CN121160791A - Application of OsvWA36 gene in regulating rice grain shape and yield - Google Patents

Abstract

The invention discloses an application of OsvWA gene in regulating rice grain type and yield, belonging to the technical field of agricultural biological genetic engineering, wherein the application of OsvWA gene in regulating rice grain type and yield is disclosed, and the nucleotide sequence of OsvWA gene is shown as SEQ ID NO. 1. The invention provides a new choice for increasing the grain length and the yield of rice, and the application is to integrate OsvWA gene into the rice through an over-expression vector to increase the grain length and the yield of the rice, which has great application value for rice breeding.

Description

Application of OsvWA gene in regulation of rice grain type and yield

Technical Field

The invention belongs to the technical field of agricultural biological genetic engineering, and particularly relates to application of OsvWA gene in regulation of rice grain type and yield.

Background

Rice is one of the most important food crops worldwide, and its yield is directly related to food safety. Grain size and morphology (grain type) are core factors determining rice yield, and also affect the appearance quality and market value of rice. Therefore, the method for excavating the key genes for regulating and controlling the rice grain types and analyzing the action mechanism thereof is an important foundation for realizing high-yield and high-quality breeding of rice. At present, research and breeding application of rice grain type regulatory genes mainly have the following limitations:

first, the regulatory mechanisms are limited to traditional pathways. Most of the currently cloned granulocytes, such as GS3, GW5, etc., have a function focused on the traditional hormone signaling or cell cycle regulatory pathways. These pathways are of natural importance, but the understanding of the granulocyte-regulated network is still incomplete. In recent years, liquid-liquid phase separation is taken as a brand new mechanism for forming membraneless organelles through biological macromolecule condensation so as to efficiently regulate key physiological processes, and plays an important role in animal and plant development, however, the role of the mechanism in rice grain development and grain regulation has not been reported yet, so that the prior art is difficult to break through the regulation ceiling of the traditional path.

Second, the coordinated regulation of multiple physiological processes is inadequate. The development of ideal grain is the result of the precise synergy of multiple physiological processes such as cell division, cell expansion, cell wall construction, nutrient transport and deposition. It is known that how many genes singly regulate a specific process, such as promoting cell division or affecting hormone levels, is difficult to achieve synergistic optimization of the above-described multiple processes. Such single regulation is liable to cause the paradox of grain increase but quality reduction, such as occurrence of problems of empty grain, increased chalkiness or insufficient hardness, and the like, and cannot realize the synergistic improvement of high yield and quality.

Thirdly, molecular breeding lacks efficient and accurate markers. Although molecular marker assisted selection has been widely applied to rice breeding, the existing research of association between natural variation and traits of many grain genes is not deep enough, and there is a lack of molecular markers which are universally effective in breeding populations and closely linked to excellent grain types. The breeder still highly depends on phenotype selection in actual work, has long period and low efficiency, is difficult to accurately polymerize a plurality of micro-effective grain genes, and restricts the breeding efficiency of a new high-yield high-quality rice variety.

Therefore, the novel rice grain type/yield key genes which have a brand new regulation mechanism, can cooperate with multiple physiological paths and have definite natural variation are mined, so that not only can the rice grain type genetic regulation theory be perfected, but also key technical support can be provided for breaking through the bottleneck of the existing breeding technology and cultivating novel rice varieties with high yield and high quality, and the novel rice grain type/yield key genes have great theoretical research value and agricultural practice significance.

Disclosure of Invention

The invention aims to provide an application of OsvWA gene in regulating and controlling rice grain type and yield, and provides a new choice for increasing rice grain length and yield, wherein the application is to integrate OsvWA gene into rice through an over-expression vector to increase rice grain length and yield, which has great application value for rice breeding.

In order to solve the technical problems, the invention adopts the following technical scheme:

The OsvWA gene is applied to regulation of rice grain type and yield, and the nucleotide sequence of the OsvWA gene is shown as SEQ ID NO. 1.

Preferably, the amino acid sequence of the protein encoded by the OsvWA gene is shown as SEQ ID NO. 2.

Preferably, the grain length and yield of rice are increased by up-regulating the expression level of OsvWA gene or the activity or content of OsvWA gene-encoded protein.

The invention also provides an over-expression vector for increasing the grain length and yield of rice, which comprises the OsvWA gene.

The invention also provides a strain for increasing the grain length and yield of rice, which comprises the over-expression vector.

The present invention also provides a method for growing rice plants with increased grain length and increased yield, comprising the steps of:

Introducing and expressing the OsvWA gene into a rice receptor to obtain a transgenic rice plant with grain length and yield higher than those of the rice receptor.

