Application of OsCPN10a gene in regulation and control of vigor, grain shape and drought tolerance of rice seeds

Abstract

The invention discloses application of an OsCPN10a gene in regulation and control of vigor, grain shape and drought tolerance of rice seeds, and belongs to the technical field of rice gene engineering. The invention particularly relates to the research of the OsCPN10a gene in the aspects of regulating and controlling the germination rate, the germination potential, the germination index, the grain length, the grain width, the thousand grain weight, the drought tolerance of the rice seeds and the like. Experimental results show that the grain shape of the OsCPN10a knockout mutant seed is obviously lengthened and widened, and the grain shape of the OsCPN10a overexpression seed is opposite; the yield of the Ocpn10a mutant is higher than that of a TNG67 plant; overexpression of the OsCPN10a has the potential of improving the drought resistance of the rice. The invention reveals that OsCPN10a affects genetic mechanisms such as rice seed vigor, grain length, grain width and storability, provides important gene resources and theoretical basis for genetic improvement of rice, and is beneficial to cultivation of a new rice variety with better grain shape and higher yield.

Description

Technical Field

[0001] The present invention belongs to the technical field of rice genetic engineering, and in particular relates to the application of the OsCPN10a gene in regulating rice seed vigor, grain shape and drought resistance. Background Art

[0002] Seed germination begins with water absorption and swelling, followed by the resumption of seed metabolism. The radicle then breaks through the surrounding tissues, including the endosperm and seed coat, to complete germination. The quality of seed germination depends critically on the quality of stored mRNA, protein stability, and DNA integrity. During seed germination, intracellular storage substances such as fat, protein, and soluble sugars are broken down and used to synthesize new macromolecular compounds, primarily proteins and nucleic acids, with the associated expenditure of energy. The level of storage protein in seeds is directly related to seed germination and seedling growth, providing a source of nitrogen for normal metabolism and development.

[0003] Molecular chaperones, also often called heat shock proteins (HSPs), are present in the plastids, mitochondria, and cytoplasm of all eukaryotes and eubacteria. They are a class of proteins that help other proteins fold and assemble correctly, refold denatured proteins, prevent misfolding, and, when necessary, unfold or degrade abnormal proteins to maintain cellular protein homeostasis, which is essential for cell survival. Molecular chaperones play a crucial role in maintaining protein homeostasis (proteostasis). They are involved in a variety of biological processes, including protein synthesis, protein transport across membranes, assembly and disassembly of protein complexes, and responses to cellular stress. In plants, molecular chaperones participate in various metabolic pathways and play important roles in plant growth and development, metabolic regulation, and environmental stress. They are primarily divided into two distinct types: type I and type II chaperones. Type I chaperones (HSP110, HSP100, HSP90, HSP80, Hsp70, Hsp60, Hsp40 and other heat shock proteins and CPN60) often work together with co-chaperones. Plant plastid and mitochondrial co-chaperones include Cpn21 and Cpn10. The co-chaperone of HSP60 is HSP10; the co-chaperones of Cpn60 are Cpn10 (10kDa chaperonin) and CPN20. Cpn60 protein is very important to plants because it is involved in the folding of many chloroplast protein polypeptides. The OsHSPl8.2 gene identified by Kaur et al. using omics mainly affects rice seed vigor, lifespan and seedling establishment by regulating the accumulation of reactive oxygen species (ROS). Currently, little is known about the functions of co-chaperone family members in rice.

[0004] The 10-kDa co-chaperone protein (CPN10) is a common co-chaperone protein in plant plastids and mitochondria. It has a molecular weight of approximately 10 kDa and is similar to Gro ES in bacteria. It can form a heptamer ring structure. CPN10 is a multifunctional protein. In Arabidopsis, it includes CPN10(1), CPN10(2), Plastid CPN10(1) and Plastid CPN10(2). It plays an important role in regulating the maintenance of mitochondrial and chloroplast structures. Zheng et al. knocked down Cpn10(1) in Arabidopsis mitochondria, leading to mitochondrial dysfunction, further confirming the importance of CPN10 in maintaining mitochondrial homeostasis. Hemmingsen et al. found that the similarity between Arabidopsis mitochondrial CPN10(1) and rice mitochondrial CPN10 was 67%. It is known that the monocotyledonous model plant rice has two OsCPN10 genes. Whether this type of gene is involved in the response of rice to biotic and abiotic stresses has not been reported. Therefore, it is of great significance to explore the function and regulatory mechanism of rice OsCPN10 genes. Summary of the Invention

[0005] This study uses wild-type, knockout, and overexpression rice lines as materials, combined with plant physiology and biochemistry, molecular biology, and other techniques, to preliminarily explore the function of OsCPN10a protein in rice response to biological processes such as seed germination, artificial aging, and drought stress, providing assistance for further analysis of the molecular mechanism of OsCPN10 in rice response to aging stress.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an application of the OsCPN10a gene in regulating rice seed vigor, grain shape and drought resistance, wherein rice seed vigor, grain shape and drought resistance are regulated by gene knockout or gene overexpression. The amino acid sequence of the protein encoded by the OsCPN10a gene is shown in SEQ ID NO.1, and the nucleotide sequence of the OsCPN10a gene is shown in SEQ ID NO.2.

