Application of rice gene OsNAL12 in regulating grain shape and increasing thousand-grain weight of rice

Abstract

The application belongs to the field of plant genetic engineering, and particularly relates to application of a rice gene in regulating rice grain shape and increasing 1000-grain weight. A gene capable of increasing rice flag leaf width and promoting rice grain shape to become larger is obtained through whole genome association analysis and cloning screening. The leaf width trait candidate gene is subjected to CRISPR knockout identification, and a gene OsNAL12 capable of regulating rice leaf shape and grain shape is obtained. Phenotype identification on the mutant and overexpression material shows that deletion of OsNAL12 can lead to narrow flag leaf, small seed and reduced 1000-grain weight, and overexpression of OsNAL12 can lead to wide flag leaf, large seed and increased 1000-grain weight, proving the function and application approach of the gene in regulating rice grain shape and increasing 1000-grain weight. The nucleotide sequence of the gene is shown as SEQ ID NO:1, the protein encoded by the gene is shown as SEQ ID NO:2, and the promoter sequence is shown as SEQ ID NO:3.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant genetic engineering technology, and particularly relates to application of rice gene OsNAL12 in regulating rice leaf shape and improving thousand-grain weight. The present application identifies a gene OsNAL12 regulating leaf shape and grain shape by adopting whole-genome association analysis method on leaf width traits, and confirms the function of the gene in regulating rice leaf shape and grain shape and the application value of the gene in improving rice yield by constructing mutant and overexpression materials of the gene and analyzing leaf width and grain type phenotypes and thousand-grain weight of the materials. BACKGROUND

[0002] Photosynthesis is an important way for plants to obtain energy, and photosynthesis of rice cannot be separated from its leaves. Leaves are the "source" of rice yield and an important direction for tapping yield improvement potential. Studies have shown that most of the carbohydrates in rice grains come from photosynthesis of the upper three leaves (sword leaf, second leaf from the top, and third leaf from the top) (Li et al., Genetic dissection of the source-sink relationship affecting fecundity and yield in rice. MoL Breeding, 1998, 4:419-426), and there is a clear positive correlation between the morphology of the upper three leaves and the yield of rice, that is, there is a "source-sink" cooperation relationship (Zhang B. QTL analysis of morphological and yield-related characters of rice sword leaf under different conditions (in Chinese). Dissertation for Master's Degree. Beijing: Chinese Academy of Agricultural Sciences, 2014). Improving the leaf shape of rice with the aid of molecular markers is conducive to the improvement of rice yield.

[0003] Rice yield is a complex agronomic trait, which is determined by panicle number, grain number per panicle, seed setting rate and 1000-grain weight. Among them, the heredity of 1000-grain weight is the most stable, with a coefficient of variation of 40%-60%. The research of International Rice Research Institute has confirmed that increasing grain weight can increase rice yield by more than 30%, which is the most effective way to increase yield (Liu et al. Research progress on cloning and regulation mechanism of rice grain shape genes. Chinese Journal of Rice Science, 2018, 32(1): 1-11). The grain weight of rice is determined by grain shape (grain length, grain width, and grain thickness), which is a quantitative trait controlled by multiple genes. So far, more than 60 grain shape regulatory genes have been cloned (Liu et al. Research progress on cloning and regulation mechanism of rice grain shape genes. Chinese Journal of Rice Science, 2018, 32(1): 1-11), such as TGW6, GS3, GW2, GS5, qSW5 / GW5, GW8 / SPL16, GL3.1 / qGL3 / qGL3-1, GIF1, EP2 / DEP2 / SRS1, FLO(a) / FLO2, etc. These genes determine grain size by regulating cell proliferation and cell expansion, and their functions are involved in plant hormone, G protein signal, MAPK signal, epigenetic modification, protein degradation, and other signal transduction pathways (Zheng et al. Molecular functions of genes related to grain shape in rice. Breeding Science, 2015, 65: 120-126) (Li N, Li Y. Signaling pathways of seed size control in plants. Current Opinion in Plant Biology, 2016, 33: 23-320). The cloning of grain shape genes provides gene resources for rice molecular design breeding, and has important significance for guiding rice high-yield breeding (Huang H Y, Qian Q. Progress in genetic research of rice grain shape and breeding achievements of long-grain shape and good quality japonica rice. Chinese Journal of Rice Science, 2017, 31(6): 665-672).