Preferably, the introducing is performed by connecting the OsvWA gene to the downstream of the rice promoter, constructing an over-expression vector, and transforming the rice by an agrobacterium-mediated method.

The invention also provides a method for cultivating rice plants with increased grain length, which is to modify OsvWA genes as described in the OsvWA genes by a gene editing technology and enhance the liquid-liquid phase separation capability of OsvWA gene coding proteins.

The invention also provides a DNA molecular marker for detecting rice grain length characters, which is designed based on single nucleotide polymorphism sites related to grain length characters in the OsvWA gene coding region.

The invention also provides a method for breeding rice varieties with target grain length, which comprises the following steps of detecting OsvWA gene haplotypes of rice to be detected by using the molecular marker, and selecting rice with haplotypes related to the target grain length for breeding.

Compared with the prior art, the invention has the following advantages and technical effects:

1. The invention discloses an application of OsvWA gene in regulating rice grain type and yield, which is verified by constructing osvwa mutant, anaplerotic strain and phenotype verification of over-expression strain, wherein OsvWA is a positive regulating key gene of rice grain length, the grain length of osvwa mutant is obviously reduced compared with wild type, the grain length of anaplerotic strain can be restored to wild type level, the grain length of over-expression strain is further increased compared with wild type, the regulating effect is directly related with thousand grain weight (the grain length is a core influencing factor of thousand grain weight), a clear target point is provided for rice yield improvement, compared with the traditional grain type improvement relying on polygenic polymerization, the regulating target is more accurate, and the yield gain predictability is stronger.

2. The cytological regulation and control basis is revealed, the precise direction is provided for granule improvement, and scanning electron microscope experiments prove that the extension of the glume epidermal cells of the osvwa mutant is blocked, and the effect of OsvWA on granule length by regulating and controlling the extension process of the glume epidermal cells is clear.

3. The tissue specificity is high, the negative effect of non-target tissues is avoided, and the qRT-PCR and Western-Blot experiments prove that OsvWA is high in rice spike (key tissue for grain development), and the expression level in root, stem, leaf and other nutritional organs is low.

4. The novel regulation and control mechanism of liquid-liquid phase separation (LLPS) is found, the limitation of the traditional path is broken through, in-vitro protein aggregate observation, in-vivo fluorescence fusion experiments and FRAP experiments prove that OsvWA forms a dynamic and recoverable membraneless aggregate through the IDR (intrinsic disorder region) -mediated LLPS, the novel regulation and control mechanism provides a novel path for rice grain type regulation and control, the traditional framework which depends on hormone signals (auxin and cytokinin) or cell cycle genes in the prior art is broken through, the dynamic property of the LLPS can realize 'flexible regulation and control' on the grain development process, abnormal grain development caused by rigid regulation and control is avoided, and the adaptability and stability of regulation and control are improved.

5. The natural mutation sites are excavated, high-efficiency molecular breeding is enabled, haplotype analysis of 3K rice genome groups shows that 11 natural haplotypes are related to grain length in OsvWA coding regions, the grain length of a rice sample carrying Hap6 is obviously shorter than that of haplotypes such as Hap1, hap4 and the like, the detection can be directly converted into molecular markers, and the molecular markers are used for rapid screening of rice germplasm resources and directional polymerization of excellent grain shape characters, and compared with a traditional breeding mode, the grain shape improvement period can be shortened, and the breeding efficiency is greatly improved.

In summary, the invention not only defines the core role of OsvWA in rice grain type and yield regulation, but also reveals a brand-new LLPS regulation mechanism and multi-channel cooperative mode, and the tissue specificity, dynamic regulation and natural variation usability of the LLPS regulation mechanism become ideal targets for high-yield and high-quality rice breeding, and have important practical significance for solving the problems of low precision, quality defect, long breeding period and the like in the existing grain type improvement, and providing key technical support for guaranteeing grain safety.

The technical scheme of the invention is further described in detail through the drawings and the embodiments.

Drawings

FIG. 1 is a diagram showing an expression pattern of OsvWA, wherein A in FIG. 1 is a qRT-PCR analysis diagram of transcripts of each tissue OsvWA in a seedling stage and a reproductive growth stage of Syringa oblata (DXB), and B in FIG. 1 is a diagram showing the result of Western blot analysis of protein abundance of different tissues OsvWA36 in the development process of DXB;

FIG. 2 is a map of the subcellular localization of OsvWA, wherein A in FIG. 2 is a confocal microscopy image showing OsvWA-GFP distribution on the nucleus and B in FIG. 2 is a confocal microscopy image showing the differential localization of OsvWA-GFP to FM4-64 stained membrane systems;

