knockout mutant plant and the OsGAmyb gene knockout mutant plant have reduced biomass and reach obvious difference. In fig. 7 and 8, the data were analyzed for variables (ANOVA) using SPSS software, and for significance of differences at the 0.05 level using Duncan's, asterisks indicate significant differences between the different groups compared to the control group (p <0.05, p < 0.01).

Experimental example 2: field planting experiment

The field planting experiments were performed using the over-expressed plants (example 1), nipponlily (Ni) and the knockout plants (comparative example 1). Three lines of over-expression plants of OsTCP21 gene, three lines of OsTCP21 gene knockout plants and a control Nipponbare (Ni) are randomly selected from a field, and are placed into a small bucket for photographing, so that the over-expression plants are found to have increased tillering and the gene knockout plants are found to have reduced tillering (figure 9). Randomly selecting one from the three strains of the over-expression plant, the three gene knockout strains and the control Nipponbare, arranging all the grouted rice seeds with shells into a circle, finding that the seeds of the over-expression plant are enlarged than the circle of the control Nipponbare, and the seeds of the gene knockout plant are reduced than the circle of the control Nipponbare (figure 10). The single plant yield and the single plant tillering number of the over-expression plants, the gene knockout plants and the control group are counted, statistics shows that the single plant yield and the single plant tillering number of the over-expression plants are obviously increased compared with the control group, but the gene knockout plants are obviously reduced (figure 11).

The field planting experiment was carried out using the over-expressed plants (example 2), Nipponbare (Ni) and knockout plants (comparative example 2) according to the contents of the previous paragraph of this example, and the experimental results are shown in FIGS. 12, 13 and 14. Over-expression plants have increased tillering, and gene knockout plants have decreased tillering (fig. 12). The over-expressed plant seeds increased in size compared to the circle of control nipponica, and the knockout plant seeds decreased in size compared to the circle of control nipponica (fig. 13). The yield and tiller number of the over-expression plants are obviously increased compared with those of the control group, but the gene knockout plants show obvious reduction (figure 14).

In FIGS. 11 and 14, the hollow bar-shaped columns represent the yield per plant (g), and the solid bar-shaped columns represent the tiller number per plant. Data were analyzed for variables (ANOVA) using SPSS software, and for significance of differences at the 0.05 level using Duncan's, lower case letters indicating significant differences between groups (. about.. indicates p <0.05,. about.. indicates p < 0.01). The results show that the tillering number of normal rice can be increased after the OsTCP21 gene is improved and the number of seeds of a single rice plant is increased, so that the yield of the single rice plant is increased. By knocking out the OsTCP21 gene, the tillering number of the rice is reduced, the number of seeds of a single rice plant is reduced, the thousand kernel weight is reduced, the single-plant yield of the rice is reduced, and the single-plant yield of the rice is seriously influenced. After the OsGAmyb gene is improved and expressed, the tillering number of normal rice can be increased, the number of single rice seeds is increased, the biomass is increased, and therefore the single rice yield is increased. The OsGAmyb gene is knocked out, so that the tillering number of rice is reduced, the number of seeds of a single rice plant is reduced, the thousand kernel weight is reduced, the single-plant yield of the rice is reduced, and the single yield of the rice is seriously influenced.

Experimental example 3: upstream regulatory factor of OsTCP21 gene and OsGAmyb gene

In the process of researching the influence of miR319 on rice development, three miR319 knockout plants STTM319-1, STTM319-2 and STTM319-3 are constructed, and three miR319 overexpression plants OE319a-1, OE319a-2 and OE319a-3 are constructed. The base parts (rhizome junction, also at the tillering bud extension) of the miR319 knockout plant, the miR319 overexpression plant and the wild type plant are taken, RNA is extracted and is reversely transcribed into cDNA, the expression quantity of the OsTCP21 gene and the OsGAmyb gene in the plants is detected by real-time fluorescence quantitative PCR, and the results are shown in fig. 15 and fig. 16. Experimental results show that miR319 overexpression can cause the expression quantity of the OsTCP21 gene and the OsGAmyb gene to be reduced, and miR319 knockout can cause the expression quantity of the OsTCP21 gene and the OsGAmyb gene to be increased. This indicates that both the OsTCP21 gene and the OsGAmyb gene are simultaneously regulated by miR 319. The data in fig. 15 and 16 were analyzed for variables (ANOVA) using SPSS software, and for significance of differences at the 0.05 level using Duncan's, asterisks indicate significant differences between the different groups compared to the control group (p <0.05, p <0.01, p < 0.001).