[0008] The regulating seed activity is regulating seed germination rate, germination potential and germination index; the regulating seed grain shape is regulating seed grain length, grain width and thousand-grain weight.

[0009] The gene knockout is carried out by using OsCPN10a as the target gene, using the CRISPR / Cas9 gene editing system to construct a knockout vector of the target gene, transforming it into rice, and cultivating the rice.

[0010] The construction method of the knockout vector is:

[0011] Select a target and design gRNA, perform PCR amplification using the pCBC-MT1T2 plasmid as a template, and after recovery, use T4 ligase to ligate it with the pHUE411 vector to obtain a ligation product, which is then transformed into Escherichia coli, the plasmid is extracted, and then sequencing verification is performed.

[0012] The gRNA sequence is shown as SEQ ID NO. 3 or 4.

[0013] The gene overexpression is to use OsCPN10a as the target gene, connect the target gene with a vector by seamless cloning and homologous recombination to construct an overexpression vector, transform the vector into rice, and cultivate the rice.

[0014] The method for constructing the gene overexpression vector is as follows:

[0015] Upstream and downstream primers were designed for OsCPN10a gene for PCR amplification. The binary vector pRHVcGFP and the amplified product were digested with HindIII and KpnI as restriction sites. Then, the target fragment was connected with the linearized vector by homologous recombination to obtain a recombinant vector.

[0016] The upstream primer sequence is shown in SEQ ID NO.11, and the downstream primer sequence is shown in SEQ ID NO.12.

[0017] In a second aspect, the present invention provides the use of the OsCPN10a gene in breeding drought-resistant rice varieties. The nucleotide sequence of the OsCPN10a gene is shown in SEQ ID NO.2.

[0018] In a third aspect, the present invention provides a method for breeding drought-resistant rice varieties, wherein an expression vector containing the OsCPN10a gene is transformed into a target plant for overexpression to obtain the drought-resistant rice; the nucleotide sequence of the OsCPN10a gene is shown in SEQ ID NO.2.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] To elucidate the function of OsCPN10 in rice, we cloned OsCPN10 (Os03g0366000) from rice embryos and constructed knockout and overexpression transgenic lines. Using wild-type, knockout, and overexpression rice lines, we employed plant physiology, biochemistry, and molecular biology to preliminarily explore the role of OsCPN10a in rice responses to seed germination, artificial aging, and drought stress. The results are as follows:

[0021] (1) There were certain differences in morphological characteristics between wild-type and transgenic rice lines. TNG67, Oscpn10a, and OE showed significant differences in grain length and width. The seeds of the OsCPN10a knockout mutant showed significantly longer and wider grains, while the opposite was true for seeds of the OsCPN10a overexpression mutant. In the field, the yield of the Oscpn10a mutant was higher than that of the TNG67 plant.

[0022] (2) The present invention found that knocking out OsCPN10a resulted in delayed seed germination and slow seedling growth, while seeds overexpressing OsCPN10a germinated earlier than the wild type. After artificial aging treatment, the germination rate of Oscpn10a seeds was significantly reduced. This indicates that the function of OsCPN10a is necessary for normal early seed germination. During seed germination, the amount of ABA accumulated in the seeds of the OsCPN10a knockout mutant was 41% higher than that of TNG67 at 8 h after germination. Under ABA treatment, the seed germination rate, root length and shoot length of Oscpn10a were significantly reduced, indicating that OsCPN10a has a negative regulatory effect on ABA accumulation. Compared with wild-type plants, the increase in ABA accumulation in the Oscpn10a knockout mutant is associated with the increased expression of ABA biosynthesis genes ZEP1, NCED1, NCED3, NCED4 and NCED5. Further analysis found that the expression levels of ABA marker genes NCEDs and ABIs in the embryos of the knockout strain were higher than those in the wild type, while the expression levels of NCEDs and ABIs in the overexpression strain were lower than those in the wild type, indicating that OsCPN10a plays a negative regulatory role in the ABA signal transduction pathway.

[0023] (3) Under drought stress, the survival rate of OsCPN10a-overexpressing plants at the seedling stage was significantly higher than that of wild-type plants, while the survival rate of Oscpn10a-knockout mutants was significantly lower than that of wild-type plants. These results indicate that OsCPN10a overexpression has the potential to improve drought resistance in rice.

[0024] In summary, molecular biology and genetics studies have revealed the genetic control mechanisms by which OsCPN10a influences rice seed vigor, grain length, grain width, and storage stability, providing important genetic resources and a theoretical basis for rice genetic improvement. These findings will help breed new rice varieties with improved grain shape and higher yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1These are the results of knocking out and overexpressing OsCPN10a in the Tainong 67 background material in Example 4 (Figure A is a schematic diagram of the OsCPN10a genome; Figure B is an alignment of the OsCPN10a protein sequences of TNG67 and the OsCPN10a knockout line; Figure C is a photograph of plants of TNG67, the Oscpn10a mutant, and the OsCPN10a overexpression line; and Figure D is an analysis of the relative expression of the OsCPN10a gene in TNG67 and the OsCPN10a overexpression line).

