Rice gene OsMYB63L and application of rice gene OsMYB63L in regulation and control of ear branching and yield

Abstract

The invention relates to a rice gene OsMYB63L and application of the rice gene OsMYB63L in regulation and control of spike branching and yield, and belongs to the technical field of plant gene breeding, the nucleotide sequence of the gene OsMYB63L is as shown in SEQ ID NO.1, the amino acid sequence of protein coded by the gene is as shown in SEQ ID NO.2, overexpression of the gene OsMYB63L promotes development of the spike branching of rice, then the yield of rice is increased, a transgenic technology is utilized, and the yield of the rice is increased. Overexpression is carried out on the rice gene OsMYB63L, and a rice plant with increased ear branch number and increased yield can be obtained. The invention discovers and verifies that the rice gene OsMYB63L has the function of regulating and controlling the ear branching number and the yield of rice. And a new gene resource and a technical path are provided for molecular breeding of high-yield rice.

Description

Technical Field

[0001] This invention belongs to the field of plant gene breeding technology, specifically relating to a rice gene OsMYB63L and its application in regulating panicle branching and yield. Background Technology

[0002] Among the components of rice yield, the number of panicle branches is a key trait determining the number of grains per panicle and has a significant impact on the final yield. Increasing the number of panicle branches is one of the important ways to achieve high rice yield (Sakamoto T, Matsuoka M. Identifying and exploiting grain yield genes in rice. Curr Opin Plant Biol. 2008). However, the development of panicle branches is precisely regulated by multiple gene networks, and its molecular mechanisms are not yet fully understood. Therefore, identifying and utilizing key genes that regulate the increase of panicle branches in rice is of great significance for breeding new high-yielding rice varieties.

[0003] The gene OsMYB63L is a MYB transcription factor composed of 340 amino acids. This family has a variety of physiological and biochemical functions and is widely involved in plant development and metabolic regulation. However, there are few reports on the correlation between OsMYB63L and the number of panicle branches and rice yield. Therefore, this invention proposes a rice gene OsMYB63L and its application in regulating panicle branching and yield. Summary of the Invention

[0004] The purpose of this invention is to provide a rice gene OsMYB63L and its application in regulating panicle branching and yield in order to solve the above-mentioned problems.

[0005] The present invention achieves the above objectives through the following technical solutions: This invention provides an application of the rice gene OsMYB63L in regulating panicle branching and yield.

[0006] As a further optimization of the present invention, the nucleotide sequence of the gene OsMYB63L is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0007] As a further optimization of the present invention, overexpression of the gene OsMYB63L promotes panicle branching development in rice, thereby increasing rice yield.

[0008] As a further optimization of the present invention, the rice gene OsMYB63L was overexpressed using transgenic technology to obtain rice plants with increased panicle branch number and higher yield.

[0009] As a further optimization of the present invention, the method for overexpressing the rice gene OsMYB63L to obtain rice plants with increased panicle branching and higher yield includes the following steps: (1) Construct a recombinant plasmid pCAMBIA1300-Ubi-GFP-Flag-OsMYB63L containing the rice OsMYB63L gene; (2) The recombinant plasmid pCAMBIA1300-Ubi-GFP-Flag-OsMYB63L was transferred into Agrobacterium competent cells. The rice callus was infected by Agrobacterium-mediated transformation, and rice seedlings were obtained. After the sequencing was correct, the seedlings were cultured and propagated to obtain rice plants with increased panicle branch number and increased yield.

[0010] The beneficial effects of this invention are as follows: This invention cultivates plants overexpressing the rice gene OsMYB63L by infecting rice callus tissue with a recombinant plasmid containing the OsMYB63L gene via Agrobacterium-mediated infection. Phenotypic observation and trait statistics of wild-type and overexpressing plants revealed that, compared to wild-type plants, the overexpressing plants showed improvements in core yield traits such as primary and secondary branch number, thousand-grain weight, grains per panicle, and grain weight per plant. This verifies that the rice gene OsMYB63L has the function of regulating panicle branch number and yield in rice. This discovery provides a new direction for the development of OsMYB63L gene function and offers new gene resources and technical pathways for molecular breeding of high-yield rice. Attached Figure Description