[0004] In the ubiquitin degradation pathway, E3 ligases are key regulatory components because they mediate the specific binding of ubiquitin to substrates (Morrea Le F.E., WaLden H..types of ubiquitin Ligases, Cell, 2016, 165:248). GW2 encodes a RING class E3 ubiquitin ligase in rice, which negatively regulates seed size by ubiquitination EXPANSIN-LIKE1 (Choi et al., GW2 functions as an E3 ubiquitin ligase for rice expansin-like1. Int. J. MoL. Sci, 2018, 19:7). CLG1 also encodes a RING class E3 that can degrade GS3 to regulate rice grain size (Yang et al., The RING E3 Ligase CLG1 targets GS3 for degradation via the endosome pathway to determine grain size in rice. Molecular plant, 2021, 14(10): 1699-1713). LARGE2 encodes a HECT domain E3 ubiquitin ligase, which negatively regulates grain size and panicle size, and is important for increasing rice yield and high-yield breeding (Huang et al., The LARGE2-APO1 / APO2 regulatory module controls panicle size and grain number in rice. Plant Cell, 2021, 33(4): 1212-1228). These studies have demonstrated that E3 ligases, as important components of the ubiquitination pathway, play an important role in regulating rice grain size. Perhaps we can improve rice grain size by exploring potential natural variations of E3 ubiquitin ligases from populations to improve rice yield.

[0005] Rice is an important food crop and model organism. In today's extreme climate, it is important to increase rice production to increase rice reserves to cope with the reduction in production caused by adverse weather. The present invention relates to OsNAL12, which encodes a RING class E3 ubiquitin ligase. The function of this gene in rice has not been reported. Through population haplotype analysis, it was found that this gene has an important function in seed size and thousand-grain weight. Therefore, identifying the function of this gene in regulating rice seed size and increasing yield is of great significance for agricultural production and breeding of new varieties with increased yield. SUMMARY

[0006] The purpose of the application relates to the application of a RING family E3 ubiquitin ligase OsNAL12 in regulating rice grain shape and yield. Because the gene is located by leaf width GWAS, it is named as OsNAL12 gene according to the rice naming order. The application identifies and clones a fragment containing OsNAL12 gene, and the deletion of the fragment will lead to leaf narrowing and rice seed size reduction, thereby reducing the thousand grain weight. Overexpression of the fragment can lead to wide rice leaves, large seeds and increased thousand grain weight. The nucleotide sequence of the OsNAL12 gene is shown in SEQ ID NO: 1, and the sequence length is 1506 bp. The amino acid sequence of the gene is shown in SEQ ID NO: 2, which contains 501 AA.

[0007] The application includes the isolation and cloning of the target fragment, and the transformation of the rice genome by the agrobacterium infection method, and the determination of the positive plants of the overexpression plants and the deletion mutants, and the determination of the sword leaf width, seed size phenotype (such as grain length, grain width) and thousand grain weight, and the determination of the OsNAL12 which can regulate the thousand grain weight and yield by regulating the seed size.

[0008] The expression vector carrying the OsNAL12 gene of the application can be introduced into plant cells by using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation and other conventional biological technology methods (Weissbach, Method for Plant Molecular Biology VIII, Academy Press, New York, 1998, pp. 411-463; Geiserson and Corey, Plant Molecullar Biology (2nd Edition), 1998). Various plant hosts such as rice can be transformed by using the expression vector containing the OsNAL12 gene of the application to cultivate high-yield plant varieties.

