CN116814674A - Application of MYB transcription factor OsMYBR17 gene for promoting tillering and ear development simultaneously in improving rice yield - Google Patents

Abstract

The invention belongs to the technical field of plant genetic engineering, relates to the technical field of crop breeding, and specifically relates to the application of a MYB transcription factor OsMYBR17 gene that simultaneously promotes tiller and panicle development in increasing rice yield. The protein encoded by the OsMYBR17 gene is an amino acid sequence such as SEQ ID The protein shown in NO.1; or it is a protein with equivalent activity obtained by substituting, replacing and/or adding one or several amino acids to the amino acid sequence shown in SEQ ID NO.1. This technical solution has discovered for the first time a gene that positively regulates tillering and panicle development at the same time, which can promote rice tillering, increase the number of tillers, the panicle length of a single panicle branch, the weight of a single panicle, and the number of grains in a single panicle. thereby increasing rice yield. Overexpression of this gene can be applied to the genetic improvement of rice plant type and yield, creating conditions for increasing rice yield and cultivating new plant type rice.

Description

OsMYBR17, a MYB transcription factor that promotes both tiller and panicle development, is Application in increasing rice yield

Technical field

The invention belongs to the field of plant genetic engineering technology, relates to the field of crop breeding technology, and specifically relates to the application of a MYB transcription factor OsMYBR17 gene that simultaneously promotes tiller and panicle development in increasing rice yield.

Background technique

Rice (Oryza sativa L.) is one of the most important food crops in the world, and its yield determines the world's food security. The yield of rice is mainly determined by three agronomic traits, namely the number of panicles per plant, the number of grains per panicle and the weight of a single grain. Among them, the number of panicles per plant is closely related to the tillers of rice. Rice tillers are special branches containing spikes in monocotyledonous plants. Their formation can be mainly divided into two stages, namely the initiation of tiller buds and the elongation of tiller buds (O.Leyser, Regulation of shoot branching by auxin.8( 2003):541-545.). Usually, rice tiller buds mainly occur in the axils of each leaf on the main stem of rice, but not all tiller buds can form effective tillers, affecting the yield of a single rice plant. Only the axillary buds located in the ungerminated basal internode can develop into effective tillers, thereby producing effective panicles and affecting the yield of individual rice plants (Y.Wang, J.Li, The plant architecture of rice (Oryza sativa). Plant Molecular Biology, 59(2005):75-84.). Therefore, the final tiller number of rice depends on the initial number of tiller buds and the number of panicle branch differentiations. Therefore, if you want to increase the yield of a single rice plant, the most important thing is to increase the number of initial tiller buds and panicle branch differentiation.

Tiller and panicle development, as two independent development processes in rice, can both affect the final yield of rice. Previous studies have revealed that many genes exhibit pleiotropic effects in rice tiller and panicle development, and these genes are co-regulated in different parts. For example, PAY1 plays an important role in the core pathway of rice panicle development, encoding a protein related to auxin transport. It positively regulates panicle secondary branches and negatively regulates rice tillers (L. Zhao, L. Tan, Z. Zhu ,et al,PAY 1improves plant architecture and enhances grain yield inrice,The Plant Journal.83(2015)528-536.). IPA1 binds to DEP1 at the panicle to increase the number of branches, and binds to TB1 at the base to inhibit rice tillering, thereby regulating the rice plant architecture (G.Li, H.Zhang, J.Li, et al, Genetic control of panicle architecture in rice, Crop .J.9(2021)590-597.). The Ghd8 gene in rice can simultaneously regulate rice tillers, plant height, and number of panicle branches. However, the gene regulates tillers and panicle branches in opposite directions (W.H.Yan, P.Wang, H.X.Chen, et al ,A major QTL,Ghd8,playspleiotropic roles in regulating grain productivity,plant height,and heading date in rice,Mol.Plant.4(2011)319-330.). There are many other genes that are regulated simultaneously on panicles and tillers, including APO2/RFL, RCN1, RCN2 and OsCOL13 (Y.Lu, M.Chuan, H.Wang, et al, Genetic and molecular factors in determining grain number per panicle of rice, Front.PlantSci.13(2022).) etc. However, these genes have opposite effects on the regulation of tillering and panicle development. Promoting panicle development in tillers inhibits panicle development, whereas promoting panicle development inhibits tillering. There are few reports of genes that simultaneously promote the regulation of tillering and panicle development in rice.