[0026] Figure 2 Figure 5 shows the effect of the OsCPN10 mutant on seed shape in Example 5 (Figure A shows mature seeds of TNG67, Oscpn10a mutant and OsCPN10a overexpression line, Bar = 5 mm; Figures BG show grain width, grain length, 1000-grain weight, grain yield per plant, seed setting rate and number of effective tillers per plant of TNG67, Oscpn10a mutant and Oscpn10a overexpression line; Figure H shows scanning electron microscopy observation of the outer surface of the hull of mature seeds, Bar = 100 μm; Figure IK shows cell length, width and number analysis; Figures LN show the relative mRNA expression levels of OsDEP1, OsGS3 and OsGW2 in the embryos of mature seeds of TNG67, Oscpn10a mutant and OsCPN10a overexpression line).

[0027] Figure 3 This is a functional analysis of the OsCPN10a gene during seed germination in Example 6 (Figure A is a photo of seed germination of TNG67, Oscpn10a knockout mutant and overexpression line; Figures BD are a comparison of seed germination potential, germination rate and germination index of TNG67, Oscpn10a knockout mutant and overexpression line).

[0028] Figure 4 Comparison of the vigor of rice seeds of TNG67, Oscpn10a mutant, and Oscpn10a overexpression line under artificial aging in Example 6 (Figure A shows the germination of seeds of TNG67, Oscpn10a mutant, and Oscpn10a overexpression line 14 days after artificial aging treatment for 7, 14, and 21 days; Figure B shows the germination potential, germination rate, and germination index of seeds of TNG67, Oscpn10a mutant, and Oscpn10a overexpression line 7, 14, and 21 days after artificial aging treatment; Figure C shows the appearance of rice grains of TNG67, Oscpn10a mutant, and OE line 21 days after artificial aging, Bar = 100 μm; Figure D shows a scanning electron microscope image of a cross-section of rice grains of TNG67, Oscpn10a mutant, and OE line 21 days after artificial aging, Bar = 10 μm & 5 μm).

[0029] Figure 5Comparison of the vigor of rice seeds of TNG67, Oscpn10a mutant, and Oscpn10a overexpression line under natural aging conditions in Example 6 (Figure A shows the germination of seeds of TNG67, Oscpn10a mutant, and Oscpn10a overexpression line 10 days after natural aging for 6 months; Figure B shows the germination potential, germination rate, and germination index of seeds of TNG67, Oscpn10a mutant, and OE line 10 days after natural aging for 6 months).

[0030] Figure 6 This is the study on the drought tolerance of OsCPN10a and rice in Example 7 (wherein, Figure A shows the growth of the Oscpn10a knockout mutant, OE line, and TNG67 under normal nutrient solution culture and the phenotypic characteristics of the plants rewatered 3 days after 3 weeks of drought treatment, Bars = 10 cm; Figure B shows the statistical analysis of the plant survival rate of TNG67, Oscpn10a knockout mutant, and OE line rewatered 3 days after 3 weeks of drought treatment; Figures C and G show the antioxidant activity of rice leaves of TNG67, Oscpn10a knockout mutant, and OE line before and after drought stress). DETAILED DESCRIPTION

[0031] To better illustrate the present invention, the following embodiments are listed. Obviously, the embodiments described are only part of the present invention, not all of the embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without making any creative efforts are also within the scope of protection of the present invention.

[0032] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0033] The amino acid sequence of the protein encoded by the rice OsCPN10a gene is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2;

[0034] SEQ ID NO.1:

[0035] MAARRLIPSMNRVLVEKLLQPNKSAGGILLPETTKQLNSAKVVAVGPGERDR DGKLIPVSLKEGDTVLLPEYGGTEVKLAEKEYLLFREHDILGRLEE*

[0036] SEQ ID NO.2:

[0037] ATGGCGGCGAGGAGGCTGATCCCGTCGATGAACCGGGTGCTGGTGGAGAA

[0038] GCTGCTGCAGCCCAACAAGAGCGCCGGCGGCATCCTCCTCCCGGAGACCA

[0039] CCAAGCAGCTGAATTCTGCAAAAGTAGTGGCTGTTGGTCCTGGCGAACGT

[0040] GACAGGGATGGCAAACTGATCCCTGTATCTTTGAAAGAAGGTGACACCGTT

[0041] CTGCTACCTGAGTATGGAGGAACTGAAGTGAAGCTTGCTGAGAAAGAGTA

[0042] CCTTCTTTTCAGAGAGCACGACATACTCGGACGGCTTGAGGAGTAA

[0043] Example 1: Knockout of OsCPN10a gene based on CRISPR / Cas9 technology

[0044] To generate clustered regularly interspaced short palindromic repeat (CRISPR)-OsCPN10a transgenic plants with knockout of the OsCPN10a gene, single guide RNA (sgRNA) sequences for OsCPN10a, specifically CRISPR / Cas9-specific targeting sites, were designed using the CRISPR-PLANT online website. Based on target prediction results, two gRNA targets were selected within the first exon of the OsCPN10a gene: target-1: gtcgatgaaccgggtgctgg (SEQ ID NO. 3) and target-2: cctgcttggtggtctccggg (SEQ ID NO. 4). Off-target analysis of the selected targets was then performed using the CRISPRRGEN Tools website. Knockout primers were designed. The designed primer sequences were Cpn10-F: AATAATGGTCTCAGGCGTCGATGAACCGGG TGCTGGGTTTTAGAGCTAGAAATAGC (SEQ ID NO. 5); PCBC-MT1-R (universal): ATTATTGGTCTCTGCTTCTTGGTGCCGC (SEQ ID NO. 6); CPN10-MT2-F: ATATATGGTCTCaAAGCCCCGGAGACCACCAAGCAGGGTTTTAGAGCTAGA AATAGC (SEQ ID NO. 7); PCBC-MT2(m6b)-R (universal): AATAATGGTCTC aAACACAAGCGGCAGCGCGCG (SEQ ID NO. 8).

[0045] Amplification of the gRNA target junction fragment used a 100-fold dilution of the pCBC-MT1T2 plasmid (laboratory-maintained, described in the following literature: https: / / link.springer.com / article / 10.1186 / s12870-014-0327-y) as a template. Primers containing the BsaI restriction site were used with 2× Primer Star Max high-fidelity enzyme to amplify the desired fragments. After determining the fragment size, the fragments were recovered by gel extraction. The PCR reaction system and procedure are shown in Tables 1 and 2 below:

[0046] Table 1 PCR reaction system

[0047]

[0048] Table 2 PCR reaction procedure

[0049]

[0050] The amplified fragment was recovered and ligated into the cloning vector.

[0051] The pHUE411 vector was used to establish an enzyme digestion-ligation system to obtain the ligation product. The enzyme digestion and ligation reaction system and reaction procedures are shown in Tables 3 and 4 below:

[0052] Table 3 Enzyme digestion and ligation reaction system

[0053]

[0054]

[0055] Table 4 Enzyme digestion and ligation reaction procedures

[0056]

[0057] The ligation product was transformed into Escherichia coli competent cells, wherein the sequencing primers were universal primers for the pHUE411 vector, and the specific primer sequence information was OsU3-FD3: GACAGGCGTCTTCTACTGGTGCTAC (SEQ ID NO. 9); TaU3-RD: CTCACAAATTATCAGCACGCTAGTC (SEQ ID NO. 10).

[0058] Extraction of recombinant plasmid: The correctly sequenced colonies were picked into 5 mL of LB liquid medium containing Kan antibiotics and cultured overnight. The recombinant plasmid was extracted using a plasmid extraction kit and then transformed into Agrobacterium competent cells EHA105.

[0059] Example 2 Overexpression of OsCPN10a gene based on CRISPR / Cas9 technology

[0060] To construct an OsCPN10a overexpression vector, primers CPN10-Pro-F: ttgaaacactcccagtgatattagg (SEQ ID NO. 11) and CPN10-Pro-R: cgacgggat cagcctcct (SEQ ID NO. 12) were designed according to the method of Example 1. The full-length coding sequence of OsCPN10a was amplified from the Nipponbare genome, the target fragment was purified and recovered, and cloned into the binary vector pRHVcGFP (WGL28) (from the following document: https: / / link.springer.com / article / 10.1186 / s12284-018-0220-7, originally named pRHVcGFP) by seamless cloning, i.e., the recombinant pUBI:OsCPN10a-GFP plasmid.

[0061] The pRHVcGFP (WGL28) vector was selected with HindIII and KpnI as restriction sites, and the vector plasmid was double-digested. The vector restriction enzyme digestion reaction system is shown in Table 5 below:

[0062] Table 5 Enzyme digestion reaction system

[0063]

[0064]

[0065] The enzyme digestion conditions are 37° C. for 3.5 h or overnight, and the digestion products are subjected to 1% agarose gel electrophoresis and the gel is cut and recovered.

[0066] The target fragment was connected to the linearized vector by homologous recombination. The reaction system is shown in Table 6 below:

[0067] Table 6 Ligation reaction system

[0068]

[0069] Ligation conditions were 37°C for 30 minutes. Transformation was performed according to the transformation system and procedures described in Example 1, using the sequencing primers cpn10-ver-F: CCCGTCACCCACTCACCCGTCT (SEQ ID NO. 13) and cpn10-ver-R: CACCGCTCTATGTCCCAAACCTC (SEQ ID NO. 14). After correct sequencing, the recombinant plasmid was extracted according to the kit instructions and transformed into Agrobacterium competent cells EHA105.