[0009] The application will be further described below in combination with the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0010] Figure 1 : The results of using NAL12 gene to regulate rice leaf width. The figure mark explanation: Figure 1 The A figure in the figure is the editing condition of the osnal12 CRISPR mutant of rice. Figure 1 The B and C figures in the figure are the widest part of the sword leaf of the osnal12 mutant and WT (abbreviation of Wildtype, namely wild type, same below) are photographed. It is shown that the leaf of the mutant is obviously narrower than the wild type (n=20, Bar=1cm); Figure 1Figure D in the figure shows the changes in OsNAL12 expression in the overexpression lines detected by real-time quantitative PCR. The overexpression of OsNAL12 in the two lines was about 7-fold. The rice actin1 gene was used as the internal reference gene. The data are the average of three replicates plus the standard error. Figure 1 Figure E in the figure shows the content of OsNAL12 protein in overexpressing plants. Because overexpressing NAL12 incorporates the HA tag, the detection of the HA tag revealed that NAL12 protein is indeed highly expressed in rice. Rice ACTIN was used as an internal control protein. Figure 1 The F and G images in the figure are taken and statistically analyzed at the widest point of the sword blade of OE-NAL12 (OE-6, OE-15) and WT (Wild type). Figure 1 The G plot in the figure represents the statistical analysis of leaf width in NAL12 overexpressing plants (n=10, Bar=1cm); the data difference analysis was performed using one-way ANOVA and Tukey's t-test, with P<0.05.

[0011] Figure 2 Results of using the OsNAL12 gene to regulate rice grain shape. (Figure labels are explained.) Figure 2 Figures A and B in the figure show the observation and photographs of the granule shape of two families of the nal12 mutant. According to the statistics, the granule length and granule width of the nal12 mutant were significantly reduced (n=39, Bar=1cm). Figure 2 The D and G plots in the image show that the granule shape of the two families overexpressing OsNAL12 was observed and photographed. Statistical analysis showed that the granule length and width of the overexpressing OsNAL12 were significantly increased (n=20, Bar=1cm). One-way ANOVA and Tukey's t-test were used for data difference analysis, with P<0.05.

[0012] Figure 3 Effects of the OsNAL12 gene on the thousand-grain weight of rice. (Figure labels are explained.) Figure 3 Figure A in the figure shows the thousand-grain weight of the osnal12 CRISPR mutant; Figure 3 Figure B in the figure shows the thousand-grain weight of the OsNAL12 overexpression line.

[0013] Figure 4 Analysis of the expression pattern of the OsNAL12 gene. Figure label explanation: pOsNAL12:GUS expression site is mainly in the immature embryo (…). Figure 4 Figure A in the middle), young sheath ( Figure 4 (Figure B in the diagram), root () Figure 4 Figure B in the diagram), young spikelet branches ( Figure 4 Figures I, J, and M), stamens ( Figure 4 (K diagram in the image) and husk (Figure 4 M plot) in FIG. 6.

[0014] Figure 5 : OsNAL12 is a functional E3 ubiquitin ligase. FIG. legend: The ubiquitination band was activated by adding OsNAL12 protein in the presence of E1, E2 and ubiquitin. It is proved that NAL12 is a functional E3 ubiquitin ligase.

[0015] Figure 6 : CRISPR knockout NAL12 vector map (for example 1).

[0016] Figure 7 : NAL12 overexpression vector map (original vector) (for example 1).

[0017] Figure 8 : OsNAL12 expression pattern analysis vector (for example 4).

[0018] Figure 9 : OsNAL12 protein expression vector (for example 5). DETAILED DESCRIPTION

[0019] Explanation of the sequence listing:

[0020] The sequence listing SEQ ID NO: 1 is the nucleotide sequence of the cloned DNA fragment containing the coding region of OsNAL12 gene, and the sequence length is 1506 bp.

[0021] The sequence listing SEQ ID NO: 2 is the amino acid sequence encoded by OsNAL12 gene, and it encodes 501 amino acids.

[0022] The following examples define the present application and describe the methods of obtaining and identifying the osnal12 CRISPR mutant, cloning the DNA fragment containing the complete coding region of OsNAL12 gene, constructing OsNAL12 overexpression rice and verifying the function of OsNAL12 gene. Based on the following description and these examples, those skilled in the art can determine the essential characteristics of the present application, and can make various changes and modifications to the present application without departing from the spirit and scope of the present application, so as to make it suitable for different uses and conditions.