The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

SEQUENCE LISTING

<110> Guizhou university

<120> application of transcription factor in rice breeding and rice breeding method

<130> 2021/3/2

<160> 16

<170> PatentIn version 3.5

<210> 1

<211> 553

<212> PRT

<213> Oryza sativa L.

<400> 1

Met Tyr Arg Val Lys Ser Glu Ser Asp Cys Asp Met Ile His Gln Glu

1 5 10 15

Gln Met Asp Ser Pro Val Ala Asp Asp Gly Ser Ser Gly Gly Ser Pro

20 25 30

His Arg Gly Gly Gly Pro Pro Leu Lys Lys Gly Pro Trp Thr Ser Ala

35 40 45

Glu Asp Ala Ile Leu Val Asp Tyr Val Lys Lys His Gly Glu Gly Asn

50 55 60

Trp Asn Ala Val Gln Lys Asn Thr Gly Leu Phe Arg Cys Gly Lys Ser

65 70 75 80

Cys Arg Leu Arg Trp Ala Asn His Leu Arg Pro Asn Leu Lys Lys Gly

85 90 95

Ala Phe Thr Ala Glu Glu Glu Arg Leu Ile Ile Gln Leu His Ser Lys

100 105 110

Met Gly Asn Lys Trp Ala Arg Met Ala Ala His Leu Pro Gly Arg Thr

115 120 125

Asp Asn Glu Ile Lys Asn Tyr Trp Asn Thr Arg Ile Lys Arg Cys Gln

130 135 140

Arg Ala Gly Leu Pro Ile Tyr Pro Thr Ser Val Cys Asn Gln Ser Ser

145 150 155 160

Asn Glu Asp Gln Gln Cys Ser Ser Asp Phe Asp Cys Gly Glu Asn Leu

165 170 175

Ser Asn Asp Leu Leu Asn Ala Asn Gly Leu Tyr Leu Pro Asp Phe Thr

180 185 190

Cys Asp Asn Phe Ile Ala Asn Ser Glu Ala Leu Pro Tyr Ala Pro His

195 200 205

Leu Ser Ala Val Ser Ile Ser Asn Leu Leu Gly Gln Ser Phe Ala Ser

210 215 220

Lys Ser Cys Ser Phe Met Asp Gln Val Asn Gln Thr Gly Met Leu Lys

225 230 235 240

Gln Ser Asp Gly Val Leu Pro Gly Leu Ser Asp Thr Ile Asn Gly Val

245 250 255

Ile Ser Ser Val Asp Gln Phe Ser Asn Asp Ser Glu Lys Leu Lys Gln

260 265 270

Ala Val Gly Phe Asp Tyr Leu His Glu Ala Asn Ser Thr Ser Lys Ile

275 280 285

Ile Ala Pro Phe Gly Gly Ala Leu Asn Gly Ser His Ala Phe Leu Asn

290 295 300

Gly Asn Phe Ser Ala Ser Arg Pro Thr Ser Gly Pro Leu Lys Met Glu

305 310 315 320

Leu Pro Ser Leu Gln Asp Thr Glu Ser Asp Pro Asn Ser Trp Leu Lys

325 330 335

Tyr Thr Val Ala Pro Ala Leu Gln Pro Thr Glu Leu Val Asp Pro Tyr

340 345 350

Leu Gln Ser Pro Ala Ala Thr Pro Ser Val Lys Ser Glu Cys Ala Ser

355 360 365

Pro Arg Asn Ser Gly Leu Leu Glu Glu Leu Ile His Glu Ala Gln Thr

370 375 380

Leu Arg Ser Gly Lys Asn Gln Gln Thr Ser Val Ile Ser Ser Ser Ser

385 390 395 400

Ser Val Gly Thr Pro Cys Asn Thr Thr Val Leu Ser Pro Glu Phe Asp

405 410 415

Met Cys Gln Glu Tyr Trp Glu Glu Gln His Pro Gly Pro Phe Leu Asn

420 425 430

Asp Cys Ala Pro Phe Ser Gly Asn Ser Phe Thr Glu Ser Thr Pro Pro

435 440 445

Val Ser Ala Ala Ser Pro Asp Ile Phe Gln Leu Ser Lys Val Ser Pro

450 455 460

Ala Gln Ser Thr Ser Met Gly Ser Gly Glu Gln Val Met Gly Pro Lys

465 470 475 480

Tyr Glu Pro Gly Asp Thr Ser Pro His Pro Glu Asn Phe Arg Pro Asp

485 490 495

Ala Leu Phe Ser Gly Asn Thr Ala Asp Pro Ser Val Phe Asn Asn Ala

500 505 510

Ile Ala Met Leu Leu Gly Asn Asp Leu Ser Ile Asp Cys Arg Pro Val

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Leu Gly Asp Gly Ile Met Phe Asn Ser Ser Ser Trp Ser Asn Met Pro

530 535 540

His Ala Cys Glu Met Ser Glu Phe Lys

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<210> 2

<211> 1662

<212> DNA

<213> Oryza sativa L.