[0070] Example 3 Agrobacterium-mediated genetic transformation of rice callus

[0071] All transgenic rice plants were obtained through Agrobacterium-mediated transformation of rice callus.

[0072] (1) Callus induction and subculture

[0073] Preparation: Select approximately 100 freshly harvested, plump seeds (TNG67), carefully remove the shells, and place them in a sterilized flask, double-distilled water, and filter paper.

[0074] Disinfection: Transfer the prepared seeds to a sterilized Erlenmeyer flask. Rinse the seeds 4-5 times with sterile, single-distilled water until free of impurities. Disinfect the seed surface with sterile 75% ethanol for 2 minutes. Sterilize the seeds with 2.5% sodium hypochlorite in a shaker at room temperature for 20 minutes. Rinse with sterile water until no sodium hypochlorite remains. Transfer the seeds to a Petri dish containing filter paper and air dry.

[0075] Induction and subculture of callus tissue: Move the air-dried seeds to NEB induction medium, place them in a 28℃ light incubator to induce callus tissue for about 12 days, then remove the buds and subculture. Thereafter, subculture once every 14 days (the time can be adjusted according to the state of the callus tissue). At the late stage of the second generation, bright yellow calli can be selected for Agrobacterium infection.

[0076] (2) Agrobacterium transformation of rice callus

[0077] Strain activation: aspirate an appropriate amount of the strain stored at -80°C and streak it onto a solid culture medium containing Rif and Kan antibiotics, and culture it in the dark at 28°C for 2 days.

[0078] Callus pre-culture: Select rice callus with bright yellow color and good condition and culture it in NEB medium at 28℃ for 4 days.

[0079] Agrobacterium infection: Rinse the bacteria with sterile AAM, transfer the washed bacteria to a sterile 50 mL centrifuge tube, measure the OD of the bacterial solution using a spectrophotometer, and adjust the OD of the bacterial solution with AAM. 600 The concentration of the culture medium was 0.3, and the cells were incubated in the dark at 28°C for 1 hour. The callus tissue, which had been pre-cultured for 4 days, was transferred to an Agrobacterium culture medium containing the overexpression / knockout plasmid. The infection time was 5-10 minutes, with gentle shaking several times. After infection, the infected callus tissue was transferred to a Petri dish containing several sheets of filter paper. After absorbing the excess culture medium, the callus tissue was transferred to a Petri dish lined with one or two sheets of filter paper. After the callus tissue surface was air-dried, the callus tissue was transferred to the co-culture medium and incubated in the dark at 28°C for 1 day.

[0080] Screening and differentiation of resistant callus into seedlings: Callus cultured in the dark for one day is transferred to the first-generation screening medium containing Car and Hyg, with each generation screened every 14 days. New callus will emerge during the screening process. Once grown, it is transferred to a pre-differentiation medium and cultured in the light at 28°C until seedlings emerge. When the seedlings reach 3-4 cm and have roots, they are transferred from the pre-differentiation medium to a rooting medium and cultured in the light for a period of time.

[0081] Hardening off the seedlings: After the root systems of the seedlings are well developed, remove them and place them in nutrient solution for hardening.

[0082] Example 4 Transgenic seedling screening

[0083] When transgenic seedlings reach five leaves, DNA is extracted from leaves using the CTAB method. PCR amplification using specific primers is performed and the DNA is sent to the company for sequencing. Plants that test positive are transplanted to peat soil and incubated in a greenhouse for three and a half months. T0-generation transgenic rice seeds are harvested. These T0-generation transgenic rice seeds are then cultivated in field soil, and genomic DNA is again extracted from leaves at the same location across all lines.

[0084] The OsCPN10a knockout mutant was generated by PCR amplification of DNA from T0 seeds and subsequent generations of leaves using the specific primers cpn10-ver-F: CCCGTCACCC ACTCACCCGTCT (SEQ ID NO. 15) and cpn10-ver-R: CACCGC TCTATGT CCCAAACCTC (SEQ ID NO. 16). Sequencing results were compared with the original sequences using SnapGene software to identify positive transformed materials. Rice lines harboring mutations or deletions in the target gene loci were selected for subsequent experiments.

[0085] The transgenic plants expressing the overexpression vector were analyzed for copy number by qRT-PCR using primers conum-sfu2af-F / conum-sfu2af-R and conum-HYG-F / conum-HYG-R. DNA from seeds of the T0 generation and leaves of subsequent generations was detected. The specific sequence information is shown in Table 7 below:

[0086] Table 7 Sequence information

[0087]