FIG. 3 shows that OsvWA can undergo liquid-liquid phase separation (LLPS) in vitro and in vivo, wherein A in FIG. 3 is the result of an in vitro LLPS assay for purifying recombinant His-OsvWA36 protein, B in FIG. 3 is the dynamic formation process of OsvWA-GFP in vivo, i.e., aggregates in tobacco leaf epidermal cells transiently expressing 35S:: osvWA-GFP, C in FIG. 3 is the result of analysis of Fluorescence Recovery (FRAP) after photobleaching of the aggregates of OsvWA-GFP, and D in FIG. 3 is the quantitative result of FRAP recovery kinetics;

FIG. 4 is a graph of a grain size analysis of osvwa36 mutant versus control plant, wherein A in FIG. 4 is a representative image of the grain size of Wild Type (WT) and osvwa mutant grains, B in FIG. 4 is a representative image of the grain size of Wild Type (WT) and osvwa mutant grains, C in FIG. 4 is a statistical analysis of the grain size of Wild Type (WT) and osvwa36 mutant grains, D in FIG. 4 is a statistical analysis of the grain size of Wild Type (WT) and osvwa36 mutant grains, E in FIG. 4 is a Thousand Grain Weight (TGW) of WT and osvwa mutants;

FIG. 5 is a graph showing analysis of the length and lignin deposition of the osvwa36 mutant glume cells, wherein A in FIG. 5 is a graph of the small ear of the WT and osvwa mutant before flowering, B in FIG. 5 is a Scanning Electron Microscope (SEM) image of the WT and osvwa36 mutant glume outer and inner epidermal cells, C in FIG. 5 is a statistical analysis of the length of the WT and osvwa mutant glume outer epidermal cells, D in FIG. 5 is a statistical analysis of the width of the WT and osvwa mutant glume outer epidermal cells, E in FIG. 5 is a statistical analysis of the length of the WT and osvwa mutant glume inner epidermal cells, F in FIG. 5 is a statistical analysis of the width of the WT and osvwa mutant glume inner epidermal cells, G in FIG. 5 is a phloroglucinol-hydrochloric acid staining to detect lignin deposition of the small ear of the WT and osvwa mutant before and 1 day after flowering (magenta);

FIG. 6 is a graph showing a grain size analysis of OsvWA gene complementation lines, wherein A in FIG. 6 is representative of the grain sizes of Wild Type (WT), osvwa mutant and seed of the complementation line, B in FIG. 6 is representative of the grain widths of Wild Type (WT), osvwa mutant and seed of the complementation line, C in FIG. 6 is a statistical grain size analysis of Wild Type (WT), osvwa mutant and seed of the complementation line, and D in FIG. 6 is a statistical grain width analysis of Wild Type (WT), osvwa mutant and seed of the complementation line;

FIG. 7 is a graph of genetic verification of grain length function of OsvWA gene regulatory in the context of ZH11, A in FIG. 7 is a representative image of grain length of ZH11, osvwa ΔIDR mutants (CR-1, CR-2) and OsvWA over-expressed (OE) lines, B in FIG. 7 is ZH11, osvwa36 Representative grain width images of IDR mutants (CR-1, CR-2) and OsvWA over-expression (OE) lines, C in FIG. 7 being a graph of grain length statistical analysis results, and D in FIG. 7 being a graph of grain width statistical analysis results;

FIG. 8 is a diagram showing the natural mutation analysis result of OsvWA genes, wherein A in FIG. 8 is a diagram showing the comparison analysis of Hap1 and Hap6, and B in FIG. 8 is a diagram showing the comparison analysis of Hap4 and Hap 6.

Detailed Description

The technical scheme of the invention is further described below through the drawings and the embodiments.

Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs.

Test material sources:

wild Type (WT) the Guangxi high quality maintainer line indica rice variety "Syringb" was selected, and further functional verification was performed using genetic material of the "Zhonghua 11 (ZH 11)" background.

3K rice genome population material data were derived from public database (https:// v1. Rmbreding. Cn/Genotype/haplotype), whole set 3K-RG germplasm containing OsvWA coding region sequence was selected and GL in RFGB phenotype was selected for analysis.

Molecular cloning reagents Trizol reagent (Invitrogen), reverse transcription kit (Invitrogen), high fidelity DNA polymerase (vazyme), restriction enzymes (EcoRI, bamHI, NEB), homologous recombinase (vazyme).

The vectors include CRISPR/Cas9 vector, over-expression vector (pCAMBIA 1300-35S-GFP) and prokaryotic expression vector (pET-28 a).

Antibodies OsvWA polyclonal antibody (general biosome (Wuhan) technology Co., immune Rabbit source), actin antibody (Proteintech), his antibody (Proteintech)

In the present invention, other test materials and instruments are conventional in the art and are commercially available unless otherwise specified.