Molecular breeding is the application of molecular biology technology to breeding at the molecular level. It is a breeding method that is different from traditional hybrid breeding. Molecular breeding includes transgenic breeding, which is the application of genetic engineering to breeding work. A breeding method that produces new varieties with certain requirements through gene introduction. Transgenic breeding of rice to increase the number of initial tiller buds and panicle branch differentiation is of great significance to increasing rice yield. However, in fact, most genes either only regulate rice tillering or panicle development. A few genes can simultaneously regulate rice tillering and panicle development, but the regulation trends of the above two events are opposite. When such genes are used in genetic breeding, they may improve one trait while having a negative impact on other traits, which is not advisable. In the practice of transgenic breeding, it is very critical to select the target gene for transgenic breeding. There is an urgent need to screen and study genes related to rice development to find genes that can simultaneously positively regulate rice tiller and panicle development. This resulted in a molecular breeding method that can significantly increase rice yield.

Contents of the invention

The present invention intends to provide an application of the MYB transcription factor OsMYBR17 gene that simultaneously promotes tiller and panicle development in increasing rice yield, so as to solve the difficulty in the prior art of improving rice yield by simultaneously promoting tiller and panicle development of rice plants. question.

In order to achieve the above objects, the present invention adopts the following technical solutions:

The application of OsMYBR17 gene in rice planting. The protein encoded by OsMYBR17 gene is a protein with an amino acid sequence as shown in SEQ ID NO.1; or it is a protein with an amino acid sequence as shown in SEQ ID NO.1 with one substitution, substitution and/or addition. Or a protein with equivalent activity obtained from several amino acids.

This solution also provides a rice planting method for increasing rice yield by overexpressing the OsMYBR17 gene in rice plants; the nucleotide sequence of the OsMYBR17 gene is shown in SEQ ID NO.2.

The principles and advantages of this program are:

After increasing the protein expression of OsMYBR17 gene in rice plants, the tiller number, biomass, panicle branch differentiation number and rice number of each rice plant were significantly increased at the same time, thereby increasing the yield of each rice plant. Knockout of this gene simultaneously reduces the number of tillers, biomass, and panicle branch differentiation per rice plant, and plays a negative regulatory role in the yield of a single rice plant. Without affecting the activity of the OsMYBR17 protein (that is, not in the active center of the protein), those skilled in the art can make various substitutions, add and/or delete one or several amino acids to the amino acid sequence shown in SEQ ID NO. Functionally equivalent amino acid sequences. Therefore, the OsMYBR17 protein also includes proteins with equivalent activity obtained by substituting, replacing and/or adding one or several amino acids to the amino acid sequence shown in SEQ ID NO.1. In addition, taking into account the degeneracy of codons and the preference of codons in different species (for example, synonymous mutations according to codon preference), those skilled in the art can use codons suitable for expression of specific species as needed.

The inventor cloned the OsMYBR17 gene cDNA sequence from rice Zhonghua 11 (ZH11). By constructing an OsMYBR17 gene overexpression vector and introducing the overexpression vector into Zhonghua 11 (ZH11), an OsMYBR17 gene overexpression plant was obtained. The length, biomass and panicle branch differentiation of a single rice plant were similar to those of Zhonghua 11. ratio, were significantly improved. By constructing a gene knockout vector for the OsMYBR17 gene, the knockout vector was introduced into Zhonghua 11 (ZH11), and a gene knockout plant for the OsMYBR17 gene was obtained. The number of tillers per rice plant and the yield per plant were similar to those of Zhonghua 11 (ZH11). ratio, were significantly reduced.

The above studies have shown that overexpression of the OsMYBR17 gene in rice plants can increase rice yield, specifically by simultaneously increasing the number of tillers, biomass, panicle branch differentiation and number of rice grains per plant.