[0011] Figure 1 Phylogenetic analysis of the OsMYB63L gene; Figure 2 Map of the overexpression vector; Figure 3 Plant height phenotypes of wild-type NPB and OsMYB63L overexpressing rice; Figure 4 The results show the gene expression levels of wild-type NPB and rice overexpressing the OsMYB63L gene. Figure 5 Phenotypic and yield statistics for wild-type NPB and OsMYB63L overexpressing rice (A: primary and secondary branch phenotypes of rice; B: rice yield); Figure 6 The phenotypic statistics for wild-type NPB and OsMYB63L-overexpressing rice are as follows: (A: Rice plant height; B: Number of primary branches; C: Number of secondary branches; D: 1000-grain weight; E: Number of grains per panicle; F: Grain weight per plant). Figure 7The cellulose content of the branch cell wall and the expression level of the OsCesA5 gene were determined in wild-type NPB and OsMYB63L overexpressing rice (A: cellulose content of rice branch cell wall; B: expression level of cellulose synthesis-related gene OsCesA5 in rice). Detailed Implementation

[0012] The present application will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0013] 1. Experimental materials Unless otherwise specified, all methods used in this invention are conventional methods known to those skilled in the art, and all reagents and materials used are commercially available products.

[0014] 2. Experimental Methods 2.1 Phylogenetic analysis of the OsMYB63L gene To clarify the evolutionary position of OsMYB63L within the MYB transcription factor family and its phylogenetic relationships with homologous genes, we constructed a phylogenetic tree. Homologous protein sequences of the MYB family were selected from species such as rice and Arabidopsis thaliana. Multiple sequence alignment was performed using MEGA12 software. A phylogenetic tree was constructed using the neighbor-joining method, and a bootstrap test with 1000 replicates was conducted to assess node support.

[0015] The results are as follows Figure 1 As shown, OsMYB63L clusters with MYB transcription factors known to be involved in cell wall synthesis in rice (such as OsMYB58 / 63), indicating that it belongs to the MYB subfamily related to cell wall synthesis regulation in evolution.

[0016] 2.2. Obtaining the OsMYB63L overexpression line and identifying its expression level 2.2.1 Obtaining the OsMYB63L overexpression line The OsMYB63L gene (DNA sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2) was recombined into the overexpression vector pCAMBIA1300-Ubi-GFP-Flag to obtain the OsMYB63L overexpression vector. The vector map is shown below. Figure 2As shown. The constructed overexpression vector was transformed into Agrobacterium, and the target gene was transferred into mature callus tissue of NPB using Agrobacterium-mediated rice transgenic technology. After co-culture, screening, and differentiation and rooting processes, transgenic T0 generation rice seedlings were obtained. After positive identification by RT-qPCR, three overexpressing plants, OsMYB63L-OE-1, OsMYB63L-OE-2, and OsMYB63L-OE-3, were detected by sequencing analysis.

[0017] 2.2.2 Identification of OsMYB63L overexpression line expression level From wild-type NPB and 3 overexpressing plants (see...) Figure 3 Total RNA was extracted from rice (using the Novizan FastPure Universal Plant Total RNA Isolation Kit), and reverse transcribed into cDNA [using the Novizan HiScript IV RT SuperMix for qPCR (+gDNA wiper) kit]. Subsequently, RT-qPCR experiments were performed (according to the Novizan ChamQ SYBR qPCR Master Mix instructions). The primers used in the RT-qPCR experiment were: SEQ ID NO.3: 5'-GAGGAGGGAGCAGAGTGGTG-3'; SEQ ID NO.4: 5'-TGAGAGAACGGCGGGTGAT-3'; The results of the identification are as follows Figure 4 As shown, compared with wild-type NPB, the expression levels of the OsMYB63L gene in all three overexpression lines were significantly increased, indicating that rice lines overexpressing the OsMYB63L gene were obtained.