Example 1

Sterilizing the seeds with 75% ethanol for 30s, sterilizing with 2.5% sodium hypochlorite for 20min, washing with sterile water for 5 times, and concentrating to obtain a solution of 30Dark sprouting for 2 days, transferring into wood village B nutrient solution (pH 5.5), and placing into incubator (28)Photoperiod ofDark, light intensity 300molm^-2 S ^-1, culturing with the humidity of 70 percent, and transplanting to the field (conventional water and fertilizer management) after the seedling stage.

Microorganism culture E.coli (DH 5)BL 21) in LB medium 37Shaking culture (200 rpm), agrobacterium (GV 3101) in LB medium 28Shaking culture (180 rpm).

Creation of osvwa36 mutants and transgenic lines:

CRISPR/Cas9 mutant (osvwa) construction:

The sgRNA was designed based on the OsvWA coding region sequence (MSU_Locus: LOC_Os11g 45990), using CRISPR-P2.0 tool to design the sgRNA1 and the sgRNA2, the sgRNA1 sequence is shown as SEQ ID NO:3, and the sgRNA2 sequence is shown as SEQ ID NO: 4.

SEQ ID NO:3:5'-GATTGTGGGGAAGATGGGTGTGG-3'。

SEQ ID NO:4:5'-GCATAAGCGCAGTGCCACCACGG-3'。

Vector construction cloning of the sgRNA1 and sgRNA2 sequences intoBsaI cleavage site of vector, transformation DH5Competent cells were picked and positive clones were sequenced for validation.

Agrobacterium transformation the correct recombinant vector was transformed into Agrobacterium GV3101 and transformed into Syringa oblonga B by the hundred-Grating Gene technology (Jiangsu Co., ltd.).

Mutant identification, namely extracting regenerated plant leaf DNA, and using identification primers F and R, wherein the F sequence is shown as SEQ ID NO. 5, and the R sequence is shown as SEQ ID NO. 6.

SEQ ID NO:5:ACTGCTCCAGTTTTCCTTTGAA。

SEQ ID NO:6:TGGTATTCTAGCACGGAGGAGT。

PCR amplification is carried out, and amplified products are sequenced, and homozygous mutants (such as vw-1 deleted by 1bp and vw-2 deleted by 2 bp) are screened out.

(5) Osvwa36 mutant of flower 11 background in the hundred gene technology (Jiangsu) limited subscription and identification.

Construction of OsvWA36 anaplerotic and overexpressing lines

(1) Vector construction the OsvWA coding region sequence was cloned into pRHVcGFP vector (driven by the ubiquitin promoter), fused with GFP tag and sequenced.

(2) The transgenic line is obtained by referring to the agrobacterium-mediated rice transformation method, a reverse complement vector is transferred into osvwa mutant, an over-expression vector is transferred into Zhonghua 11, a positive line is obtained by hygromycin screening, qRT-PCR verifies OsvWA expression quantity, and primers comprise qRT-PCR-F and qRT-PCR-R, wherein the qRT-PCR-F sequence is shown as SEQ ID NO. 7, and the qRT-PCR-R sequence is shown as SEQ ID NO. 8.

SEQ ID NO:7:TATAGCTCCGGCTTGTTGGA。

SEQ ID NO:8:AAGGTGTGGACGGGGTACTT。

OsvWA36 gene is the nucleotide sequence shown in SEQ ID NO. 1.

SEQ ID NO:1:

ATGGGGCAACTCCATAATTTGATGCTTCTGCTGCCGTGCCTGATCTTCTCTACGCTACTGCGCACAGAGGCGATGAGTGTAGCTCCAGTGAAAGTGAGCACCACACCCATCTTCCCCACAATCCCAAGAGGTCAGACGAACAAGGACTTCCAGGTGCTACTGCGCGTCGAGGCGCCGCCGGCGGCCGATCTCAACAGCCATGTCCCCTTAGACGTAGTCGCGGTGCTTGATGTCAGCGGCAGCATGAATGATCCGGTGGCGGCGGCGTCGCCGAAGAGCAATCTGCAGGGGTCGAGGTTGGATTTGCTCAAGGCGTCCATGAAGTTCGTCATCAGGAAGCTTGATGATGGTGATCGCCTCTCCATCGTGGCGTTTAACGATGGACCCGTCAAGGAATATAGCTCCGGCTTGTTGGATGTTTCCGGCGATGGCCGGAGCATCGCCGGAAAAAAGATTGACCGGCTTCAGGCCCGTGGTGGCACTGCGCTTATGCCAGCCCTGGAGGAGGCCGTCAAGATCCTTGATGAGCGGCAAGGCAGCAGCCGGAACCACGTAGGGTTCATCCTCCTCCTCACCGACGGCGACGACACGACCGGATTCCGGTGGACCCGCGACGCCATCCATGGCGCCGTCGCCAAGTACCCCGTCCACACCTTCGGCCTGGGCGCGTCCCACGACCCGGAGGCGCTGCTCCACATCGCGCAAGGATCGCGCGGCACCTACTCCTTCGTCGACGACGACAACCTCGCCAACATCGCCGGCGCCCTCGCCGTGTGCCTTGGCGGGCTCAAGACCGTCGCCGCCGTCGACACGCGCGTCAGCCTCAAGGCCGCCGAGCTAAGCGGCGGCGGCGCGCGGATAGTGCGCGTAGACTCCGGCGGCTACGAGAGCAGCGTTGCTTGCGGCGGGGCCTCCGGTGAGGTCGTCGTCGGCGTGCTCTACGCCGGCGAGGTGAAGAACTTCGTCGTCCACCTCCACGTGCCGGCTGCTTCGTCAACGACCTTGACCTTCTCGTCGGTGGAGTGCGGCGGCTACTACGACGCCGCCACGGTCTGCGACCACTGCCATCACCGTCACCAGCAGCAGCTGCTCGCCGTCGGCTACTCGTACAGCCACGCTCCGGGCGCCGCCGCTGCAGCGGTGTCCGTCGAAGGGCACGGCGTGTTCGTCGAGAGGCCCGAGGTGGCGGCCGTCTTCGTCTCCGTCGACGGCGTCGGCGTCGGCGGCGGCCGACAGCGACAAATCCCCCTCCCCTCCCCCGTCGTGATGCAGCACATGGTCCGGTTCGAGCTGCTGGAACTCGTCGCCGGCTTCGCGGAAGCCGAGATGGCGTCGAAGCCGGCGGCGACGACGACGCAGCCGCGCGCCGCCGACGTGCTGCAGGGCAAGTGGGAGGAGTTCCGGCGATCCCGGCAGTTCTGGGGCGGCGTCGAGCTGGACGGCGTGGAGAAGGAGGTGGACGCCATGGTGGCCAGCCTCAGGAGCGGGCTAGCCTACGTCATCTCGTGGGTGTCGAGCCACCAGATGCAGCGCGCCACCGCCATGGGCTCGCCGGAGAAGGTGGTGGCCGAGTTCATGACTCCGGCGATGGTGATCATGGTGGAGGAGGCGCGGAAGCTACCGCCACCACCGCCGCCGCCGCCGGCAGCTGCTGAGGCGGCGAGAGAGAGGCCCGGCGGCTGCGATGGCGGCGACGATATCCATCACGTGATCCGGCAGCGGCTTGAGCTGTGGTCGAAGGTGAGACGCGAGGTGCCGCTCATGTACCAGCCGTCGCCGGAGCAGGAAGACGTCCAGCTGACCGCCGTGTTCCGGGAGGCGTCGCTGGAGGCCATCGACCGAGCAATGCACCACGACATCTACCTGGCCGTTGTGCACGTGAGCAACCAGAGGCGATGCTGA.

The amino acid sequence of the protein coded by OsvWA gene is shown as SEQ ID NO. 2.

SEQ ID NO:2:

MGQLHNLMLLLPCLIFSTLLRTEAMSVAPVKVSTTPIFPTIPRGQTNKDFQVLLRVEAPPAADLNSHVPLDVVAVLDVSGSMNDPVAAASPKSNLQGSRLDLLKASMKFVIRKLDDGDRLSIVAFNDGPVKEYSSGLLDVSGDGRSIAGKKIDRLQARGGTALMPALEEAVKILDERQGSSRNHVGFILLLTDGDDTTGFRWTRDAIHGAVAKYPVHTFGLGASHDPEALLHIAQGSRGTYSFVDDDNLANIAGALAVCLGGLKTVAAVDTRVSLKAAELSGGGARIVRVDSGGYESSVACGGASGEVVVGVLYAGEVKNFVVHLHVPAASSTTLTFSSVECGGYYDAATVCDHCHHRHQQQLLAVGYSYSHAPGAAAAAVSVEGHGVFVERPEVAAVFVSVDGVGVGGGRQRQIPLPSPVVMQHMVRFELLELVAGFAEAEMASKPAATTTQPRAADVLQGKWEEFRRSRQFWGGVELDGVEKEVDAMVASLRSGLAYVISWVSSHQMQRATAMGSPEKVVAEFMTPAMVIMVEEARKLPPPPPPPPAAAEAARERPGGCDGGDDIHHVIRQRLELWSKVRREVPLMYQPSPEQEDVQLTAVFREASLEAIDRAMHHDIYLAVVHVSNQRRC.