[0088] Experimental results: To investigate the function of the OsCPN10a gene, we constructed OsCPN10a knockout mutants and overexpression transgenic lines using CRISPR / Cas9 in the Tainong 67 background. Figure 1After propagation, we obtained four T2 generation homozygous OsCPN10a knockout strains, namely Oscpn10a-1, Oscpn10a-2, Oscpn10a-3, and Oscpn10a-4. Their mutation sites were all located in the first exon of the OsCPN10a gene. Oscpn10a-1 had a single base "G" insertion in the sequence of the first target site of OsCPN10a; the Oscpn10a-2 mutant had an "A" insertion in the sequence of the first target site of the OsCPN10a gene and an 11-bp deletion (SEQ ID NO.21: -CTCCTCCCGGA-) in the sequence of the second target site of the OsCPN10a gene; Oscpn10a-3 had "G" deletions in both the first and second target sites of OsCPN10a; and Oscpn10a-4 had a 52-bp deletion in the first exon of OsCPN10a. Based on these nucleotide sequences, the amino acid sequence of OsCPN10a was predicted to contain only 61, 28, 60, and 53 amino acids in Oscpn10a-1, Oscpn10a-2, Oscpn10a-3, and Oscpn10a-4, respectively. These mutations all resulted in premature termination of OsCPN10a translation. These results suggest that these four Oscpn10a mutants likely lack a functional OsCPN10a protein.

[0089] Compared with TNG67, the mRNA expression levels of OsCPN10a increased by 29-fold and 92-fold in the OsCPN10-OE1 (OE-1) and OsCPN10-OE2 (OE-2) overexpression lines, respectively.

[0090] The progeny of these homozygous mutants were used in subsequent experiments.

[0091] Example 5 Observation of mutant phenotypes

[0092] Seeds of wild-type TNG67, mutants Oscpn10a-2 and Oscpn10a-4, and transgenic rice of different overexpression lines were planted in the field, and agronomic traits such as grain length, grain width, and yield of rice were evaluated at the tillering and maturity stages.

[0093] Experimental results: Seed shape and size are key factors affecting yield and are also target traits for genetic engineering and molecular breeding. Studying the genetic effects of seed shape is of great significance for high-yield rice breeding. This study analyzed the effect of the OsCPN10a gene on yield. Figure 2. The results showed that the seeds of the Oscpn10a knockout mutant were significantly longer. Compared with the wild-type TNG67, the seed length of the Oscpn10a-2 and Oscpn10a-4 knockout mutants increased by 6.55% and 8.23%, respectively, and the grain width increased by 7.96% and 9.61%, respectively, while the seed length of OE-1 and OE-2 decreased by 12.41% and 6.55%, respectively, and the grain width decreased by 11.81% and 11.74%, respectively. The rice kernels after removing the husk also showed a similar phenotype to the seeds. The thousand-grain weight of OE-1 and OE-2 was 8.21% and 10.92% lower than that of TNG67, respectively, while the thousand-grain weight of Oscpn10a-2 and Oscpn10a-4 increased by about 10%. In addition, the single-plant yield data showed that under field conditions, overexpression or knockout of OsCPN10a would lead to a decrease or increase in single-plant yield. The yield per plant of Oscpn10a-2 and Oscpn10a-4 increased by 14.54% and 11.71%, respectively. In contrast, the yield per plant of OE-1 and OE-2 decreased significantly by 17.33% and 22.16%, respectively. To reveal the possible causes of the changes in seed shape, the outer surface of the glumes of mature seeds was observed using scanning electron microscopy (SEM). The results showed that the length and width of the glumes of seeds overexpressing OsCPN10a were significantly reduced, while the length and width of the glumes of Oscpn10a-2 and Oscpn10a-4 seeds were significantly increased. Compared with TNG67 plants, the total number of cells along the longitudinal direction of the outer surface of the glumes of the Oscpn10a-2 and Oscpn10a-4 knockout mutants and the OE line also showed significant differences. These results suggest that Oscpn10a negatively regulates seed grain length and width.

[0094] Example 6 Vitality Evaluation of OsCPN10a Mutant Seeds

[0095] Seeds of TNG67, OsCPN10a knockout mutants, and overexpression lines were germinated under natural conditions at room temperature, after artificial aging for 0, 7, 14, and 21 days, and after natural storage for six months. The seeds were then analyzed for storability and vigor. Seed moisture content before and after aging was analyzed using a Lesdekar moisture meter, and apparent amylose content (AAC) and protein content were determined using kits from Solebol.

[0096] To investigate changes in seed structure before and after aging in transgenic seeds expressing or knocking out OsCPN10a, cross-sections of seeds were observed using a scanning electron microscope. The samples were fixed in 2.5% (w / v) glutaraldehyde in 0.1 M PBS for 2 h and then washed three times with 0.1 M PBS. After dehydration with a gradient of ethanol at 4°C, the samples were critical point dried, coated with palladium gold using a sputter coater, and observed under a scanning electron microscope (JSM-6390LV, JEOL).

[0097] Experimental results: Seed vigor is a key factor affecting seed germination and longevity. This study evaluated the seed vigor of mature seeds of OsCPN10a knockout mutants and OsCPN10a overexpression lines. Figure 3-5 The results showed that compared with TNG67, the GP and GI of Oscpn10a-2 and Oscpn10a-4 seeds significantly decreased after 3 days of germination, while those of OE-1 and OE-2 seeds significantly increased (P<0.05). The GP of Oscpn10a-2 and Oscpn10a-4 seeds decreased by 35.48% and 62.37%, respectively, while the GP of OE-1 increased by 17.20%. However, no significant difference in germination rate was observed among TNG67, the Oscpn10a knockout mutant, and the OsCPN10a overexpression line at 7 days (P>0.05).