[0023] Example 1: Construction of OsNAL12 CRISPR mutant and overexpression material and identification of leaf shape phenotype

[0024] The gene sequence of OsNAL12 gene was obtained from the rice gene database Rice Data( http: / / www.ricedata.cn / gene / ) in the rice gene database Rice Data( http: / / crispr.hzau.edu.cn / CRISPR2) Pick one target site. The vector construction of CRISPR mutant strains can refer to the relevant literature (Xie Kabin et al. Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA-processing system. PNAS. 2015, 112: 3570-3575). Limited by the length of the specification, it will not be described here. The target sites selected in the CRISPR-Pv2.0 website are as follows:

[0025] Target site 1: CC GCC GCC GTC GTC GCC GGT

[0026] The constructed CRISPR vector OsNAL12-CRISPR is transferred into rice variety "Zhonghua 11" (a publicly used rice variety provided by China Rice Research Institute, that is, a conventional scientific research resource) by Agrobacterium-mediated rice genetic transformation method (the specific steps are as follows). After pre-culture, infection, co-culture, selection of hygromycin-resistant callus, differentiation, rooting, seedling, transplanting, transgenic plants are obtained. The above-mentioned Agrobacterium-mediated rice (Zhonghua 11) genetic transformation method (system) is improved on the basis of the method reported by Hiei et al. (or refer to the relevant transgenic rice patent literature or non-patent literature of Huazhong Agricultural University) (Hiei et al. Efficient transformation of rice, Oryza sativa L., mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, Plant J, 6: 271-282, 1994).

[0027] According to the above-mentioned gene editing target site, the primer is designed, and the editing of OsNAL12 gene in the mutant is detected. The primers (OsNAL12-CR-F: 5'-ACGAGAGCCTAATTAAGGC-3' and OsNAL12-CR-R: 5'-AGTCGAACGGGTCCTTCCT-3') are used for specific PCR amplification of OsNAL12 gene. The amplified PCR product is sequenced, and whether it contains Cas9 is detected. The sequencing results show that in the osnal12-1 CRISPR homozygous Cas9-free mutant family, 2 bases are deleted in the OsNAL12 gene, and in the osnal12-14 CRISPR homozygous Cas9-free mutant family, 47 bases are deleted in the OsNAL12 gene at the target site Figure 1Figure 1A, i.e. the OsNAL12 gene in the two mutant families is mutated. The width of the mature leaf in the field is measured at the widest part of the leaf blade, and the nal12 mutant shows a narrower leaf.

[0028] The gene sequence of OsNAL12 is obtained from the rice gene database Rice Data( http: / / www.ricedata.cn / gene / ). The primers OE-NAL12-F (5'-GGGGTACCATGAGCCACTACACAATGCATG-3') and OE-NAL12-R (5'-ATTTGCGGCCGCTCACAGGTGTCCTCGGATCCACAG-3') are designed to amplify the gene sequence of rice OsNAL12. The amplified fragment is digested by KPN1 and NOT1 and then ligated to the overexpression vector (pCAMBIA1301U, a commonly used plasmid (vector), UBQ10 promoter) to fuse the HA tag. Finally, the correctly sequenced vector is electroporated into the Agrobacterium competent cell EHA105, and the positive clones are detected and stored. The transformation process is the same as the mutant screening.

[0029] The expression level of OsNAL12 in the young panicle of rice is detected by real-time fluorescent quantitative PCR, and the protein at this stage is extracted to detect the HA tag signal intensity by WB. It can be found that the gene and protein expression levels of OsNAL12 in OE-6 and OE-15 lines are significantly increased (see Figure 1D, Figure 1 Figure 1E). The width of the mature leaf in the field is measured at the widest part of the leaf blade. The two NAL12 overexpression lines show a wider leaf (see Figure 1F, Figure 1 Figure 1G). The construction of the plasmid (vector) in this example can also be referred to in Figure 1 (CRISPR knockout NAL12 vector map) and Figure 6 (overexpression NAL12 vector map (i.e. the original vector). Figure 7

[0030] The specific genetic transformation steps of this example are as follows:

[0031] ​(1) Electroporation: The final CRISPR target vector OsNAL12-CRISPR (Ma et al., A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants, Mol Plant, 2015, 8: 1274-1284) and overexpression vector UBQ10-OsNAL12 (Tang et al., MODD Mediates Deactivation and Degradation of OsbZIP46 to Negatively Regulate ABA Signaling and Drought Resistance in Rice, Plant Cell, 2016, 28: 2161-2177) were electroporated into Agrobacterium EHA105 strain with 1800v voltage, spread on LA medium with corresponding resistance selection, and positive clones were selected for the following transformation of callus.

