Gene osep a1 for regulating grain shape of rice and application thereof

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to a gene OsEPA1 for regulating rice grain type and application thereof. The application provides a gene OSEPA1 for regulating rice grain type and a coding sequence thereof, and provides application of the gene in regulating rice grain type. The application provides a new gene resource for yield and quality breeding of rice.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a gene OSEPA1 that regulates rice grain shape and its application. Background Technology

[0002] Grain type is an important factor affecting rice yield and quality traits. Further research on QTL identification and candidate gene cloning for grain type, as well as the analysis of their mechanisms of action, is of great significance for improving rice yield and quality.

[0003] The main determinants of rice yield include the number of effective tillers per plant, the number of filled grains per effective panicle, and grain weight, with grain weight being closely related to grain shape. Current research indicates that most cloned grain shape-related genes control rice grain shape by influencing the development of glume cells. To better study the molecular mechanisms and genetic regulatory networks of rice grain shape gene regulation, these genes can be classified into the following signaling and regulatory pathways: ubiquitin-proteasome signaling pathway, G protein signaling pathway, mitogen-activated protein kinase signaling pathway, plant hormone regulatory pathway, transcription factor regulatory pathway, and epigenetic regulatory pathway (Li et al., 2019).

[0004] Currently, with the rapid development of rice molecular breeding technology, an increasing number of cloned grain shape-related genes are being applied to rice variety improvement. Therefore, the cloning and functional analysis of grain shape genes provide important genetic resources for rice molecular design breeding. Current research should continue to explore genes regulating grain shape and elucidate the regulatory mechanisms of rice yield formation, providing genetic and theoretical basis for rice variety improvement. Through the development and utilization of superior allele germplasm resources, combined with transgenic technology, the successful transformation of superior alleles into breeding results can be achieved efficiently and rapidly. Summary of the Invention

[0005] The purpose of this invention is to provide a gene OSEPA1 that regulates rice grain shape and its application, providing a new gene resource for rice yield and quality breeding.

[0006] This invention provides one of the following technical solutions:

[0007] OSEPA1 is a gene that regulates rice grain shape, and its DNA sequence is shown in SEQ ID No. 1.

[0008] Furthermore, the cDNA coding region of the gene OSEPA1 that regulates rice grain shape has a length of 2166 bp, and the cDNA sequence is shown in SEQ ID No. 2.

[0009] Furthermore, the gene OSEPA1 that regulates rice grain shape encodes 721 amino acids, and its amino acid sequence is shown in SEQ ID NO 3.

[0010] The present invention provides the following second technical solution:

[0011] Application of the rice gene OSEPA1 in regulating rice grain shape.

[0012] Furthermore, the regulation involves overexpressing the OsEPA1 gene to increase the width of rice grains; and deleting the OsEPA1 gene to decrease the width of rice grains.

[0013] The present invention provides the following third technical solution:

[0014] The application of products overexpressing the OSEPA1 gene in regulating rice grain width, wherein the nucleotide sequence of the OSEPA1 gene is shown in SEQ ID No. 1 and the amino acid sequence it encodes is shown in SEQ ID NO. 3.

[0015] The present invention provides the following fourth technical solution:

[0016] A method for regulating rice seed grain shape involves constructing a gene knockout vector or gene overexpression vector using the encoding gene OsEPA1 as the target gene, and then transforming the gene knockout vector or gene overexpression vector into rice using transgenic technology to change the rice grain shape.

[0017] Furthermore, a vector for knocking out the target gene was constructed using the CRISPR / Cas9 gene editing system.

[0018] Furthermore, the overexpression vector is a recombinant expression vector pEGOEPubi-H; the recombinant expression vector is introduced into the plant via Agrobacterium-mediated transformation.

[0019] The present invention provides the following fifth technical solution:

[0020] The application of the above-mentioned gene OSEPA1, which regulates rice grain shape, in the breeding of wide-grained rice varieties.

[0021] Furthermore, the application of gene OSEPA1-related biological materials in the cultivation of transgenic rice, wherein the biological materials include nucleotide sequences containing the encoding gene OSEPA1 and / or recombinant vectors and recombinant engineered bacteria containing the encoding gene OSEPA1.

[0022] Furthermore, the above application utilizes transgenic technology to cultivate transgenic rice with wide grains, thereby altering the rice grain shape. The methods and steps include:

[0023] (1) Using the encoding gene OsEPA1 as the target gene, a gene overexpression vector was constructed;

[0024] (2) Transform the gene overexpression vector into rice to obtain transgenic rice with wide grains.