Example 2

OsvWA36 expression pattern analysis:

Detection of tissue expression specificity by qRT-PCR

(1) Sample collection, namely selecting root (3-leaf stage), leaf (3-leaf stage) and spike (young spike stage and flowering stage) of Syzygium aromaticum B rice (Guangxi high-quality maintainer line), performing biological repetition on each tissue for 3 times, and performing liquid nitrogen quick freezing to obtain-80And (5) preserving.

(2) RNA extraction and reverse transcription, namely extracting total RNA by using Trizol reagent, and detecting RNA purity by using Nanodrop 2000=1.8-2.0), 1% Agarose gel to verify RNA integrity, yfxScript CDNA SYNTHESIS KIT (YIFEIXUE, china) was used to verify RNA integrity 1G RNA is reverse transcribed into cDNA.

(3) QRT-PCR reaction, wherein OsActin is used as an internal reference, and the primer comprises OsActin-F and OsActin-R, wherein the sequence of OsActin-F is shown as SEQ ID NO. 9, and the sequence of OsActin-R is shown as SEQ ID NO. 10.

SEQ ID NO:9:GAGTATGATGAGTCGGGTCCAG。

SEQ ID NO:10:ACACCAACAATCCCAAACAGAG。

OsvWA36 specific primers including OsvWA-36-F and OsvWA-R were used for real-time fluorescent quantitative PCR (instrument: CFX96, bio-Rad), reaction system (20L):SYBR Premix Ex Taq II 10L, upstream and downstream primers each 0.4L、cDNA 2L、ddH₂O 7.2L reaction procedure 95Pre-denaturation 30s,95Denaturation 5s,60Annealing for 30s,40 cycles, and dissolution profile analysis confirmed the specificity. Wherein the OsActin-F sequence is shown in SEQ ID NO. 11, and the OsActin-R sequence is shown in SEQ ID NO. 12.

SEQ ID NO:11:TATAGCTCCGGCTTGTTGGA。

SEQ ID NO:12:AAGGTGTGGACGGGGTACTT。

(4) Calculation of results byThe relative expression amount was calculated by the method, and the result is shown as A in FIG. 1.

As is clear from A in FIG. 1, osvWA has the highest expression level in the ear, especially the ear (1 DAF) of one day of flowering is 45 times of the expression level of the leaves of the booting stage, and the expression level in the root and the stem is low.

Western-Blot detection protein accumulation:

(1) Protein extraction by adding 1mL of RIPA protein extraction buffer (containing 1mM PMSF and protease inhibitor mixture) into 0.5 g of each tissue sample, ice-bath grinding, and centrifuging 12000 rpm to 15 min (4) ) Taking the supernatant.

(2) Protein quantification and electrophoresis BCA method for measuring protein concentration, 30 is takenG protein sample addition 5SDS loading buffer, 95Denaturation for 5 min, 10% SDS-PAGE gel electrophoresis (concentrate gel 80V, isolate gel 120V) until bromophenol blue reached the bottom of the gel.

(3) Transfer and immunodetection of gel protein transfer to PVDF membrane (200 mA,90 min), 5% skim milk blocking 1h (room temperature), addition of OsvWA36 polyclonal antibody (1:2000 dilution), 4Incubation overnight, TBST washing 3 times (10 min each), HRP-labeled goat anti-rabbit secondary antibody (1:5000 dilution) added, incubation at room temperature for 1h, and ECL chemiluminescent kit development (instrument: chemisoc XRS+, bio-Rad) after TBST washing 3 times, results are shown in FIG. 1, B.

As can be seen from FIG. 1B, osvWA protein was found to be substantially consistent with the mRNA expression pattern in ears with high accumulation, particularly in ears with one day of flowering (1 DAF).

Example 3

Subcellular localization:

(1) Sections were made using leaf sheaths of transgenic plants (OE-VW-2) overexpressing OsvWA-GFP fusion gene and stained with DAPI or FM 4-64.

(2) Fluorescence observation laser confocal microscope observation of GFP fluorescence signal, DAPI signal and RFP signal, the results are shown in FIG. 2.

The results showed that OsvWA-GFP formed punctate green fluorescent aggregates in the cytoplasm, with some punctate aggregates expressed on the nucleus (purple) (A in FIG. 2), which did not overlap with the red fluorescence of the FM4-64 membrane stain (B in FIG. 2).