[0032] (2) Callus induction: The mature rice seeds were dehulled, then treated with 70% ethanol for 1 minute, and 0.15% HgCl2 for 15 minutes for surface sterilization; the seeds were washed with sterilized water for 4-5 times; the sterilized seeds were placed on the induction medium (composition see below); the inoculated callus induction medium was cultured in the dark for 4 weeks at 25±1°C.

[0033] (3) Callus subculture: the bright yellow, compact and relatively dry embryogenic callus was selected and placed on the subculture medium (composition see below) for culture in the dark for 2 weeks at 25±1°C.

[0034] (4) Pre-culture: the compact and relatively dry embryogenic callus was selected and placed on the pre-culture medium (composition see below) for culture in the dark for 2 weeks at 25±1°C.

[0035] (5) Agrobacterium culture: the Agrobacterium EHA105 (from CAMBIA, commercial strain, carrying the vectors OsNAL12-CRISPR and UBQ10-OsNAL12 of the present application) was pre-cultured on LA medium with corresponding resistance selection (composition see below) for 2 days at 28°C; the Agrobacterium was transferred to the suspension medium (composition see below) and cultured on a 28°C shaker for 2-3 hours.

[0036] (6) Agrobacterium infection: Transfer pre-cultured calli to a sterile flask; adjust Agrobacterium suspension to OD600 0.8-1.0; soak calli in Agrobacterium suspension for 30 minutes; transfer calli to sterile filter paper and blot dry; then place on co-cultivation medium (composition below) for 3 days at 19-20°C.

[0037] (7) Callus washing and selection: Wash calli with sterile water until no Agrobacterium is visible; soak in sterile water containing 400 ppm carbenicillin (CN) for 30 minutes; transfer calli to sterile filter paper and blot dry; transfer calli to selection medium (composition below) and select for 2-3 times, 2 weeks each time (400 ppm carbenicillin for the first selection, 250 ppm for the second and subsequent selections, and 250 ppm hygromycin).

[0038] (8) Differentiation: Transfer resistant calli to pre-differentiation medium (composition below) and incubate in the dark for 5-7 weeks; transfer pre-differentiated calli to differentiation medium (composition below) and incubate under light at 26°C.

[0039] (9) Rooting: Trim roots produced during differentiation; then transfer to rooting medium and incubate under light at 26°C for 2-3 weeks.

[0040] (10) Transplanting: Wash roots of residual medium and transfer seedlings with good root systems to a greenhouse, while keeping the soil moist for the first few days.

[0041] Medium components and their formulations: (1) Abbreviations for reagents and solutions: Abbreviations for plant hormones used in the medium of the present application are as follows: 6-BA (6-Benzylamino Purine); CN (Carbenicillin); KT (Kinetin); NAA (Napthalene acetic acid); IAA (Indole-3-acetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid); AS (Acetosringone); CH (Casein Enzymatic Hydrolysate); HN (Hygromycin B); DMSO (Dimethyl Sulfoxide); N6max (N6 macro component solution); N6mix (N6 micro component solution); MSmax (MS macro component solution); MSmix (MS micro component solution). (2) Formulations of main solutions:

[0042] 1) Preparation of N6 medium macroelement stock solution [10X concentrate]

[0043]

[0044] Dissolve one by one, then make up to 1000 mL at room temperature.

[0045] 2) Preparation of N6 medium microelement stock solution [100X concentrate]

[0046]

[0047] Dissolve at room temperature and make up to 1000 mL.

[0048] 3) Preparation of iron salt (Fe 2+ -EDTA) stock solution (100X)

[0049] Prepare 800 mL of double distilled water and heat to 70°C, add ethylenediamine tetraacetic acid disodium salt (Na2EDTA·2H2O) 3.73 g, dissolve thoroughly, then keep in a 70°C water bath for 2 hours, make up to 1000 mL, and store at 4°C for future use.

[0050] 4) Preparation of vitamin stock solution (100X)

[0051]

[0052] Make up to 1000 mL with water, and store at 4°C for future use.

[0053] 5) Preparation of MS medium macroelement stock solution (10X)

[0054]

[0055] Dissolve at room temperature and make up to 1000 mL.

[0056] 6) Preparation of MS medium microelement stock solution (100X)

[0057]

[0058] Dissolve at room temperature and make up to 1000 mL.

