阅读说明：本技术 水稻生长素糖基转移酶基因的应用 (Application of oryza sativa auxin glycosyl transferase gene ) 是由 王州飞 何永奇 赵佳 于 2019-10-09 设计创作，主要内容包括：本发明公开了生长素糖基转移酶基因的应用。所述的基因OsIAGLU核苷酸序列如SEQ ID NO.1所示,以及编码相应的蛋白氨基酸序列如序列表SEQ ID NO.2所示。本发明在水稻中首次报道了OsIAGLU基因能调控水稻种子活力,通过试验表明突变该基因影响正常条件种子萌发及幼苗建成能力。证明本发明OsIAGLU基因调控了水稻种子活力,利用该基因有助于筛选和培育高种子活力水稻品种,有利于直播稻生产。(The invention discloses an application of an auxin glycosyl transferase gene. The nucleotide sequence of the gene OsIAGLU is shown as SEQ ID NO.1, and the amino acid sequence of the coded corresponding protein is shown as the sequence table SEQ ID NO. 2. The OsIAGLU gene is reported to regulate the activity of rice seeds for the first time in rice, and experiments show that the mutation of the gene influences the seed germination and seedling establishment capability under normal conditions. The OsIAGLU gene of the invention is proved to regulate and control the rice seed vigor, and the gene is beneficial to screening and cultivating rice varieties with high seed vigor and is beneficial to direct seeding rice production.)

1. Application of oryza sativa auxin glycosyltransferase gene OsIAGLU in screening or cultivating paddy rice varieties with high seed vigor.

2. The use of the oryza sativa auxin glycosyltransferase gene OsIAGLU of claim 1 in genetic engineering for improving germination viability of rice seeds and promoting growth of seedlings.

Technical Field

The invention belongs to the technical field of seed biology, and relates to an application of a rice auxin glycosyl transferase gene.

Background

Rice (Oryza sativa L.) is one of the longest food crops in the cultivation history in China. In recent years, with the development of economy, rural labor is increasingly in short supply, and direct-seeded rice production becomes more and more common. The direct seeding rice production has high requirements on seed sowing quality, and poor seed quality can cause slow seed germination speed, low field seedling rate, uneven seedling growth vigor and even seriously affect crop yield. Auxin is an important factor for regulating and controlling the growth and development of plants, few reports are made on the research on regulating and controlling the germination of rice seeds and the growth of seedlings by using auxin glycosyltransferase genes, and no report is made on the application of screening and cultivating high-activity rice varieties by using the auxin glycosyltransferase genes. Therefore, the auxin glycosyl transferase gene OsIAGLU participating in the regulation and control of rice seed germination and seedling growth is utilized to provide help for screening and cultivating high-activity rice varieties, and has important significance for direct-seeded rice production.

Disclosure of Invention

The invention aims to provide separation, cloning, functional verification and application of an auxin glycosyltransferase gene OsIAGLU for controlling rice seed germination and seedling growth.

The purpose of the invention can be realized by the following technical scheme:

a gene OsIAGLU from the oryza sativa auxin glycosyltransferase, the nucleotide sequence of which is shown in SEQ ID NO. 1.

The amino acid sequence of the protein coded by the oryza sativa auxin glycosyltransferase gene OsIAGLU is shown in SEQ ID NO. 2.

The rice OsIAGLU gene or protein disclosed by the invention is applied to screening or breeding of rice varieties with high seed vigor.

The rice OsIAGLU gene mutant obtaining and gene function verification method comprises the following steps:

(1) obtaining the nucleotide sequence and the amino acid sequence of the OsIAGLU gene of the rice;

(2) designing a target site and a primer thereof, carrying out PCR amplification by taking pCBC-MT1T2 as a template, purifying and recovering a PCR product, and obtaining an MT1T2-PCR vector;

(3) constructing the MT1T2-PCR glue recovery product with the target fragment of the OsIAGLU gene obtained in the step (2) onto a pHUE411 vector to obtain a pHUE411+ MT1T2-PCR vector;

(4) transforming the plasmid with the OsIAGLU gene target fragment obtained in the step (3) into agrobacterium; transforming the agrobacterium with the transformation plasmid into rice;

(5) screening and identifying rice mutants, and identifying seed vitality under normal conditions.