<400> 2

atgtatcggg tgaagagcga gagcgactgc gatatgatcc atcaggagca gatggactcg 60

ccggtggccg acgacggcag cagcgggggg tcgccgcacc gcggcggcgg gcccccgctg 120

aagaaggggc catggacgtc ggcggaggac gccatcctgg tggactacgt gaagaagcac 180

ggcgagggga actggaacgc ggtgcagaag aacaccgggc tgttccggtg cggcaagagc 240

tgccgcctcc ggtgggcgaa ccacctgagg cccaacctca agaagggggc cttcaccgcc 300

gaggaggaga ggctcatcat ccagctccac tccaagatgg ggaacaagtg ggctcggatg 360

gccgctcatt tgccagggcg cactgataat gaaataaaga attactggaa tactcgaata 420

aagagatgcc agcgagctgg cctacccatc tatcctacca gcgtatgcaa tcaatcctca 480

aatgaagatc agcagtgctc cagtgatttt gactgtggcg agaatttgtc aaacgatctt 540

ctgaatgcaa atggtcttta cctaccagat tttacctgtg acaatttcat tgctaattca 600

gaggctttac cttatgcacc acatctttca gccgtttcta taagcaatct ccttggccag 660

agctttgcat caaaaagctg tagcttcatg gatcaggtaa accagacagg gatgctaaaa 720

cagtctgatg gtgtgcttcc tggattgagc gataccatca acggtgtgat ttcctcggtg 780

gatcaattct caaatgactc tgagaagctc aagcaggctg tgggttttga ctatctccat 840

gaagccaact ctaccagcaa gattattgca cctttcgggg gtgcacttaa tggcagccat 900

gcctttttaa atggcaattt ctctgcttct aggcccacaa gtggtccttt gaagatggag 960

ctcccttcac tccaagatac tgaatctgat ccaaacagct ggctcaagta cactgtagct 1020

cctgcgttgc agcctactga gttagttgat ccctacctgc agtctccagc agcaacccct 1080

tcagtgaaat cagagtgcgc gtcgccaagg aatagtggcc ttttggaaga gttgattcat 1140

gaagctcaga ccctaagatc cgggaagaac caacagacat ctgtgataag ttctagttct 1200

tctgtcggta cgccatgtaa tactacggtt cttagcccag agtttgatat gtgtcaggaa 1260

tactgggaag aacaacatcc tggtccattc ctcaatgact gtgctccttt cagtggcaat 1320

tcattcactg aatccacccc tcctgttagc gctgcatcgc ctgacatctt tcagctctcc 1380

aaagtttccc cagcacaaag cacttcaatg ggatctggag agcaagtaat ggggcctaaa 1440

tatgaacctg gggacacttc acctcatcct gaaaacttca ggccagatgc attgttttct 1500

gggaatacag ctgatccatc agttttcaac aatgccatag caatgcttct gggcaatgac 1560

ttgagtatcg attgcagacc tgttcttggc gacggtatca tgttcaattc ttcctcgtgg 1620

agcaacatgc cacacgcctg tgaaatgtca gaattcaaat ga 1662

<210> 3

<211> 445

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<213> Oryza sativa L.

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Met Glu Leu Ala Gly Ser Asn Asn Lys Arg Gly Arg Val Arg Gly Pro

1 5 10 15

