CN116286878B - Application of paddy rice OsJAZ5 gene - Google Patents
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Abstract
The invention discloses riceOsJAZ5Application of the gene. Rice plantOsJAZ5The CDS sequence of the gene is shown as SEQ ID NO. 1. The invention reports for the first time in riceOsJAZ5Can regulate and control the seed vitality, and experiments show that after the gene is mutated, the seed vitality is obviously reduced, and the gene is excessively expressed to improve the seed vitality. Demonstration of the inventionOsJAZ5The gene regulates the activity of rice seeds, and the gene is used for screening and cultivating high-activity varieties and facilitating the production of direct-seeding rice.
Description
Technical Field
The invention belongs to the technical field of seed biology, and relates to riceOsJAZ5Application of the gene.
Background
Rice [ (Oryza sativa L.)Oryza sativaL.) is one of the most important food crops in our country. The direct seeding cultivation method of rice has the advantages of labor saving, cost saving, good economic benefit and the like, and the production area is enlarged year by year in recent years. But has the problems of slow germination, difficult full seedling, heavy grass damage, easy lodging and the like in direct seeding production. The high-activity seeds have the characteristics of quick germination and seedling formation, high seedling growth potential, strong stress resistance and the like in fields, and have an important effect on guaranteeing the production of direct seeding rice. Jasmonate ZIM domain protein (JAZ) is a Jasmonic Acid (JA) signal inhibitor in rice, and is involved in various biological processes such as plant biotic stress and abiotic stress resistance, but the application of the jasmonic acid ester ZIM domain protein in genetic improvement of rice has not been reported.
Disclosure of Invention
The invention aims to provide jasmonate ZIM structural domain protein OsJAZ5 isolated cloning, functional verification and application for regulating and controlling the activity of rice seeds.
The aim of the invention can be achieved by the following technical scheme:
the gene nucleotide sequence and the amino acid sequence of the protein coded by the gene are shown in the following websites: http:// price. Uga. Edu/cgi-bin/sequence_display. Cgiorf=loc_os04g32480.1.
The rice of the inventionOsJAZ5The application of the gene in screening or cultivating rice varieties with high seed vigor.
The rice isOsJAZ5The gene can be applied to improving the activity of rice seeds and promoting the production of direct-seeding rice.
The application is preferably over-expressionOsJAZ5The gene can improve the activity of rice seeds.
The rice of the inventionOsJAZ5Construction of Gene mutant vector and Gene functionThe verification comprises the following steps:
(1) Obtaining riceOsJAZ5Nucleotide sequence of the gene;
(2) Designing target sites and primers thereof, performing PCR amplification by taking pCBC-MT1T2 as a template, and purifying and recovering PCR products to obtain MT1T2-PCR vectors;
(3) The belt obtained in the step (2) is provided withOsJAZ5Constructing MT1T2-PCR gel recovery products of target fragments of genes on a pHUE411 vector to obtain a pHUE411+MT1T2-PCR vector;
(4) The belt obtained in the step (3) is provided withOsJAZ5Transforming agrobacterium with plasmid of gene target fragment; transforming the agrobacterium with the transformation plasmid into rice;
(5) Screening and identifying rice mutants, and carrying out germination phenotype identification.
Furthermore, in the step (1), cDNA of flower 11 in rice is used as a template, the sequence of the gene is cloned by PCR, the upstream primer sequence of the PCR is shown in a sequence table SEQ ID NO.2, and the downstream primer sequence is shown in a sequence table SEQ ID NO. 3.
Further, the rice constructed in the step (2)OsJAZ5CRISPR/Cas9 mutant gRNA target sequenceOsJAZ5Target fragment of 19 bp in the gene) is shown in a sequence table SEQ ID NO.4; the primer sequences for PCR amplification by taking pCBC-MT1T2 as a template are shown as SEQ ID NO.5 and SEQ ID NO.6 of the sequence table;
in the step (3), the pHUE411 vector is digested with BsaI, and the pHUE411+MT1T2-PCR vector is obtained by homologous recombination.
In the step (5), the upstream primer sequence for screening the homozygous mutant is shown in SEQ ID NO.7 of the sequence table, and the downstream primer sequence is shown in SEQ ID NO.8 of the sequence table.