Further, in the step (1), a primer sequence of the gene is cloned by using rice cDNA as a template through PCR, wherein an upstream primer sequence of the PCR is shown as SEQ ID NO.3, and a downstream primer sequence is shown as SEQ ID NO. 4.

Further, the rice OsIAGLUCRISPR/Cas9 mutant gRNA target sequence (target fragment of 19bp in OsIAGLU gene) constructed in the step (2) is shown as a sequence table SEQ ID NO.5 and SEQ ID NO. 6; the primer sequence for PCR amplification by taking pCBC-MT1T2 as a template is shown in a sequence table SEQ ID NO.7/SEQ ID NO.8 and SEQ ID NO.9/SEQ ID NO. 10;

in step (3), the pHUE411 vector is digested with BsaI, and the pHUE411+ MT1T2-PCR vector is obtained by the homologous recombination method.

In the step (5), the upstream primer sequence used for screening the homozygous mutant is shown as SEQ ID NO.11, and the downstream primer sequence is shown as SEQ ID NO. 12. The rice seed vitality identification comprises seed germination and seedling growth under normal conditions.

The method for detecting the OsIAGLU gene expression level in the seed germination stage is characterized by comprising the following steps of:

(1) taking rice seeds in different germination stages;

(2) RNA was extracted from each sample using a TransZol Plant kit (Transgen, www.transgen.com);

(3) by usingII Reverse transcription of cDNA formed by Vazyme Biotech Co., Ltd kit, using it as template;

(4) and (3) analyzing by using fluorescent quantitative PCR, and detecting a primer sequence by using the fluorescent quantitative PCR, wherein the upstream primer sequence is shown as SEQ ID NO.13 of the sequence table, and the downstream primer sequence is shown as SEQ ID NO.14 of the sequence table.

Further, in the step (1), the rice seeds are cultured in a 10mL distilled water culture dish at 25 ℃, and are sampled after 0, 4, 12, 24, 36, 48, 60 and 72 hours of imbibition; the seeds were rapidly ground into powder after freezing treatment with liquid nitrogen and the samples were stored at-80 ℃ and sampled in triplicate at each time point.

In the step (4), a rice reference gene OsActin primer is adopted, the sequence of the upstream primer is shown as a sequence table SEQ ID NO.15, and the sequence of the downstream primer is shown as a sequence table SEQ ID NO. 16.

Has the advantages that: the OsIAGLU gene is separated and cloned from rice, and the function of the gene in the aspect of seed vigor regulation is identified, so that the OsIAGLU gene has important significance for screening or breeding high-vigor rice varieties.

The invention has the following advantages:

(1) the OsIAGLU gene is obtained by separating and cloning from rice, and the gene is proved to participate in the regulation and control of rice seed vigor for the first time by constructing a CRISPR/Cas9 mutant.

(2) The invention provides a foundation for screening rice varieties with high seed vigor, provides important gene resources for improving the rice seed vigor, and has important significance for direct seeding rice production.

Drawings

FIG. 1: seed vigor expression of rice OsIAGLU mutant under normal conditions

FIG. 2: expression condition of rice OsIAGLU gene in seed germination process

Detailed Description

The invention is further explained by combining the attached drawings and specific embodiments, the methods used in the embodiments are all conventional methods without special description, and the primers and sequencing are completed by Guangzhou Tianyihui Gene technology, Inc.; various restriction enzymes, ligase, DNA Marker, Tag DNA polymerase, dNTPs and the like used in the experiment are purchased from Guangzhou Shuicheng Biotech limited; the reverse transcription kit is purchased from Novozan Biotechnology Co., Ltd; the plasmid extraction kit, the gel recovery kit and the genome extraction kit are purchased from Meiji Biotechnology Ltd, and the methods are carried out according to the specifications.