In this project, the inventor discovered a new function of the gene and used this function for rice breeding, achieving unexpected technical effects. Regarding the OsMYBR17 gene, the OsMYBR17 gene belongs to the MYB family, is a MYB transcriptional regulator, and encodes a MYB domain. The MYB family is a relatively abundant family of transcription factors in plants, and most of its members regulate plant stress responses. Although some MYB family members have been reported to be involved in the regulation of tillering, for example, the OsMYB110 gene was reported to regulate rice plant height while affecting tillering (CN111187789B, application of a rice MYB transcription factor and its recombinant expression vector). However, existing reports that OsMYB110 independently regulates tillering do not suggest its impact on panicle development. Although it has also been reported that some MYB family members are involved in the regulation of panicle development. For example, the SFL gene belonging to the MYB family regulates floral organs and thus affects panicle development. It may be a key gene that controls the signaling pathway of panicle development. Through phenotypic observation, it was found that after editing this gene in rice, plants will be short and will not form ears. However, the SFL gene independently affects the panicle development process and has no effect on tillers (Hu Guang. Analysis of gene editing and cell-to-cell movement of MYB transcription factors controlling rice panicle development. Yangtze University, 2020.). Moreover, the SFL gene regulates panicle development through the regulation of floral organs, and does not regulate panicle development by regulating panicle branch differentiation. The mechanism of action is different from the OsMYBR17 gene of this technical solution.

Therefore, in the current technology, there is no report that MYB transcription factors can positively regulate tillering and panicle development at the same time. The reported MYB transcription factors, if related to rice tiller or panicle development, independently regulate the two developmental processes. At the same time, there are no reports in the prior art on how the OsMYBR17 gene can improve rice yield. The inventor's research found that the OsMYBR17 gene can not only increase rice yield, but also positively regulate the tillering and panicle development processes of rice. This shows that the inventor has discovered new properties and new functions of the MYB gene, and has used the newly discovered OsMYBR17 gene to It is applied to the practical operation of rice breeding to obtain new rice plants and increase rice yield.

To sum up, the beneficial effects of this program are as follows:

Overexpression of the cloned OsMYBR17 gene significantly increased the number of tillers, biomass and panicle branch differentiation per rice plant at the same time, indicating that the OsMYBR17 gene is more effective in improving rice plant type and increasing yield per rice plant. Therefore, improving OsMYBR17 through genetic engineering technology Gene expression can genetically improve rice plant shape and yield.

The successful cloning of the OsMYBR17 gene has confirmed that MYB transcription factors not only play a role in plant adversity response and stress, but also play an important role in plant tillering, panicle development and plant growth and development. This can enrich the understanding of plant MYB genes and provide insights into plant differentiation. Genetic improvement of branches and yields has a great promotion effect.

Further, the nucleotide sequence of the OsMYBR17 gene is shown in SEQ ID NO. 2.

Furthermore, the OsMYBR17 gene was used to increase the number of tillers in rice.

Furthermore, the OsMYBR17 gene was used to promote rice panicle development.

Furthermore, promoting rice panicle development includes increasing the number of panicle branch differentiations, the panicle length of a single panicle branch, the weight of a single panicle, and the number of panicle grains in a single panicle.

Further, the OsMYBR17 gene was used to increase rice yield.

Using the above technical solution, based on the function of the OsMYBR17 gene discovered by the inventor, it can be used in rice breeding. The purpose of rice selection is to obtain rice plants with higher tiller number, biomass, panicle branch differentiation number, rice number or single plant yield. The expression of OsMYBR17 gene can be improved through overexpression technology to obtain rice plants with excellent properties such as high number of tillers per plant, high biomass, high number of panicle branch differentiations, large number of rice grains, and high yield per plant.

Furthermore, the transcription level of OsMYBR17 gene in rice is increased, or the expression level of OsMYBR17 protein is increased.

Using the above technical solution, in rice plants overexpressing the OsMYBR17 gene, the expression level of OsMYBR17 protein increases, and at the same time, the number of rice tillers, panicle branch differentiation number and single plant yield are increased.

Further, the cDNA of the OsMYBR17 gene was cloned from wild-type rice, and then the cDNA of the OsMYBR17 gene was integrated into the pCAMBIA-1306 vector to obtain the overexpression vector OsMYBR17-p1306; the overexpression vector OsMYBR17 was obtained using the Agrobacterium-mediated genetic transformation method. -p1306 was introduced into wild-type rice, and after screening and cultivation, OsMYBR17 gene overexpression plants were obtained.

Furthermore, the nucleotide sequences of the primers used to clone the cDNA of the OsMYBR17 gene are shown in SEQ ID NO. 3 and SEQ ID NO. 4.

Description of the drawings

Figure 1 is a statistical histogram of real-time fluorescence quantitative PCR of over-expression plants in Example 1 of the present invention (A) and a schematic diagram of gene knockout target design and gene sequence alignment of knockout plants (B).