The rice of the inventionOsJAZ5The vector construction and gene function verification of the gene overexpression material comprise the following steps:
(1) Obtaining riceOsJAZ5Nucleotide sequence of the gene;
(2) Performing PCR amplification by using cDNA of rice middle flower 11 as a template, purifying and recovering PCR products to obtainOsJAZ5A gene CDS fragment;
(3) Obtaining the product in step (2)OsJAZ5Carrying out PCR amplification on the gene CDS fragment by using a primer with a homologous recombination joint, and purifying and recovering a PCR product to obtain the CDS fragment with the homologous recombination joint;
(4) The belt obtained in the step (3) is provided withOsJAZ5Constructing a target fragment of the gene on a pUN1301 body to obtain a recombinant plasmid;
(5) The belt obtained in the step (4) is provided withOsJAZ5Transforming agrobacterium with plasmid of gene target fragment; transforming the agrobacterium with the transformation plasmid into rice;
(6) Rice plantOsJAZ5Screening a gene overexpression material and identifying a seed vitality phenotype.
Furthermore, in the step (2), the primer sequence of the gene is cloned by using rice cDNA as a template through PCR, wherein the upstream primer sequence of the PCR is shown in a sequence table SEQ ID NO.2, and the downstream primer sequence is shown in a sequence table SEQ ID NO. 3.
Further, in step (3) use is made ofOsJAZ5The gene CDS fragment is used as a template for PCR cloning a primer sequence of the gene with the homologous recombination joint, the upstream primer sequence of the PCR is shown in a sequence table SEQ ID NO.9, and the downstream primer sequence of the PCR is shown in a sequence table SEQ ID NO. 10.
Further, in the step (4), pUN1301 vector was digested with KpnI and SacI, and the recombinant vector was obtained by the homologous recombination method.
In the step (6), the upstream primer sequence for screening positive over-expression materials by using hygromycin resistance tags is shown in a sequence table SEQ ID NO.11, and the downstream primer sequence is shown in a sequence table SEQ ID NO. 12. And (5) identifying the vigor of the rice seeds.
Advantageous effects
(1) The invention is proved for the first time by mutants and over-expression materialsOsJAZ5The gene is involved in the vigor regulation of rice seeds.
(2) The invention provides a foundation for cultivating high-activity rice varieties, also provides important gene resources for improving the production of direct-seeding rice, and has important significance for production.
Therefore, the jasmonate ZIM structural domain protein OsJAZ5 involved in the regulation of the vigor of rice seeds is utilized, so that the method is helpful for screening and cultivating high-vigor rice varieties and has important significance for direct-seeding rice production.
Drawings
Fig. 1: rice plantOsJAZ5And (5) phenotype identification of mutant materials.
Fig. 2: rice plantOsJAZ5And (5) phenotype identification of the over-expression material.
Detailed Description
The invention is further illustrated by the accompanying drawings and the specific examples, the methods used in the examples are all conventional methods, and the primers and sequencing are completed by the Guangzhou Tianyihui gene technology Co., ltd; various restriction enzymes, ligases, etc., used in the experiments were purchased from Guangzhou Shuohuang biosciences, inc.; plasmid extraction kit, gel recovery kit and genome extraction kit were purchased from Beijing Kang Runcheng Biotechnology Inc., and the methods were all performed by reference to the instructions.
Example 1: gene cloning
PCR cloning by using cDNA of flower 11 in japonica rice variety as templateOsJAZ5The CDS sequence of the gene and the upstream primer sequence of PCR are shown in a sequence table SEQ ID NO.2 and the downstream primer sequence is shown in a sequence table SEQ ID NO. 3. Sequencing of amplification products and http:// price. Uga. Edu/cgi-bin/sequence_display. Cgiorf=LOC_Os04g32480.1OsJAZ5The gene sequences are identical.
Example 2: mutant construction
ScreeningOsJAZ5And (5) gene targets. The target sequence is shown as SEQ ID NO.4, and the primer is designed by the target sequence, and the primer structure is shown as sequence lists SEQ ID NO.5 and SEQ ID NO.6. And (3) performing four-primer PCR amplification by taking pCBC-MT1T2 as a template, and purifying and recovering a PCR product to obtain the MT1T2-PCR vector. And (3) cutting the pHUE411 vector by BsaI, and constructing the MT1T2-PCR gel recovery product on the pHUE411 vector by using a homologous recombination method to obtain the pHUE411+MT1T2-PCR vector.