Asn Asp Asp Asp Asp Asp Ala Gly Glu Pro Asp Ala Lys Arg His His

20 25 30

His Gln Leu Leu Leu Pro Trp Pro Gln Gln Gln Gln Gln Gln His Pro

35 40 45

Ala Ser Arg Ile Tyr Arg Val Ser Arg Ala Ser Gly Gly Lys Asp Arg

50 55 60

His Ser Lys Val Tyr Thr Ala Lys Gly Ile Arg Asp Arg Arg Val Arg

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Leu Ser Val Ser Thr Ala Ile Gln Phe Tyr Asp Leu Gln Asp Arg Leu

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Gly Tyr Asp Gln Pro Ser Lys Ala Ile Glu Trp Leu Ile Lys Ala Ala

100 105 110

Ala Ala Ala Ile Asp Lys Leu Pro Ser Leu Asp Thr Ala Ser Phe Pro

115 120 125

Thr Asn Pro Ala Ser Ser Ala Ala Val Ala Ala Ala Ala Ala Pro Pro

130 135 140

Leu Pro His Ala Glu Arg Glu Gln Gln Gln Gln Leu Thr Lys Ser Gly

145 150 155 160

Cys Ser Ser Thr Ser Glu Thr Ser Lys Gly Ser Val Leu Ser Leu Ser

165 170 175

Arg Ser Glu Ser Arg Val Lys Ala Arg Glu Arg Ala Arg Glu Arg Ser

180 185 190

Ser Ala Ala Ala Ala Ala Ala Ser Lys Asp Ala Gly Asp Asp Ala Ala

195 200 205

Thr Pro Thr Ala Pro Thr Ala Ala Pro Ala Ser Ser Gln Ala Ala Ser

210 215 220

Phe Thr Glu Leu Leu Thr Gly Met Ala Ala Ala Asn Ala Ser Pro Ala

225 230 235 240

Asp His Lys Gln Gln Gln Ala Trp Gln Pro Met Thr Val Ala Ala Ala

245 250 255

Thr Ala Asp Tyr Ile Gly Phe Ala Ala Ala Ala Ala Pro His Thr Gln

260 265 270

Pro Arg Lys Ser Ala Ala Gly His His Ser Ala Met Pro His Thr Phe

275 280 285

Ala Ser Pro Ala Pro His Leu Ala Asn Ile Thr Pro Ile Ala Met Ala

290 295 300

Pro Ala Gln His Phe Thr Leu Thr Pro Ala Ala Ala Glu His His Ala

305 310 315 320

Glu Met Thr His Tyr Ser Phe Asp His Phe Met Pro Val His Ala Ala

325 330 335

Ala Ala Ala Ala Ala Ala Ala Ser Thr Pro Ala Gly Gly Asp Tyr Asn

340 345 350

Leu Asn Phe Ser Met Ser Ser Gly Leu Val Gly Val His Ser Arg Gly

355 360 365

Thr Leu Gln Ser Asn Ser Gln Ser His Leu Ser Ser His His His His

370 375 380

His His Gln Gln Gln Gln Gln Gln Gln Gln Leu Gln Arg Leu Ser Ala

385 390 395 400

Pro Leu Asp Ala Pro Asn Ile Pro Phe Leu Phe Ser Pro Ala Ala Ala

405 410 415

Pro Thr Ala Ala Asp Thr Gln Phe Ala Ala Ala Leu Gln Leu Trp Asp

420 425 430

Gly Phe Arg His Ala Asp Ile Lys Glu Lys Gly Lys His

435 440 445

<210> 4

<211> 1338

<212> DNA

<213> Oryza sativa L.