Example 4

Liquid-liquid phase separation (LLPS) characterization of OsvWA 36:

1. In vitro LLPS validation (recombinant protein aggregate observations)

(1) Recombinant protein expression and purification, cloning OsvWA coding region into pET-28a vector (His tag), converting colibacillus BL21 (DE 3), picking up monoclonal and inoculating it in LB culture medium (containing 50Kanamycin), 37Culturing to OD 600=0.6, adding 0.5mM IPTG,16Inducing 16 h, collecting thallus, ultrasonic crushing (power 300W, work 3s stop 5s, total 30 min), purifying recombinant protein by Ni-NTA affinity chromatography column (GE HEALTHCARE), and checking purity (purity > 90%) by SDS-PAGE.

(2) LLPS condition optimization purified OsvWA-His protein (final concentration 2M,4M and 8M) was dissolved in Tris-HCl buffer (20 mM, pH 7.0) containing 0.2M NaCl, incubated at room temperature for 30min, and aggregate formation was observed using a laser confocal microscope (LSM 980, zeiss) (excitation wavelength 488nm, emission wavelength 520 nm), image J counted for aggregate number, and the results are shown as A in FIG. 3.

As can be seen from FIG. 3A, 8 under the conditions of 0.2M NaCl and pH 7.0M OsvWA-His protein aggregates were most efficient (30-40 per field of view).

2. In vivo LLPS validation (fluorescent fusion protein observations)

(1) Fusion vector transformation OsvWA-GFP fusion gene (vector pRHVcGFP-OsvWA-GFP) was transformed into Nicotiana benthamiana by the Agrobacterium-mediated method described above, expressed 48 h and observed.

(2) Fluorescence observation laser confocal microscopy observed GFP fluorescence signal, indicating that punctate aggregates were membraneless and that movement, fusion and separation occurred (B in FIG. 3), confirming LLPS properties in vivo.

3. Fluorescence bleaching recovery assay (FRAP):

(1) FRAP procedure the OsvWA-GFP aggregates described above were selected, the aggregate areas were bleached using the bleaching mode of a laser confocal microscope (488 nm laser, 50% power), and then fluorescence recovery images were taken every 10s for 60s.

(2) Recovery rate calculation Image J software analyzed the fluorescence intensity of the bleached area to 100% of the pre-bleaching fluorescence intensity and calculated the relative fluorescence intensity at different time points, as shown by C in fig. 3 and D in fig. 3.

As can be seen from C in FIG. 3 and D in FIG. 3, the fluorescence recovery rate reaches 50% -60% after bleaching for 60 seconds, and the OsvWA aggregate is proved to have dynamic recovery capability and be a membraneless dynamic aggregate.

Example 5

OsvWA36 functional verification of regulatory grain type:

particle and yield related index determination

(1) And (3) material planting, namely planting 30 strains of Wild Type (WT) and osvwa mutant (vw-1 and vw-2) in a field according to a random block design, and performing conventional water and fertilizer management.

(2) Index measurement, namely harvesting main ears in the mature period, selecting 30 full seeds for each strain, measuring the grain length and the grain width by using a WANSHEN SC-S system, selecting 1000 full seeds, and measuring thousand seed weight by using an electronic balance (precision is 0.001 g).

(3) Results analysis statistical analysis was performed using GRAPHPAD PRISM software and t-test was performed to compare the differences between the strains, and the results are shown in figure 4.

As can be seen from FIG. 4, osvwa mutant had a significantly shorter grain length than WT (A in FIG. 4 and C in FIG. 4) and a significantly lower thousand grain weight (E in FIG. 4).

Example 6

Glume epidermal cell morphology observation and glume lignin deposition analysis:

1. Glume epidermis cell morphology observation (scanning electron microscope)

(1) Sample preparation selecting WT (Syringa oblonga B) and osvwa mutant glume before flowering, cutting 1mm in the middle2Mm fragments, immediately put into 2.5% glutaraldehyde fixative (pH 7.2,0.1M phosphate buffer, preparation), 4Fixed for 12 hours, and sent to the Wuhan Seville company for scanning electron microscope scanning and analysis.

(2) Statistics 5 fields were selected for each sample, and Image J software counted the length, width and number of cells per field of epidermal cells, the results are shown in FIG. 5.

As can be seen from B in FIG. 5 and F in FIG. 5, osvwa mutant glume epidermal cell length was significantly shortened compared to WT, confirming that cell elongation was hindered.

2. Glume lignin deposition analysis

(1) Sample preparation, namely selecting WT (clove B) and osvwa mutant spikelets before and one day of flowering to prepare freehand slices;

(2) Staining with phloroglucinol-hcl, immediately observation with a microscope and photographing, the results are shown as G in fig. 5.

As can be seen from G in FIG. 5, the osvwa mutant had more lignin deposition (magenta) on the spikelet before and 1 day after flowering than the wild type (FIG. 5G).