[0059] 7) 2,4-D stock solution, 6-BA stock solution, naphthalene acetic acid (NAA) stock solution, indole acetic acid (IAA) stock solution: all 1 mg / mL.

[0060] 8) Glucose stock solution: 0.5 g / mL.

[0061] 9) Preparation of AS stock solution: weigh AS 0.392 g, DMSO 10 mL.

[0062] (3) Medium formula for genetic transformation of rice

[0063] 1) Callus induction medium

[0064]

[0065]

[0066] Add distilled water to 900 mL, adjust pH to 5.9 with 1 N KOH, boil and make up to 1000 mL, dispense into 50 mL flasks (25 mL / flask), seal and sterilize.

[0067] 2) Subculture medium

[0068]

[0069] Add distilled water to 900 mL, adjust pH to 5.9 with 1 N KOH, boil and make up to 1000 mL, dispense into 50 mL flasks (25 mL / flask), seal and sterilize.

[0070] 3) Pre-culture medium

[0071]

[0072]

[0073] Add distilled water to 250 mL, adjust pH to 5.6 with 1 N KOH, seal and sterilize. Before use, heat to dissolve the medium and add 5 mL of glucose stock and 250 μL of AS stock, dispense into Petri dishes (25 mL / dish).

[0074] 4) Co-culture medium

[0075]

[0076] Add distilled water to 250 mL, adjust pH to 5.6 with 1 N KOH, seal and sterilize. Before use, heat to dissolve the medium and add 5 mL of glucose stock and 250 μL of AS stock, dispense into Petri dishes (25 mL / dish).

[0077] 5) Suspension medium

[0078]

[0079] Add distilled water to 100 mL, adjust pH to 5.4, dispense into two 100 mL flasks, seal and sterilize. Before use, add 1 mL of glucose stock and 100 μL of AS stock.

[0080] 6) Selection medium

[0081]

[0082]

[0083] Add distilled water to 250 mL, adjust pH to 6.0, seal and sterilize. Dissolve medium before use, add 250 μL HN and 400 ppm CN, dispense into Petri dishes (25 mL / dish).

[0084] 7) Pre-differentiation medium

[0085]

[0086] Add distilled water to 250 mL, adjust pH to 5.9 with 1 N KOH, seal and sterilize. Dissolve medium before use, add 250 μL HN and 200 ppm CN, dispense into Petri dishes (25 mL / dish).

[0087] 8) Differentiation medium

[0088]

[0089]

[0090] Add distilled water to 900 mL, adjust pH to 6.0 with 1 N KOH. Boil and make up to 1000 mL, dispense into 50 mL Erlenmeyer flasks (50 mL / flask), seal and sterilize.

[0091] 9) Rooting medium

[0092]

[0093] Add distilled water to 900 mL, adjust pH to 5.8 with 1 N KOH. Boil and make up to 1000 mL, dispense into rooting tubes (25 mL / tube), seal and sterilize.

[0094] The Q-PCR reaction system was as follows: 2 μL cDNA (50 ng / μL), 5 μL Universal SYBR Green Supermix (Bio-Rad), 0.2 μL forward primer (FP, 10 μmoL / L), 0.2 μL reverse primer (RP, 10 μmoL / L), and ddH2O was added to 10 μL. The reaction program was as follows: 95°C for 15 sec, 60°C for 15 sec, 72°C for 30 sec, 40 cycles, using Bio-Rad CFX96 Real-Time System. Finally, the data was processed using Comparative Ct method. OsNAL12 expression detection primers: OsNAL12-qF (5'-GCGGACTGGGACCTCGAGT-3') and OsNAL12-qR (5'-CAGGTGTCCTCGGATCCACAG-3'); reference gene actin7 detection primers ACTIN1-QP-F (5'-ATGAGTAACCACGCTCCGTC-3') and ACTIN1-QP-R (5'-TTCAACCCCAAGGCCAATC-3').