<400> 4

atggagctcg ccggcagcaa caacaagcgg gggagggtga gggggcccaa cgacgacgac 60

gacgacgccg gggagccgga cgccaagcgc catcaccacc agctgctgtt gccgtggccg 120

cagcagcagc agcagcagca tccggcgtcg cggatctacc gggtgtcgcg ggcctccggc 180

gggaaggacc gccacagcaa ggtgtacacg gccaagggca tccgcgaccg ccgcgtccgc 240

ctctccgtct ccaccgccat ccagttctac gacctccagg atcgtctcgg gtatgaccag 300

ccgagcaagg ccatcgagtg gctcattaag gccgccgccg ccgccatcga caagctccct 360

tcgcttgata ccgcttcctt ccccaccaac cccgcctcct ccgccgctgt tgctgctgct 420

gctgctcctc ctcttcccca tgccgaacgc gagcagcagc agcagctcac caagtccgga 480

tgcagtagca cgtcggagac cagcaagggc tccgtcctct ccctctcccg ctccgagagc 540

cgcgtcaagg ccagggagcg cgccagggag cggagcagcg cggccgccgc cgcggcatcc 600

aaggacgccg gggacgacgc cgccaccccc accgcgccca ccgccgcgcc cgcctcctcg 660

caggcggctt ccttcaccga gctactcacc gggatggccg cagccaacgc ctcacccgct 720

gaccacaagc agcagcaggc gtggcagccg atgaccgtcg ccgcggccac cgccgactac 780

atcggcttcg ccgccgccgc cgcgccgcat acgcagccgc ggaaatccgc cgcgggccat 840

cactccgcga tgccgcacac cttcgcatca cccgctcccc acctcgccaa catcaccccc 900

atcgccatgg cgcccgcgca gcacttcacc ctcacacccg ccgccgccga gcaccacgcg 960

gagatgacgc actactcgtt cgaccacttc atgcccgtcc acgcggcagc agcggcggcg 1020

gcggcggcct ccaccccggc cggcggcgac tacaacctca acttctccat gtcctcgggt 1080

cttgtgggtg tccacagtag ggggaccctt cagtccaatt cgcagtctca cctctccagc 1140

catcaccacc accatcacca gcaacagcag cagcagcagc agctgcagag gctgtctgct 1200

ccgctggatg cgcccaacat tccgttcctc ttcagcccgg ccgccgcgcc caccgccgcg 1260

gacacccagt tcgccgccgc attgcagctc tgggacgggt tccggcacgc cgacatcaag 1320

gagaagggca agcactga 1338

<210> 5

<211> 41

<212> DNA

<213> Artificial sequence

<400> 5

gagctcggta cccggggatc catggagctc gccggcagca a 41

<210> 6

<211> 44

<212> DNA

<213> Artificial sequence

<400> 6

tccaagggcg aattggtcga cgtgcttgcc cttctccttg atgt 44

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

tctcacctct ccagccatca 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<400> 8