[0098] After 21 days of artificial aging, the germination rate, germination potential, and GI of Oscpn10a-2 and Oscpn10a-4 seeds were significantly lower than those of TNG67 seeds, while those of OE-1 and OE-2 seeds were significantly higher than those of TNG67 (P < 0.05). The germination rates of Oscpn10a-2 and Oscpn10a-4 seeds aged for 21 days decreased by 65.13% and 67.61%, respectively, compared with those of TNG67, while those of OE-1 and OE-2 increased by 71.88% and 75.00%, respectively.

[0099] Comparison of polished rice from TNG67, an Oscpn10a mutant, and transgenic seeds overexpressing OsCPN10a after 21 days of artificial aging revealed that OE-1 rice had relatively normal, transparent grains, TNG67 had a translucent, dark endosperm phenotype, and Oscpn10a-4 had a completely opaque, milky white color. Scanning electron microscopic analysis of polished rice cross-sections revealed that before artificial aging, starch granules in the endosperm of TNG67, Oscpn10a-4, and OE-1 seeds were regular, polyhedral, uniform in size, and densely arranged. However, after 21 days of artificial aging, starch granules in the endosperm of Oscpn10a-4 seeds were less uniform in shape and size than those in wild-type and OE-1 seeds, and were more loosely arranged.

[0100] After 6 months of storage under natural conditions, the germination potential, germination rate, and GI of Oscpn10a mutant seeds were significantly reduced compared with wild-type seeds (P < 0.01), while those of OE-1 and OE-2 were significantly increased (P < 0.05). The results showed that seeds of OsCPN10a knockout and overexpression transgenic lines exhibited similar trends under both natural and artificial aging conditions. These results suggest that OsCPN10a plays an important role in regulating seed vigor and longevity.

[0101] The appearance of rice is influenced by multiple factors, including moisture content, apparent amylose content (AAC), and protein content. In this study, the moisture content of polished rice samples from TNG67, the Oscpn10a mutant, and the OE line was measured after artificial aging. The moisture content of all samples after 21 days of artificial aging was approximately 12%, with no significant differences (P>0.05). Analysis of the AAC content of the different samples revealed that after 21 days of artificial aging, the AAC content of OE-1 and OE-2 rice decreased compared to the wild type, while the AAC content of the Oscpn10a-2 and Oscpn10a-4 mutants increased by 20.64% and 36.12% compared to the wild type (Table 8), consistent with the dark endosperm phenotype of the rice. Further analysis of the total protein content of different samples after 21 days of artificial aging revealed no significant difference in the total protein content among TNG67, Oscpn10a mutant and OE polished rice (P>0.05). The above results indicate that the difference in transparency among TNG67, Oscpn10a mutant and OE rice may be directly related to AAC, but has no direct relationship with moisture content and protein content.

[0102] Table 8 AAC and total protein content of polished rice of TNG67, Oscpn10a mutant and OE line

[0103]

[0104]

[0105] Data represent mean ± SD (n = 3) from three independent replicates. In the table, the same lowercase letters indicate no significant difference compared with TNG67 under the same treatment; different lowercase letters indicate significant difference compared with TNG67 under the same treatment. Significant differences were analyzed by Student's t-test. Same below.

[0106] Example 7 Drought stress treatment of OsCPN10a transgenic rice seedlings

[0107] For soil drought stress, uniformly germinated seeds of wild-type and transgenic lines were transplanted into well-mixed soil (forest soil:vermiculite, 1:1 ratio) and grown under normal watering conditions for four weeks. Then, drought stress was applied, and irrigation was withheld for approximately 21 days. When all leaves had curled, watering was resumed for 10 days. The survival rate of each plant was calculated (the ratio of the number of surviving plants to the total number of treated plants in the pot). Antioxidant markers were also measured in the stems and leaves of all plants on the fifth day of drought stress.

[0108] Diaminobenzidine (DAB) is the most sensitive and commonly used chromogenic substrate for horseradish peroxidase. Due to the presence of peroxidase in plant leaves, DAB can react with H2O2 to form a yellow-brown insoluble precipitate that accumulates on the leaf surface. The darker the color, the higher the H2O2 content. Nitro blue tetrazolium (NBT) staining is a commonly used histochemical method for detecting superoxide anions (O 2- In this study, DAB and NBT staining were used to detect the H2O2 and O2 in the leaves of wild-type, OsCPN10a knockout mutant and OsCPN10a overexpressing plants treated with simulated drought for 5 days. 2- content.