[0025] The beneficial effects of this invention are:

[0026] This invention clones a major gene, OsEPA1, that positively regulates rice grain width in rice. Its loss-of-function alleles can alter rice grain shape, providing new gene resources for rice yield and quality breeding, and also providing an important theoretical basis for molecular design breeding to improve rice grain shape. Attached Figure Description

[0027] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0028] Figure 1 This is a gene mapping result of the rice gene OSEPA1 in this invention;

[0029] Figure 2 This is a graph showing the quantitative fluorescence results and promoter differences of the rice gene OSEPA1 in different varieties in this invention;

[0030] Figure 3 This is a map of the OSEPA1 gene knockout vector in this invention;

[0031] Figure 4 This is a spectrum of the OSEPA1 gene overexpression vectors used in this invention;

[0032] Figure 5 This is a diagram showing the comparison of grain width phenotype between the OSEPA1 gene knockout line and the wild-type 93-11 rice in this invention.

[0033] Figure 6 This is a comparison of the grain width phenotype of rice between the OSEPA1 overexpressing lines and the wild-type 93-11 in this invention.

[0034] in, Figure 1 In section A, the grain size of salt-tolerant rice variety 8, indica rice variety 93-11, and the near-isogenic population (EPA1) are compared. Figure 1 Figure B is a bar chart comparing the grain lengths of three types of rice. Figure 1 C is a bar chart comparing the grain width of the three types of rice; Figure 1 D in the middle represents the gene location map of OSEPA1;

[0035] Figure 2In the middle section, A represents the relative expression levels of salt-tolerant rice variety 8 and indica rice variety 93-11. Figure 2 Figure B shows a comparison of promoter differences of OSEPA1 in salt-tolerant rice variety 8 and indica rice variety 93-11;

[0036] Figure 5 Comparison of grains in Indica rice variety 93-11 and the double knockout mutants KO1 and KO2 (Category A). Figure 5 B is a bar chart comparing the grain lengths of the three types of rice. Figure 5 C is a bar chart comparing the grain width of the three types of rice.

[0037] Figure 6 In section A, the grains of the indica rice variety 93-11 and the overexpressing transgenic lines OE7 and OE10 are compared. Figure 6 B is a bar chart comparing the grain lengths of the three types of rice. Figure 6 The bar chart in section C compares the grain widths of the three types of rice. Detailed Implementation

[0038] To clearly illustrate the technical features of this solution, the invention will be described in detail below through specific embodiments and in conjunction with the accompanying drawings. The scope of this invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to this invention without departing from its spirit and scope.

[0039] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0040] Note: 93-11 is a conventional indica rice variety.

[0041] I. Location of rice gene OSEPA1

[0042] 1.1. Initial Positioning of OSEPA1

[0043] The inventors crossed the japonica rice variety Yandao 8 with the indica rice variety 93-11 to obtain the F1 generation, and then self-pollinated to produce the F2 generation. This F2 generation was used for subsequent experiments. 2：3384 materials were randomly selected from the population as the QTL mapping population, and their grain width phenotype was recorded as the phenotypic value. PCR was performed using 117 pairs of SSR markers evenly distributed on the 12 rice chromosomes, selected from the SSR Markers Resource module of Gramene (http: / / archive.gramene.org / ), which could distinguish parental genotypes. The PCR amplification bands were recorded as genotypes, and initial localization analysis was performed using the ICIM method of QTL IciMapping software to obtain the major QTL locus qGW2.1. (See [link to relevant documentation]). Figure 1 .

[0044] The statistical method for the grain width phenotype mentioned above is as follows: 20 uniform grains from the middle of each rice panicle are selected from each material, and the results are obtained by measuring with an electronic vernier caliper and calculating the average value.

[0045] 1.2. Fine mapping of OSEPA1 and identification of candidate genes

[0046] Exchange plants were selected from 4076 accessions of the fine mapping population BC3F4 using markers RM12664 and RM12761 on both sides of the smallest interval of the initial mapping. The exchange plants were those with different genotypes at both ends. 23 homozygous exchange plants were selected from this interval for fine mapping.

[0047] The 23 exchanged individuals were analyzed using the following seven InDel markers developed near the initial localization region. Based on the marker genotypes and the grain width phenotypes of the exchanged individuals, see [link to relevant documentation]. Figure 1 In the middle D region, we finely mapped qGW2.1 to a 354kb chromosomal segment. We then used RT-qPCR to detect the expression levels of each gene within this segment and compared the sequence differences of 93-11 and Yandao 8 in each gene. (See [link to RT-qPCR]). Figure 2 .

[0048] Os02g0202800 (OsEPA1) was ultimately selected as the candidate gene for this site.

[0049]

[0050] II. Knockout of OSEPA1 gene in rice, construction of overexpression vectors, and genetic transformation

[0051] 2.1. Construction and transformation of OSEPA1 gene knockout vector

[0052] The knockout vector used in this invention is the pEGCas9Pubi-H vector (purchased from Wuhan Aidijing Biotechnology Co., Ltd.). The target 1 / 2 sequence of the target gene was designed using an online website (http: / / cbi.hzau.edu.cn / cgi-bin / CRISPR), and then constructed into a CRISPR / Cas9 vector. Figure 3 The sample was sent to Wuhan Aidijing Biotechnology Co., Ltd. for genetic transformation under the 93-11 background.