Figure 2 is a tiller bud phenotype diagram (A) and a statistical bar chart (B: first tiller bud; C: second tiller bud) of a hydroponic rice seedling in Experimental Example 1 of the present invention.

Figure 3 is a phenotypic diagram (A) of plant height and fresh weight of a hydroponic rice seedling in Experimental Example 1 of the present invention and a statistical bar chart (B: plant height; C: fresh weight).

Figure 4 is a phenotypic diagram of a single rice plant under field planting in Experimental Example 2 of the present invention.

Figure 5 is a phenotypic diagram of single panicle branches of rice under field planting in Experimental Example 2 of the present invention.

Figure 6 is a phenotypic diagram of rice yield of a single rice plant under field cultivation in Experimental Example 2 of the present invention.

Figure 7 is a statistical column chart of the yield of a single rice plant under field planting in Experimental Example 2 of the present invention.

Figure 8 is a statistical bar chart of the differentiation number of the first branch of a single panicle branch of rice grown in the field in Experimental Example 2 of the present invention.

Figure 9 is a statistical bar chart of the differentiation number of the second branch of a single panicle branch of rice grown in field in Experimental Example 2 of the present invention.

Figure 10 is a statistical bar chart of ear length of a single ear of rice grown in field in Experimental Example 2 of the present invention.

Figure 11 is a statistical bar chart of the ear weight of a single ear of rice planted in the field in Experimental Example 2 of the present invention.

Figure 12 is a statistical column chart of the number of grains per ear of rice planted in field planting in Experimental Example 2 of the present invention.

Detailed ways

The present invention will be further described in detail below with reference to examples, but the implementation of the present invention is 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 methods used are all conventional methods and can be in accordance with the described recombinant techniques (see Molecular Cloning, Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; MaXetal, Arobust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicotplants. MolPlant.2015,8(8):1274-1284.) Completed; the materials and reagents used are all commercially available.

Example 1: Construction of OsMYBR17 gene overexpression plants

Extract the RNA of rice Zhonghua 11 (ZH11) and reverse-transcribe it into cDNA. Use the primer pair F1 and R1 to amplify the cDNA of the OsMYBR17 gene by PCR (for the protein sequence of the OsMYBR17 gene, see SEQ ID NO.1, gene sequence See SEQ ID NO. 2).

SEQ ID NO.1：

Met Asp Leu Tyr Gly Ala Ala Ala Gly Gly Gly Pro Val Ala Arg Arg ProTrp Ser Lys ValGlu Asp Lys Val Phe Glu Ser Ala Leu Val Leu Cys Pro Glu AspVal Pro Asp Arg Trp Ala LeuVal Ala Ala Gln Leu Pro Gly Arg Thr Pro Gln GluAla Leu Glu His Tyr Gln Val Leu Val AlaAsp Ile Asp Leu Ile Met Arg Gly AlaVal Asp Ala Pro Gly Ser Trp Asp Asp Asn Asp Gly AsnAsp Arg Arg Gly Gly GlyGly Lys Pro Arg Gly Glu Glu Arg Arg Arg Gly Val Pro Trp Ser GluAsp Glu HisArg Leu Phe Leu Glu Gly Leu Asp Arg Tyr Gly Arg Gly Asp Trp Arg Asn IleSerArg Phe Ser Val Arg Thr Arg Thr Pro Thr Gln Val Ala Ser His Ala Gln LysTyr Phe Ile ArgGln Ala Asn Ala Gly Ala Arg Asp Ser Lys Arg Lys Ser Ile HisAsp Ile Thr Thr Pro.

SEQ ID NO.2(O.sativa v7.0|LOC_Os01g64360.1 CDS):

Atggatttgtacggcgcggcggcgggcgggggaccggtggcgaggcgaccgtggagcaaggtggaggacaaggtgttcgagagcgcgctggtgctgtgcccggaggacgtccccgaccggtgggcgctcgtcgcggcgcagctcccagggcgcacgccgcaggaggccttggagcactaccaggt gctcgtcgccgacatcgatctcatcatgcgcggcgccgtcgacgcccccgggtcctgggacgataacgacggcaacgaccgccgcggcggcggcggcaagccccgcggcgaggagcggcgccgcggcgtaccctggtccgaagacgagcacaggttgtttctcgaggggttggacaggtacgggcggggagact ggaggaacatctcgcggttctcggtgaggacgcggacgccgacgcaggtggcgagccacgcgcagaagtacttcatccggcaggccaacgccggcgcccgcgactccaagcgcaagagcatccatgacatcaccaccccttga.