[0018] 2.3 Evaluation of the yield-increasing capacity of OsMYB63L overexpression lines Observe the ear structure of wild-type NPB and three overexpression lines ( Figure 5 A), plant height, number of primary branches, and number of secondary branches were counted for wild-type NPB and three overexpression lines. Figure 6 (AC) It was found that compared with wild-type NPB, the number of primary and secondary branches in the three overexpression lines was significantly increased. This indicates that overexpression of OsMYB63L can effectively promote the development of panicle branches in rice; The key yield traits of wild-type NPB and three overexpression lines were investigated and statistically analyzed, and the results are as follows: Figure 5 B and Figure 6 As shown in DF, compared with wild-type NPB, the three overexpression lines significantly improved yield traits such as thousand-grain weight, number of grains per ear, and grain weight per plant. The results above indicate that the increase in the number of panicle branches directly leads to an increase in the number of grains per panicle, which in turn significantly increases the yield per plant. This suggests that OsMYB63L overexpression increases yield by regulating panicle development.

[0019] 2.4 Determination of cell wall components and expression analysis of cellulose synthesis-related genes in overexpression lines 2.4.1 Determination of cell wall components The cellulose content in wild-type NPB and three overexpression lines was determined according to the cellulose determination method described in Part 5 of NB / T 34057 "Determination of Chemical Composition of Lignocellulosic Biomass Raw Materials".

[0020] The results are as follows Figure 7 As shown in Figure A, compared to wild-type NPB, the cellulose content in the branches and stalks of the three overexpression lines was significantly increased.

[0021] 2.4.2 Expression analysis of OsCesA5, a gene related to cellulose synthesis OsCesA5 (LOC_Os03g62090) is an important gene in cellulose synthase and is closely related to cellulose synthesis. OsCesA5 may be a downstream regulatory gene of OsMYB63L.

[0022] Total RNA was extracted from rice (using the Novizan FastPure Universal Plant Total RNA Isolation Kit), reverse transcribed into cDNA (using the Novizan HiScript IV RT SuperMix for qPCR (+gDNAwiper) kit), and then subjected to RT-qPCR experiments (according to the Novizan ChamQ SYBR qPCR Master Mix instructions). The primer sequences used for the OsCesA5-RT-qPCR experiment (primer design derived from Wu Leiming, 2019. Study on the mechanism of OsMYBs regulating secondary cell wall synthesis and efficient enzymatic hydrolysis of lignin fibers to produce sugar) are as follows: SEQ ID NO.5: 5'-CCTTGTTAATACCCCCTACC-3'; SEQ ID NO. 6: 5'-AATCCAAACCACACTCCTCC-3'.

[0023] Analysis results as follows Figure 7As shown in Figure B, the expression level of OsCesA5 was significantly higher than that of wild-type NPB in all three OsMYB63L overexpression lines. This result suggests that OsMYB63L overexpression may promote stem cellulose accumulation by upregulating the expression of cellulose synthesis-related genes (such as OsCesA5), thereby enhancing stem strength and providing structural support for carrying more ear branches and grains. This may be one of the potential mechanisms by which it achieves synergistic improvement in ear structure and increased yield.

[0024] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. Application of a rice gene OsMYB63L in regulating panicle branching and yield.

2. The application according to claim 1, characterized in that: The nucleotide sequence of the gene OsMYB63L is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that: Overexpression of the gene OsMYB63L promotes panicle branching development in rice, thereby increasing rice yield.

4. The application according to claim 1, characterized in that: By using transgenic technology to overexpress the rice gene OsMYB63L, rice plants with increased panicle branching and yield were obtained.

5. The application according to claim 4, characterized in that: The method for overexpressing the rice gene OsMYB63L to obtain rice plants with increased panicle branching and higher yield includes the following steps: (1) Construct a recombinant plasmid pCAMBIA1300-Ubi-GFP-Flag-OsMYB63L containing the rice OsMYB63L gene; (2) The recombinant plasmid pCAMBIA1300-Ubi-GFP-Flag-OsMYB63L was transferred into Agrobacterium competent cells. The rice callus was infected by Agrobacterium-mediated transformation, and rice seedlings were obtained. After the sequencing was correct, the seedlings were cultured and propagated to obtain rice plants with increased panicle branch number and increased yield.

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