[0109] Experimental results (see Figure 6 Because OsCPN10a is induced by ABA and actively participates in ABA responses during post-germination growth, we hypothesized that Oscpn10a mutant and OsCPN10a-overexpressing plants might exhibit altered responses to drought stress. To test our hypothesis, we examined the drought stress responses of soil-grown Oscpn10a mutant and OsCPN10a-overexpressing plants. Wild-type, Oscpn10a-2, Oscpn10a-4, and OE-1 and OE-2 plants were grown in soil under normal growth conditions for four weeks and then subjected to drought treatment. After 21 days without watering, Oscpn10a mutant plants exhibited a severe wilting phenotype, while wild-type and OsCPN10a plants grew well. After rewatering, only 25.0% of Oscpn10a-4 mutant plants survived, while the survival rate of OE-2 plants exceeded 78.0%. Stomata were significantly different between wild-type and transgenic rice lines. Compared with the wild type, the stomata of Oscpn10a-2 and Oscpn10a-4 leaves were closed, while the stomata of OE-1 and OE-2 leaves were larger.

[0110] To further explore the reasons for the differences in drought tolerance between lines, we analyzed the antioxidant activity and ABA content of rice leaves from the Oscpn10a knockout mutant and the OE line. The results showed that after drought treatment, the SOD, POD, CAT activities, and MDA content of wild-type, Oscpn10a knockout, and OsCPN10a overexpressing seedlings all showed an upward trend compared to those before drought treatment, indicating that the antioxidant system of rice was activated. Compared with the wild-type under the same treatment, the MDA content of Oscpn10a-2 and Oscpn10a-4 seedlings increased significantly (P < 0.05), increasing by 24.19% and 36.23%, respectively, while the MDA content of OE-1 and OE-2 decreased significantly, decreasing by 18.66% and 21.66%, respectively. The SOD, POD, and CAT activities of Oscpn10a-2 and Oscpn10a-4 seedlings were significantly reduced. The activities of SOD, POD, and CAT decreased by 15.61%, 25.12%, and 15.77%, respectively, in Oscpn10a-2 seedlings and by 15.09%, 23.77%, and 25.98%, respectively, in Oscpn10a-4 seedlings. However, the activities of SOD, POD, and CAT in leaves of OsCPN10a-overexpressing plants were similar to those of the wild type. Furthermore, ABA levels were measured 3 and 5 days after drought stress, revealing that wild-type and OsCPN10a plants recovered rapidly from the decrease in ABA content, while Oscpn10a plants recovered more slowly.

[0111] In summary, overexpression of OsCPN10a can protect against drought stress by reducing stomatal water loss. Considering that OsCPN10a actively participates in the ABA response, we speculate that OsCPN10a may play a positive regulatory role in the ABA-dependent drought stress response.

[0112] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of the OsCPN10a gene in regulating rice seed vigor, grain shape and drought tolerance, characterized in that: Rice seed vigor, grain shape and drought resistance are regulated by gene knockout or gene overexpression. The amino acid sequence of the protein encoded by the OsCPN10a gene is shown in SEQ ID NO.1, and the nucleotide sequence of the OsCPN10a gene is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that The regulating seed activity is regulating seed germination rate, germination potential and germination index; the regulating seed grain shape is regulating seed grain length, grain width and thousand-grain weight.

3. The use according to claim 1, characterized in that The gene knockout is carried out by using OsCPN10a as the target gene, using the CRISPR / Cas9 gene editing system to construct a knockout vector of the target gene, transforming it into rice, and cultivating the rice.

4. The use according to claim 3, characterized in that The construction method of the knockout vector is: Select a target and design gRNA, perform PCR amplification using the pCBC-MT1T2 plasmid as a template, and after recovery, use T4 ligase to ligate it with the pHUE411 vector to obtain a ligation product, which is then transformed into Escherichia coli, the plasmid is extracted, and then sequencing verification is performed.

5. The use according to claim 4, characterized in that The gRNA sequence is shown as SEQ ID NO. 3 or 4.

6. The use according to claim 1, characterized in that The gene overexpression is to use OsCPN10a as the target gene, connect the target gene with a vector by seamless cloning and homologous recombination to construct an overexpression vector, transform the vector into rice, and cultivate the rice.

7. The use according to claim 6, characterized in that The method for constructing the gene overexpression vector is as follows: Upstream and downstream primers were designed for OsCPN10a gene for PCR amplification. The binary vector pRHVcGFP and the amplified product were digested with HindIII and KpnI as restriction sites. Then, the target fragment was connected with the linearized vector by homologous recombination to obtain a recombinant vector.

8. The use according to claim 7, characterized in that The upstream primer sequence is shown in SEQ ID NO.11, and the downstream primer sequence is shown in SEQ ID NO.

12.

9. Application of the OsCPN10a gene in breeding drought-resistant rice varieties, characterized in that: The nucleotide sequence of the OsCPN10a gene is shown in SEQ ID NO.

2.

10. A method for cultivating drought-resistant rice varieties, characterized in that: The expression vector containing the OsCPN10a gene is transformed into the target plant for overexpression to obtain the drought-resistant rice; the nucleotide sequence of the OsCPN10a gene is shown in SEQ ID NO.2.

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