Primer pair F1 and R1, respectively:

F1: 5'-atgaattcatggatttgtacggcgcggcggcg-3' (SEQ ID NO.3, containing restriction site EcoRI);

R1: 5'-ataagctttcaaggggtggtgatgtcatggat-3' (SEQ ID NO. 4, containing enzyme cleavage site HindIII).

Then, the cDNA of the OsMYBR17 gene was ligated into the pCAMBIA-1306 vector (pCAMBIA-1306 vector was purchased from Cambia Company) through two restriction sites EcoRI and HindIII, and the overexpression vector OsMYBR17-p1306 of the OsMYBR17 gene was constructed. The over-expression vector was introduced into the normal rice variety Zhonghua 11 using the conventional Agrobacterium EHA105-mediated genetic transformation method.

All the obtained T1 generation transgenic seedlings were cultured in normal nutrient solution plus hygromycin for one week. If the seedlings can grow normally, it means that the transgenic plants are positive plants. Transplant all transgenic positive plants into baskets with soil, water and fertilize regularly, and plant them in the field when the seedlings grow to about 15cm in height. When the seedlings grow up, individual transgenic rice plants are harvested and planted. Until homozygous transgenic plants are identified in the T2 generation, OsMYBR17 gene overexpression plants are obtained. In this example, three gene overexpression plants were prepared, namely OsMYBR17-OE1, OsMYBR17-OE4 and OsMYBR17-OE5.

The leaves of OsMYBR17 gene overexpressing plants were taken, RNA was extracted and reverse transcribed into cDNA, and the expression level of OsMYBR17 gene in overexpressing plants was detected by real-time fluorescence quantitative PCR. The results showed (Figure 1A) the expression level of OsMYBR17 gene in overexpressing plants. Compared with the control Hua 11 (the expression level of wild-type ZH11 in the figure is set as "1"), the experiment shows that the transgene is successful, the gene is successfully over-expressed, and the over-expression plant is successfully constructed. In Figure 1A, the test samples include the control wild-type Zhonghua 11 (ZH11) and OsMYBR17 gene overexpression plants of three lines OE1, OE4 and OE5; the statistical results are expressed as mean±SD, n=3. Data were analyzed using SPSS software for variable analysis (ANOVA), and Duncan's test was used for significant difference analysis. Lowercase letters are marked above the data, and different lowercase letters indicate significant differences, p<0.05.

The primer pair F2 and R2 used in real-time fluorescence quantitative PCR, their sequences are:

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

R2: 5'-ggatgaagtacttctgcgcgtg-3' (SEQ ID NO. 6).

Example 2: Construction of OsMYBR17 gene mutant plants (gene knockout)

The gene knockout vector OsMYBR17-C of the OsMYBR17 gene was constructed using the primer pair F5 and R5, where F5 and R5 are respectively:

F5: 5'-tcactcggcatggatttgtagttttagagctagaaatagcaagtta-3' (SEQ ID NO. 7);

R5: 5'-gtcgttatcgtcccaggacccgccacggatcatctgcacaactc-3' (SEQ ID NO. 8).

Gene knockout is performed using the CRISPR/Cas9 system. For the method, please refer to the existing technical literature: MaXetal, Arobust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. MolPlant.2015,8(8):1274- 1284. The gene knockout expression vector was introduced into the normal rice variety Zhonghua 11 (ZH11) using the genetic transformation method mediated by Agrobacterium tumefaciens EHA105. The mutant plants were sequenced at the T0 generation, and it was determined that the gene had been knocked out in 3 lines (Figure 1B). They continued to be independently propagated to the T1 generation, and independent mutant plant lines of the OsMYBR17 gene were obtained (all homozygous mutations). body), respectively: OsMYBR17-C5 (29bp deleted), OsMYBR17-C7 (67bp deleted) and OsMYBR17-C9 (53bp deleted).