Transforming agrobacterium with the obtained plasmid containing pHUE411+MT1T2-PCR vector; transforming agrobacterium with transformation plasmid into middle flower 11 of wild japonica rice variety; and (3) sequencing by utilizing a PCR amplification product, comparing the PCR amplification product with a wild type, and screening a homozygous mutant, wherein the upstream primer sequence is shown in a sequence table SEQ ID NO.7, and the downstream primer sequence is shown in a sequence table SEQ ID NO. 8.
Example 3: overexpression construction
PCR cloning by using cDNA of rice middle flower 11 as templateOsJAZ5The gene CDS fragment, the upstream primer sequence of the PCR is shown in a sequence table SEQ ID NO.2, and the downstream primer sequence is shown in a sequence table SEQ ID NO. 3. By means ofOsJAZ5The gene CDS fragment is used as a template for cloning the sequence of the gene with the homologous recombination joint, the upstream primer sequence of the PCR is shown as a sequence table SEQ ID NO.9, and the downstream primer sequence of the PCR is shown as a sequence table SEQ ID NO. 10. Further, pUN1301 vector was digested with KpnI and SacI, and the recombinant vector was obtained by homologous recombination.
The upstream primer sequence for screening positive over-expression materials by using hygromycin resistance tag is shown in a sequence table SEQ ID NO.11, and the downstream primer sequence is shown in a sequence table SEQ ID NO. 12.
Example 4: phenotypic identification of Gene mutants
By means of constructionOsJAZ5MutantOsjaz5-1、Osjaz5-2And wild medium flower 11 (WT) rice varieties, germination and direct seeding tests were performed. The germination test comprises the following specific steps: selecting 30 healthy and full seeds each time repeatedly, sterilizing with 75% alcohol solution for 1 min, washing with distilled water for 3 times, wiping the seed surface, placing in culture dish with two layers of filter paper laid therein, culturing at 25deg.C under light/dark conditions of 12 h, and counting germination. The test was repeated 3 times. The live broadcast method specifically comprises the following steps: repeatedly selecting 20 healthy and full seeds each time, sterilizing with 75% alcohol solution for 1 min, washing with distilled water for 3 times, standing at 25deg.C for 2 days, and sowing in nutrient soil. The results showed that the mutation compared with the wild typeOsJAZ5The germination rate, germination index and the fourth seedling rate of the gene on the third day are obviously reduced, and the seedling growth vigor is obviously weakened (figure 1).
Example 5: phenotypic identification of Gene overexpression Material
By means of constructionOsJAZ5Overexpression of OE-2, OE-9, OE-11 and wild-type Medium flower 11 (W)T) rice variety, and carrying out germination test and field direct seeding. The germination test comprises the following specific steps: selecting 30 healthy and full seeds each time repeatedly, sterilizing with 75% alcohol solution for 1 min, washing with distilled water for 3 times, wiping the seed surface, placing in culture dish with two layers of filter paper laid therein, culturing at 25deg.C under light/dark conditions of 12 h, and counting germination. The test was repeated 3 times. The field direct seeding method comprises the following steps: selecting 60 healthy and full seeds each time repeatedly, sterilizing with 75% alcohol solution for 1 min, washing with distilled water for 3 times, standing at 25deg.C for 2 days, and sowing in field. The results showed that over-expression compared to the wild typeOsJAZ5The germination rate, germination index and seedling growth vigor of the gene are obviously improved in the next day (figure 2). The gene has important function of improving seed vitality.
Claims (1)
1. Over-expressed riceOsJAZ5The gene is applied to improving the activity of rice seeds and promoting the production of direct-seeding rice, and is characterized in that the CDS sequence of the gene is shown as SEQ ID NO. 1.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114763375A (en) * | 2020-12-31 | 2022-07-19 | 中国科学院分子植物科学卓越创新中心 | Gene for regulating and controlling quality of rice grains and application thereof |
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| WO2017197322A1 (en) * | 2016-05-12 | 2017-11-16 | University Of Florida Research Foundation, Inc. | Drought tolerant plants |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114763375A (en) * | 2020-12-31 | 2022-07-19 | 中国科学院分子植物科学卓越创新中心 | Gene for regulating and controlling quality of rice grains and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| Rice Full-Length cDNA Consortium et al..Collection, mapping, and annotation of over 28,000 cDNA clones from japonica rice.Science.2003,第301卷(第5631期),第376-379页. * |
| 水稻抗旱机理和抗旱育种研究进展;杨瑰丽 等;中国农学通报;第28卷(第21期);第1-6页 * |
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