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低温寒害严重影响水稻(Oryza sativa)的生长、发育和产量。为了更好的解析水稻响应低温的分子机制,改良水稻对低温的耐受能力。本实验室前期构建了贵州特有的抗寒粳稻“告公鬼”的低温胁迫响应抑制差减文库,从文库中筛选克隆了一个冷诱导表达的基因LOC_Os03g52450。该基因含有保守的TIF[F/Y]XG氨基酸排列形式,编码水稻TIFY转录因子家族中的TIFY1b。本实验利用成簇规律间隔的短回文重复序列及其相关蛋白(clustered regularly interspaced short palindromic repeats/CRISPR-associated 9,CRISPR/Cas9)基因组编辑系统,分别构建TIFY1b及其同源基因TIFY1a(LOC_Os03g47970)的单基因以及双基因敲除载体。通过农杆菌(Agobacterium tumefaciens)介导的愈伤转化法侵染水稻品种日本晴(Nipponbare),潮霉素抗性筛选之后获得T_0代转基因植株。对转基因植株的靶位点进行测序分析发现有纯合,杂合和双等位等多种突变类型。进一步分析氨基酸序列发现突变株系的蛋白质翻译均存在着不同程度的变异。本研究成功实现了水稻转录因子TIFY1家族2个基因,TIFY1a和TIFY1b的单基因和双基因编辑,获得了一系列不同类型的tify1a和tify1b的单基因突变株系以及双基因突变株系,为进一步揭示TIFY1响应水稻低温应答的作用机制提供材料和理论依据,并为利用CRISPR/Cas9技术研究功能冗余的基因家族提供了新思路。
Chilling injury seriously affected the growth, development and yield of Oryza sativa. In order to better understand the molecular mechanism of rice response to low temperature, improve the tolerance of rice to low temperature. In our laboratory, a cryogenic stress response suppression subtractive library was constructed for Guizhou-specific japonica-resistant japonica rice in the early stage and a cold-inducible gene, LOC_Os03g52450, was screened and cloned from the library. This gene contains a conserved TIF [F / Y] XG amino acid arrangement that encodes TIFY1b in the rice TIFY transcription factor family. In this study, TIFY1b and its homologue TIFY1a (LOC_Os03g47970) were constructed by using the clustered regularly interspaced short palindromic repeats / CRISPR-associated 9 (CRISPR / Cas9) genome editing system. Of single gene and double gene knockout vectors. The rice variety Nipponbare was infected by Agrobacterium tumefaciens-mediated callus transformation and T_0 transgenic plants were obtained after hygromycin resistance screening. Sequencing and analysis of the target sites of the transgenic plants revealed that there are many types of mutations, such as homozygous, heterozygous and biallelic. Further analysis of amino acid sequences revealed that there were some variations in the protein translation of the mutant lines. In this study, single and double genes of TIFY1, TIFY1a and TIFY1b were successfully edited. A series of single-and double-gene mutant strains of tify1a and tify1b were obtained. It is revealed that TIFY1 can provide material and theoretical basis for its mechanism of response to low-temperature response in rice and provide a new idea for studying the gene family of functionally redundant genes by using CRISPR / Cas9 technology.