Experimental example 1: Hydroponics test

The seeds of the overexpression plant (Example 1), Zhonghua 11 (ZH11) and the gene knockout plant (Example 2) were soaked in distilled water on a petri dish for 3 days and cultured for 7 days, then transferred to rice nutrient solution for culture. The liquid formula refers to the conventional International Rice Research Institute formula in the prior art, and is prepared by referring to the literature method (Yoshida S, Fomo DA, Cock JH, etc. Routine procedure for growing rice plants in culture solution. In: Laboratory manual for physiological-logical studies of rice [J] ,International Rice Research Institute,61-66.). After culturing for 40 days respectively, the rice phenotype was observed, the number and biomass of rice tiller buds (i.e. seedling fresh weight) were counted, and the rice tiller buds were photographed with a fluorescent stereomicroscope. The results of tillering bud photography are shown in Figure 2A. It can be seen from the figure that compared with the control Zhonghua 11 plant, the number of tillers and the length of tillering buds in the OsMYBR17 gene overexpression plants increased, and the difference was significant. Compared with the control Zhonghua 11 plant, the number of tillers and tiller bud length of OsMYBR17 gene knockout mutant plants were reduced, and the difference was significant.

The tiller bud lengths of the hydroponic plants of Example 1 and Example 2 were counted. The statistical results are shown in B and C in Figure 2. The statistical results of rice tiller buds showed that the length of tiller buds of OsMYBR17 gene overexpressing plants increased compared with the control Zhonghua 11 plants, and the difference was significant. Compared with the control Zhonghua 11 plant, the number of tillers and tiller bud length of OsMYBR17 gene knockout mutant plants were reduced, and the difference was significant.

Biomass statistics were performed on the hydroponic plants of Example 1 and Example 2. The biomass statistics results showed (Figure 3) that the biomass of the OsMYBR17 gene overexpressing plants increased compared with the control Zhonghua 11 plant, and the difference was significant. Compared with the control Zhonghua 11 plant, the biomass of OsMYBR17 knockout mutant plants was reduced, and the difference was significant.

Among them, in Figures 2 and 3, the statistical results are expressed as mean ± SD, n = 10; the data is analyzed using SPSS software for variable analysis (ANOVA), and the Duncan test is used for significant difference analysis. The upper part of the data is marked with lowercase letters. Different Lowercase letters indicate significant differences, p<0.05.

Experimental Example 2: Field planting experiment

Field planting experiments were conducted using overexpression plants (Example 1), Zhonghua 11 (ZH11) and gene knockout plants (Example 2). Three lines of overexpressing OsMYBR17 gene plants, three lines of OsMYBR17 gene knockout plants and one control Zhonghua 11 (ZH11) were randomly selected from the field and placed in a small bucket for taking pictures. It was found that the tillers of the overexpressed plants were The number of tillers increased and the number of tillers in the gene knockout plants decreased (Figure 4). Randomly select one ear from each of the above three lines of over-expression plants, three gene knockout lines and control medium flower 11, unfold all the fully differentiated ear branches and take pictures, and find that the number of ear branch differentiation ratios of the over-expression plants The number of panicle branch differentiations in flower 11 in the control increased, while the number of panicle branch differentiations in the knockout plants was reduced compared to the number of panicle branch differentiations in flower 11 in the control (Figure 5). Randomly select one plant from the above three lines of over-expression plants, three lines of gene knockout and control medium flower 11, and arrange all the shelled and filled rice seeds in a circle. It is found that the over-expression plant seeds are more The circle of flower 11 in the control was enlarged, and the circle of flower 11 in the seeds of the knockout plant was smaller than that in the control (Figure 6). The yield per plant of overexpression plants, gene knockout plants and the control group was counted. Statistics showed that the yield per plant of overexpression plants was significantly increased compared with the control group, but the yield of gene knockout plants was significantly reduced (Figure 7). Statistics of the first branch differentiation number of panicle branches of overexpression plants, gene knockout plants and control plants showed that the first branch differentiation number of overexpression plants was significantly increased compared with the control group, but the gene knockout plants showed a significant decrease. (Figure 8). The statistics of the second branch differentiation number of panicle branches of overexpression plants, gene knockout plants and control plants showed that the number of secondary branch differentiation of overexpression plants was significantly increased compared with the control group, but the gene knockout plants showed significant differences. decreased (Figure 9). Statistics of the panicle length of overexpression plants, gene knockout plants and control plants showed that the panicle length of overexpression plants was significantly longer than that of the control group, but the gene knockout plants were significantly shorter (Figure 10). The statistics of the single panicle weight of overexpression plants, gene knockout plants and control plants showed that the single panicle weight of overexpression plants was significantly increased compared with the control group, but the gene knockout plants showed a significant decrease (Figure 11). The statistics of the grain number per panicle of overexpression plants, gene knockout plants and control plants showed that the number of grains per panicle of overexpression plants was significantly increased compared with the control group, but the gene knockout plants showed a significant decrease (Figure 12).