gtgcttgccc ttctccttga 20

<210> 9

<211> 44

<212> DNA

<213> Artificial sequence

<400> 9

gagctcggta cccggggatc catgtatcgg gtgaagagcg agag 44

<210> 10

<211> 45

<212> DNA

<213> Artificial sequence

<400> 10

tccaagggcg aattggtcga ctttgaattc tgacatttca caggc 45

<210> 11

<211> 19

<212> DNA

<213> Artificial sequence

<400> 11

cagaagaaca ccgggctgt 19

<210> 12

<211> 19

<212> DNA

<213> Artificial sequence

<400> 12

caaatgagcg gccatccga 19

<210> 13

<211> 46

<212> DNA

<213> Artificial sequence

<400> 13

gctgtggcgg tccttcccgc gttttagagc tagaaatagc aagtta 46

<210> 14

<211> 40

<212> DNA

<213> Artificial sequence

<400> 14

gcgggaagga ccgccacagc aacctgagcc tcagcgcagc 40

<210> 15

<211> 46

<212> DNA

<213> Artificial sequence

<400> 15

ggagcagatg gactcgccgg gttttagagc tagaaatagc aagtta 46

<210> 16

<211> 40

<212> DNA

<213> Artificial sequence

<400> 16

ccggcgagtc catctgctcc aacctgagcc tcagcgcagc 40

Claims

1. A method of rice breeding characterized by: improving the expression quantity of the OsGAmyb gene and/or OsTCP21 gene protein of the rice plant; the amino acid sequence of the OsGAmyb gene is shown as SEQ ID NO.1, and the amino acid sequence of the OsTCP21 gene is shown as SEQ ID NO. 3.

2. A method of breeding rice as claimed in claim 1, wherein: the nucleotide sequence of the OsGAmyb gene is shown as SEQ ID NO.2, and the nucleotide sequence of the OsTCP21 gene is shown as SEQ ID NO. 4.

3. A method of breeding rice as claimed in claim 2, wherein: the steps for improving the expression quantity of the OsGAmyb gene of the rice plant are as follows: obtaining cDNA of OsGAmyb gene from wild rice by RT-PCR, and then connecting the cDNA of OsGAmyb gene to pCAMBIA-1306 vector to obtain overexpression vector OsGAmyb-p 1306; an agrobacterium-mediated genetic transformation method is adopted to introduce an overexpression vector OsGAmyb-p1306 into wild rice, and an OsGAmyb gene overexpression plant is obtained through screening and cultivation.

4. A method of breeding rice as claimed in claim 3, wherein: the primers for obtaining the cDNA of the OsGAmyb gene from wild-type rice by using RT-PCR comprise F1 with a nucleotide sequence shown as SEQ ID NO.5 and R1 with a nucleotide sequence shown as SEQ ID NO. 6.

5. A method of breeding rice as claimed in claim 2, wherein: the steps for improving the expression quantity of the OsTCP21 gene of the rice plant are as follows: obtaining cDNA of OsTCP21 gene from wild rice by using RT-PCR, and then connecting the cDNA of OsTCP21 gene to pCAMBIA-1306 vector to obtain overexpression vector OsTCP-p 1306; an agrobacterium-mediated genetic transformation method is adopted to introduce the overexpression vector OsTCP-p1306 into wild rice, and an OsTCP21 gene overexpression plant is obtained after screening and cultivation.

6. A method of breeding rice as claimed in claim 5, wherein: the cDNA of the OsTCP21 gene obtained from wild-type rice by RT-PCR comprises F3 with a nucleotide sequence shown as SEQ ID NO.9 and R3 with a nucleotide sequence shown as SEQ ID NO. 10.

7. The application of the transcription factor in rice breeding is characterized in that: increasing the expression level of the transcription factor; the transcription factor comprises OsGAmyb protein and/or OsTCP21 protein; the amino acid sequence of the OsGAmyb protein is shown as SEQ ID NO.1, and the amino acid sequence of the OsTCP21 protein is shown as SEQ ID NO. 3.

8. Use of the transcription factor of claim 7 in rice breeding, wherein: the transcription factor is used for improving the tillering number, the biomass and the grain number of rice.

9. Use of the transcription factor of claim 8 in rice breeding, wherein: constructing the overexpression rice plant of the OsGAmyb gene.

10. Use of the transcription factor of claim 8 in rice breeding, wherein: constructing an overexpression rice plant of the OsTCP21 gene.