[0053] Target 1: 5'-GAGCCAGGGGAGGGCTCGCAGGG-3' (SEQ ID No 4);

[0054] Target 2: 5'-GGTGGCAAGCACTACCCAGCCGG-3' (SEQ ID No 5);

[0055] Transgenic plants of generation T0 were screened for hygromycin resistance, and those that survived were identified as positive plants. Subsequently, the target fragment of EPA1(1070) F / R was amplified using primers and sequenced to analyze its genotype. EPA1(1070)-F: 5'-TCCGCAGGATTTCCCTCT-3' (SEQ ID No 6);

[0056] EPA1(1070)-R: 5'-TTGCCGCATTTCATTCAC-3' (SEQ ID No 7).

[0057] 2.2. Construction and transformation of OSEPA1 gene overexpression vector

[0058] Using cDNA from leaf tissue of 93-11 as a template, the CDS fragment of OsEPA1 was amplified using primer pair EPA1 F / R and constructed into the overexpression vector pEGOEPubi-H. See [link to documentation]. Figure 4 Subsequently, it was introduced into the indica rice variety 93-11 using Agrobacterium-mediated transformation. The steps are as follows:

[0059] (1) Take mature indica rice variety 93-11 rice seeds, mechanically dehull them, select plump, sterile, high-quality seeds, and disinfect them;

[0060] (2) Inoculate the sterilized seeds into the corresponding culture medium to induce callus tissue;

[0061] (3) The transformed Agrobacterium was co-cultured with callus tissue and resistance screening was performed;

[0062] (4) Induction and rooting of resistant callus: when the seedlings grow to about 1 cm, they are transferred to a rooting culture medium to strengthen the seedlings.

[0063] PCR amplification was performed on OsEPA1 overexpressing T0 generation transgenic plants using primer pair EPA1-JD F / R. Plants that amplified a band were considered positive.

[0064] EPA1-F: 5'-ATGTCCGGCCCCGTCGTTC-3' (SEQ ID No 8);

[0065] EPA1-R: 5'-TTAGGCCCCTTCGCTGTAC-3' (SEQ ID No 9);

[0066] EPA1-JD-F: 5'-ATGTCCGGCCCCGTCGTTC-3' (SEQ ID No 10);

[0067] EPA1-JD-R: 5'-TTAGGCCCCTTCGCTGTAC-3' (SEQ ID No 11).

[0068] The Taq enzyme used in this invention is Novizan 2×Taq Master Mix (Dye Plus), and its reaction system is as follows:

[0069]

[0070]

[0071] The reaction procedure is as follows:

[0072]

[0073] III. Functional analysis of the rice gene OSEPA1

[0074] 3.1. Phenotypic analysis of OSEPA1 knockout transgenic plants

[0075] OSEPA1 was gene-edited using the CRISPR-Cas9 system in rice 93-11. Two successfully edited double-knockout mutants, KO1 and KO2, were identified by PCR. Investigation of the grain phenotype of the knockout lines revealed that OSEPA1 knockout plants had significantly reduced grain width. (See [link to relevant documentation]). Figure 5 This indicates that loss of OSEPA1 function may lead to a decrease in rice grain width.

[0076] 3.2. Phenotypic analysis of OSEPA1 overexpressing transgenic plants

[0077] The overexpressing transgenic lines OE7 and OE10, obtained by screening using the method described in section "II" above, showed significantly higher grain width in rice compared to the control group 93-11. (See section "II" above.) Figure 6This indicates that OSEPA1 may positively regulate rice grain width. Overexpression of OSEPA1 can increase rice grain width, while the grain length does not change significantly, which has certain research value in rice breeding.

[0078] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. OSEPA1 The application of genes in regulating rice grain shape is characterized by, The OSEPA1 The DNA sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence it encodes is shown in SEQ ID No. 3; the regulation is the overexpression of the gene. OsEPA1 Genes that increase the width of rice grains, or genes that are missing. OSEPA1 Genes that reduce the width of rice grains.

2. A method for regulating rice seed grain shape, characterized in that, With encoding genes OsEPA1 For the target gene, construct a gene knockout vector or a gene overexpression vector, and use transgenic technology to transform the gene knockout vector into rice to reduce the width of rice grains; or use transgenic technology to transform the gene overexpression vector into rice to increase the width of rice grains; the gene OsEPA1 The DNA sequence is shown in SEQ ID No.

1.

3. The method for regulating rice seed grain shape according to claim 2, characterized in that, The overexpression vector is the recombinant expression vector pEGOEPubi-H; the recombinant expression vector is introduced into the plant via Agrobacterium-mediated transformation.

4. Overexpression of the expression described in claim 1 OSEPA1 Application of genes in the breeding of wide-grain rice varieties.

5. The application according to claim 4, characterized in that, The method for cultivating transgenic rice with wide grains using transgenic technology to alter the grain shape of rice includes the following steps: (1) Using the coding gene OsEPA1 To construct a gene overexpression vector for the target gene; (2) Transform the gene overexpression vector into rice to obtain transgenic rice with wide grains.

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