In Figure 7-12, the statistical results are expressed as mean±SD, n=10; the data is analyzed using SPSS software for variable analysis (ANOVA), and the Duncan test is used for significant difference analysis. The data are marked with lowercase letters, and different lowercase letters represent There is a significant difference between them, p<0.05.

The above results show that increased expression of the OsMYBR17 gene can cause normal rice to not only increase the number of tillers, but also increase the number of panicle branch differentiations (the number of first branch and second branch differentiations), and the panicle length of a single panicle branch. The weight of a single ear increases, and the number of grains per ear increases. By knocking out the OsMYBR17 gene, the number of rice tillers and panicle branch differentiations are simultaneously reduced, thereby reducing the number of seeds per rice plant, thereby reducing the yield per rice plant and seriously affecting the rice yield per unit area.

Comparative example 1

The MYB (v-myb avain myeloblastosis viral oncogene homolog) transcription factor family is one of the most numerous transcription regulator families in plants and is involved in a variety of physiological and biochemical processes in plants. Most of the MYB transcription factors reported so far combine with downstream genes to initiate gene expression in order to increase the expression of downstream genes. There are also a few members that inhibit gene expression. The MYB gene encoding the MYB transcription factor is so called because it has a highly conserved MYB domain (DNA binding domain). MYB genes are divided into four subgroups according to the number of conserved domains they possess: 1R, R2R3, 3R and 4R. The only similarity between MYB genes is that they all contain a MYB domain. The role of the MYB domain is to bind to the DNA sequence of a certain gene (regulate the expression of the gene, etc.); while in other domains or sequences of the MYB gene, MYB There is no similarity between genes. Therefore, although MYB genes have the above-mentioned MYB domain in common, there are very large differences in gene sequences and protein structures between different MYB genes. Different MYB genes are involved in the regulation of cell proliferation, cell differentiation, hormone signaling, root system structure, high temperature tolerance, various abiotic stress responses, growth and development processes, etc. Even if the genes belong to the same MYB gene, they may have vastly different functions. Therefore, it is difficult to predict the function of a gene simply by whether it belongs to the MYB gene (MYB transcription factor family). Table 1 shows some of the rice MYB genes and their functions that have been reported in the prior art. It can be seen that the diversity of MYB gene functions is very high, and the gene functions cannot be inferred from one to the other.

Table 1: Rice MYB genes and their functions that have been reported in the prior art (quoted from the document "Jin Feng et al., Research Progress of Rice MYB Transcription Factors, Journal of Plant Genetic Resources, 2022; DOI: 10.13430/j.cnki.jpgr.20221220001 ”)

There are only very few documents in the prior art reporting that MYB transcription factors are involved in the regulation of rice tillering or panicle development. However, the reported MYB transcription factors cannot realize the simultaneous positive regulation of the above two events. Moreover, the MYB transcription factors reported in the prior art that are involved in the regulation of rice tillering or panicle development have their gene or protein sequences and those of this scheme. Compared with the OsMYBR17 gene or protein, the sequence similarity is very low. The function of a gene is largely determined by its material base sequence composition. When the sequence similarity is very low, it is difficult to infer whether the gene has similar functions. The following uses OsMYB110 gene and SFL gene as examples for detailed explanation.

Chinese patent CN111187789B reports the application of a rice MYB transcription factor and its recombinant expression vector. The sequence number of the rice MYB transcription factor gene OsMYB110 is LOC_Os10g0478300, which can be used to regulate rice plant height, tillering, flowering period and rice quality. Through systematic research, this invention provides the biological function of the OsMYB110 transcription factor gene and its encoded protein for the first time. The OsMYB110 overexpression material obtained by transgenic means in the present invention significantly increases the expression level of this gene in rice, resulting in a decrease in plant height, an increase in tillers, an earlier flowering period, and improved rice quality in the transgenic rice compared with the wild-type control. The protein sequence of the gene with the patent serial number LOC_Os10g0478300 (SEQ ID NO. 9, Rice Annotation Project database; https://rapdb.dna.affrc.go.jp/), and the gene protein sequence of this scheme (SEQ ID NO. .1) Comparing (NCBI Blast), only more than twenty amino acid residues can form a match, and the number of amino acid residues of the two proteins are 276aa and 173aa respectively. This shows that the sequence similarity between the two proteins is very low, and researchers in this field will not speculate based on the OsMYB110 gene that another gene with almost no sequence similarity may have the function of promoting rice tillering. In addition, in addition to promoting rice tillering, the genes of this solution also have a very obvious promoting effect on promoting rice panicle development. It can be seen that the gene functions of this solution are different from the existing technology.