[0095] The WB (Western blot) test method is as follows: the rice tissue is added into liquid nitrogen and ground, 0.100 g of powder is weighed by an analytical balance, 500 μL of extraction buffer (50 mMOL / L Tris-HCL (PH 7.5), 150 mMOL / L NaCL, 0.5% Trition X-100, 1 mMOL / L phenylmethylsulfonyl fluoride) is added, mixed, centrifuged at 4°C for 15 min at 12000 g; the supernatant is sucked into a new centrifuge tube, and the centrifugation is repeated twice, which is the total protein of the tissue, and is stored at -20°C. After the protein sample is denatured at high temperature, it is separated by 10%-12% SDS-PAGE gel electrophoresis, and the protein transfer is performed by using a Bio-Rad Mini-Protean 3 electrophoresis tank and a Mini Trans-Blot electric transfer system (wet transfer membrane for large molecular weight proteins), and the detailed method is referred to the product instruction. The Western hybridization is performed by using a Hybond-P PVDF (polyvinylidene difluoride membrane) membrane (Amersham), and the use method is performed according to the instruction. The PBS containing 0.05% Tween-20 and 2.5% non-fatty milk is pre-hybridized for 3 h to block the background. Then, the primary antibody (ABclone HA-tag) is hybridized at 4°C overnight. After being washed by PBS, the mouse secondary antibody with horseradish peroxidase (HRP) is hybridized, and is incubated at room temperature for 1-2 h. After the membrane is washed by TBST, after the developing solution (ABclone, see the operation instruction for details) is added for 30 s, the protein developing instrument is exposed and developed, and the photograph is taken.

[0096] Example 2 Identification of the influence of OsNAL12 on the grain shape of rice

[0097] The nal12 mutant seeds harvested from the Wuhan field are threshed. The grain length and width values of the seeds are obtained by camera shooting and Image J. It can be seen that the grain length and width of the mutant are significantly smaller (see FIG. A in Figure 2 Figure 2 The NAL12 overexpression seeds harvested from the Wuhan field are threshed. The grain length and width values of the seeds are obtained by camera shooting and Image J, and the statistics show that the OsNAL12 overexpression can significantly increase the grain length and width of the seeds (see FIG. D in Figure 2 Figure 2

[0098] Example 3 Thousand-grain weight statistical analysis of OsNAL12 mutant and overexpression plant

[0099] ​​​The thousand-seed weight of naL12 mutant seeds harvested from field plantings in Wuhan, Hubei Province, was analyzed. Three batches of 1000 seeds each were weighed on an analytical balance. Statistical results are shown below. Figure 3 Figure A in the table is shown. The thousand-grain weight of NAL12 overexpressing plants grown in Wuhan, Hubei Province was also analyzed. Statistical results are shown in Figure A. Figure 3 Figure B in the diagram.

[0100] Example 4: Construction and genetic transformation of OsNAL12 GUS chromosome vector

[0101] From the Rice Gene Database (Rice Data) http: / / www.ricedata.cn / gene / The promoter sequence of the OsNAL12 gene was obtained. Primers GUS-NAL12-F (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAACGATTTTCATTACGGATTCGGT-3') and GUS-NAL12-R (5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAACGATTTTCATTACGGAT-3') were designed to amplify the promoter sequence of the rice OsNAL12 gene. The amplified fragment was then ligated into the expression vector pGreen0179-GUS (Liu et al., Two Arabidopsis MYB-SHAQKYF transcription repressors regulate leaf wax biosynthesis via transcriptional suppression on DEWAX, New Phytologist, 2022, 236: 2115-2130) via a gateway. Finally, the correctly sequenced vector was electroporated into Agrobacterium competent cells EHA105, and positive clones were detected and preserved. The transformation process was the same as in Example 1.

[0102] The vector diagram used for OsNAL12 expression pattern analysis in this embodiment is shown below. Figure 8 .

[0103] Example 5: Verification of E3 ubiquitin ligase activity of OsNAL12

[0104] From the Rice Gene Database (Rice Data) http: / / www.ricedata.cn / gene / The gene sequence of OsNAL12 was obtained in the method of RT-PCR. The primers NAL12-TEDA-F (5'-ACGACAAGGCCATGGCTGATATGAGCCACTACACAATG-3') and NAL12-TEDA-R (5'-AGCTCGAATTCGGATCCGATCAGGTGTCCTCGGAT-3') were designed to amplify the gene sequence of OsNAL12. The amplified fragment was ligated to the protein expression vector (pET-32a, Novagen, 69015) by homologous recombination, and a His tag was fused. Finally, the vector with correct sequence was heat-shocked BL21. The protein was purified. Ubiquitin (Ub), ubiquitin activating enzyme (E1) and ubiquitin binding enzyme E3 were obtained from a company (Beyotime). Then, the ubiquitination band was detected by WB after reacting in the ubiquitination solution at 30 degrees for 2 hours.