Example 7

OsvWA36 functional verification of regulatory grain type:

particle and yield related index determination

(1) And (3) material planting, namely planting 30 strains of clove B, osvwa mutant (vw-1) and anaplerotic strain (Comp) in a field according to a random block design, and performing conventional water and fertilizer management.

(2) Index measurement, namely harvesting main ears in the mature period, selecting 30 full grains from each plant line, and measuring grain length and grain width by using a WANSHEN SC-S system.

(3) Results analysis statistical analysis was performed using GRAPHPAD PRISM software and t-test was performed to compare the differences between the strains, and the results are shown in figure 6.

As can be seen from FIG. 6, osvwa36 mutant grain length was significantly shortened compared to Syringa B, and the anaplerotic line grain length was restored to WT (Syringa B) level.

Example 8

OsvWA36 functional verification of regulatory grain type:

particle and yield related index determination

(1) And (3) material planting, namely planting ZH11, osvwa mutant (CR-1, CR-2) and over-expression strain (OE-VW-2, OE-VW-3) 30 strains in a field according to a random granule design, and performing conventional water and fertilizer management.

(2) Index measurement, namely harvesting main ears in the mature period, selecting 30 full grains from each plant line, and measuring grain length and grain width by using a WANSHEN SC-S system.

(3) Results analysis statistical analysis was performed using GRAPHPAD PRISM software and t-test was performed to compare the differences between the strains, and the results are shown in figure 7.

As can be seen from FIG. 7, osvwa mutant had a significantly shorter grain length than WT and the overexpressing strain had an increased grain length than WT.

Example 9

OsvWA36 natural haplotype analysis and molecular marker design:

1. Haplotype typing data were derived from public database (https:// v1. Rmbreding. Cn/Genotype/haplotype), whole set 3K-RG germplasm containing OsvWA coding region sequence was selected and GL in RFGB phenotype was selected for analysis, and the results are shown in FIG. 8.

As can be seen from FIG. 8, osvWA.sup.36 rice samples carrying Hap6 have a significantly shorter grain length than haplotypes such as Hap1, hap4, etc.

2. The molecular marker design is to design CAPS markers aiming at SNP loci different from Hap6 and Hap1, and can be used for rapidly screening excellent granular haplotype materials.

The embodiment systematically verifies the grain length positive regulation function and LLPS regulation mechanism of OsvWA through constructing mutants and transgenic lines and combining molecular biology, cytology and genetics experiments, and defines the application value of the mutant and transgenic lines in rice grain type/yield improvement, and simultaneously excavates natural haplotypes and designs molecular markers, thereby providing an operable technical means for efficient molecular breeding.

It should be noted that the above-mentioned embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solution of the present invention may be modified or substituted by the same, and the modified or substituted technical solution may not deviate from the spirit and scope of the technical solution of the present invention.

Claims (10)

1. The application of the OsvWA36 gene in regulating rice grain shape and yield, characterized in that the nucleotide sequence of the OsvWA36 gene is shown in SEQ ID NO:1. 2. The application according to claim 1, wherein the amino acid sequence of the protein encoded by the OsvWA36 gene is as shown in SEQ ID NO:2. 3. The application according to claim 1, characterized in that rice grain length and yield are increased by upregulating the expression level of the OsvWA36 gene or the activity or content of the protein encoded by the OsvWA36 gene. 4. An overexpression vector for increasing rice grain length and yield, characterized in that the overexpression vector comprises the OsvWA36 gene as described in claim 1. 5. A strain for increasing rice grain length and yield, characterized in that the strain comprises the overexpression vector of claim 4. 6. A method for cultivating rice plants with increased grain length and yield, characterized in that the method comprises the following steps: By introducing and expressing the OsvWA36 gene as described in claim 1 into a rice recipient, transgenic rice plants with grain length and yield higher than those of the rice recipient are obtained. 7. The method according to claim 6, wherein the introduction is carried out by connecting the OsvWA36 gene downstream of the rice promoter to construct an overexpression vector and transforming rice using Agrobacterium-mediated transformation. 8. A method for cultivating rice plants with increased grain length, characterized in that the method involves modifying the OsvWA36 gene as described in claim 1 using gene editing technology to enhance the liquid-liquid phase separation ability of the protein encoded by the OsvWA36 gene. 9. A DNA molecular marker for detecting grain length trait in rice, characterized in that the molecular marker is designed based on a single nucleotide polymorphism site in the coding region of the OsvWA36 gene as described in claim 2 that is associated with grain length trait. 10. A method for breeding rice varieties with a target grain length, characterized by comprising the following steps: using the molecular markers as described in claim 9 to detect the OsvWA36 gene haplotype of the rice to be tested, and selecting rice varieties with haplotypes associated with the target grain length for breeding.