There are reports in the literature that the SFL gene has the function of regulating rice panicle development, and the SFL gene belongs to the MYB gene (Hu Guang. Analysis of gene editing and intercellular movement of MYB transcription factors that control rice panicle development. Yangtze University, 2020.). In literature records, the sequence number of the SFL gene is LOC_Os07g43580. After consulting the database, its sequence is shown as SEQ ID NO.10. Compared with the gene protein sequence (SEQ ID NO. 1) of this scheme (NCBI Blast), only a few amino acid residues can form a match, and the number of amino acid residues of the two proteins are 341aa and 173aa respectively. Researchers in this field will not speculate based on the SFL gene that another gene with almost no sequence similarity may have the function of promoting rice panicle development. What's more, the SFL gene regulates panicle development through the regulation of floral organs, and does not regulate panicle development by regulating panicle branch differentiation. The mechanism of action is different from the OsMYBR17 gene of this technical solution. In addition, in addition to promoting rice panicle development, the genes of this solution also have a very obvious promoting effect on promoting rice tillering. It can be seen that the gene functions of this solution are different from the existing technology.

The above are only embodiments of the present invention, and common knowledge such as specific technical solutions and/or characteristics that are known in the scheme are not described in detail here. It should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the technical solution of the present invention. These should also be regarded as the protection scope of the present invention and will not affect the implementation of the present invention. The effect and practicality of the patent. The scope of protection claimed in this application shall be based on the content of the claims, and the specific implementation modes and other records in the description may be used to interpret the content of the claims.

Claims (10)

1. The application of the OsMYBR17 gene in rice planting, characterized in that: the protein encoded by the OsMYBR17 gene is a protein with an amino acid sequence as shown in SEQ ID NO.1; or it is a protein with a substituted amino acid sequence as shown in SEQ ID NO.1. A protein with equivalent activity obtained by substituting and/or adding one or several amino acids. 2. Application of the OsMYBR17 gene in rice planting according to claim 1, characterized in that: its nucleotide sequence is as shown in SEQ ID NO.2, or is a sequence obtained by synonymous mutation of SEQ ID NO.2 . 3. Application of the OsMYBR17 gene according to claim 1 or 2 in rice planting, characterized in that it is used to increase the number of tillers of rice. 4. Application of the OsMYBR17 gene according to claim 1 or 2 in rice planting, characterized in that it is used to promote rice panicle development. 5. Application of the OsMYBR17 gene in rice planting according to claim 4, characterized in that: promoting rice panicle development includes increasing the number of panicle branch differentiations, the panicle length of a single panicle branch, the weight of a single panicle, and the number of panicle grains of a single panicle. . 6. Application of the OsMYBR17 gene according to claim 1 or 2 in rice planting, characterized in that it is used to increase rice yield. 7. The application of the OsMYBR17 gene in rice planting according to claim 1 or 2, characterized in that the transcription level of the OsMYBR17 gene in rice is increased, or the expression level of the OsMYBR17 protein is increased. 8. A rice planting method for increasing rice yield, characterized by overexpressing the OsMYBR17 gene in rice plants; the nucleotide sequence of the OsMYBR17 gene is shown in SEQ ID NO.2. 9. A rice planting method for increasing rice yield according to claim 8, characterized in that the cDNA of the OsMYBR17 gene is cloned from wild-type rice, and then the cDNA of the OsMYBR17 gene is integrated on the pCAMBIA-1306 vector, The over-expression vector OsMYBR17-p1306 was obtained; using Agrobacterium-mediated genetic transformation method, the over-expression vector OsMYBR17-p1306 was introduced into wild-type rice. After screening and cultivation, OsMYBR17 gene over-expression plants were obtained. 10. A rice planting method for increasing rice yield according to claim 9, characterized in that the nucleotide sequence of the primer used for cloning the cDNA of the OsMYBR17 gene is such as SEQ ID NO. 3 and SEQ ID NO. 4. shown.