[0105] The specific protein purification process of the embodiment is as follows:

[0106] (1) Take two 15mL test tubes, add 10mL LB medium, 10μL bacterial strain (BL21) and 10μL corresponding antibiotic (ampicillin) to each tube, and place them in a 37°C constant temperature shaking incubator at 180rpm for overnight culture for 16h.

[0107] (2) Take 800μL of the above bacterial solution and add it to 800mL of culture medium, then add 800μL of the corresponding antibiotic (ampicillin), and place it in a 37°C constant temperature shaking incubator at 180r / min for 4h to make the culture solution OD 600 reach 0.6-0.8.

[0108] (3) Add 800μL of inducer IPTG and continue to culture in a 37°C constant temperature shaking incubator for 4h.

[0109] (4) Collect the cultured bacterial solution, centrifuge at 8000r / min at 4°C for 10min, and collect the bacterial body at the bottom of the tube.

[0110] (5) According to the test requirements, take an appropriate amount of bacterial body and add it to 10mL PBS buffer solution, and vortex.

[0111] (6) Place the mixed bacterial body in an ice water bath and use an ultrasonic instrument to break it up. Extend the ultrasonic probe of the ultrasonic instrument into the bacterial solution, making sure not to touch the bottom and walls of the tube. Each time, ultrasonically break for 20s, a total of four times, with a half-minute interval in between (the breaking time, breaking frequency and interval time can be determined according to the specific situation).

[0112] (7) The broken bacteria solution will be relatively clear. At this time, centrifuge the bacteria solution at 8000 r / min in the centrifuge at 4°C for 10 min. The centrifugation time can also be extended. After centrifugation, separate the supernatant and precipitate, and take samples respectively. Detect the target band in the supernatant or precipitate by SDS-PAGE electrophoresis.

[0113] (8) Add 2M imidazole solution to the supernatant after the bacteria solution is broken and centrifuged to make the final concentration 20mM, and the total volume of the sample is 10mL.

[0114] The sample passing through the column is preferably filtered through a 0.45μm filter to avoid clogging the column.

[0115] (9) Equilibrium buffer: 50mM phosphate buffer pH 7.4, 0.5M NaCl, containing 20mM imidazole.

[0116] (10) Elution buffer: 50mM phosphate buffer pH 7.4, 0.5M NaCl, containing 500mM imidazole.

[0117] (11) Take 1mL nickel sepharose FF or nickel NTA sepharose FF pre-packed column, equilibrate with 10mL equilibrium buffer, then take 10mL supernatant after breaking, and load at 0.5mL / min, then collect 2mL / tube.

[0118] (12) Wash away the unabsorbed sample with 15mL equilibrium buffer at a flow rate of 1-2mL / min, and collect 2mL / tube.

[0119] (13) Wash away the unabsorbed sample with 5mL elution buffer at a flow rate of 1-2mL / min, and collect 2mL / tube.

[0120] (14) Equilibrate the column with 5mL equilibrium buffer again, fill it with 20% ethanol, and seal it for next use.

[0121] (15) This method is a general method. If the effect is not good, use elution buffer containing different concentrations of imidazole (50mM, 100mM, 300mM, 500mM) to elute in sections, elute 5-10 column volumes, 1-2mL / min, and collect 2mL / tube.

[0122] (16) Take samples of the proteins eluted at each concentration gradient, and perform SDS-PAGE.

[0123] (17) According to the SDS-PAGE electropherogram, if there is protein, collect the protein for dialysis,

[0124] Ubiquitination system solution formula:

[0125]

[0126]

[0127] The vector map for expression of OsNAL12 protein in this example is shown in Figure 9 .

Claims

1. Application of rice gene OsNAL12 in regulating grain shape and increasing thousand-grain weight of rice, characterized in that, The nucleotide sequence of the gene is shown in the sequence table SEQ ID NO: 1, and the OsNAL12 gene is regulated by overexpression to control rice grain shape and increase the thousand-grain weight.

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