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TIFY1 a(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T0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.%Low temperature is one of the major environmental factors that influence rice (Oryza sativa) growth,development and production.Rice seedlings are particularly sensitive to chilling in early spring.Therefore,improvemem of chilling tolerance in rice may significantly increase rice quality and production.To identify genes participated in the regulation of plant chilling tolerance,the cold-resistant gene library was constructed through suppression subtractive hybridization (SSH) in a previous study.From the SSH library,a transcription factor TIFYlb (LOC_Os03g52450) with an conserved TIF[F/Y]XG domain,which might involve in the rice cold resistance,was isolated.To explore the function of TIFY1b and its homology gene TIFY1a (LOC_Os03g47970),the technology of "clustered regularly interspaced short palindromic repeats/CRISPRassociated protein 9 (CRISPR/Cas9)" was used to edit their genome.According to the characteristics of CRISPR/Cas9 editing system and the conservation of target genes (TIFY1a and TIFY1b),2 sites of 20 nt guide RNA (gRNA) targeted to the exon of TIFY1a were designed and named A1 and A2;2 sites of 20 nt gRNA targeted to the exon of TIFY1b were designed and named B1 and B2.A1,A2,B1 and B2 were all ligated and constructed individually to the CRISPR/Cas9 vector with single target site to create tify1a or tify1b single mutant;A1 and B1 were together ligated and constructed to another CRISPR/Cas9 vector which harbors 2 target sites to create tify1a & tify1b double mutant.Then,these recombinant plasmids were all transferred to a rice cultivar Nipponbare by Agrobacterium-mediated transformation method.T0 transgenic mutants were obtained and confirmed by hygromycin-resistance screening.Further sequencing for the genomic DNA of TIFY1a or TIFY1b gene locus in T0 transgenic lines showed 60％ mutagenesis frequency in target A1 site,87.5％ mutagenesis frequency in target A2 site,35％ mutagenesis frequency in target B1 site,75％ mutagenesis frequency in target B2 site,and 62.5％ mutant ratio of lines containing both target A1 and target B1.These results suggested that CRISPR/Cas9 systems can effectively induce site-specific mutations in T0 rice plants.Moreover,the results found various targets of mutant types presented in the T0 transgenic mutants,such as deletion of bases,insertion of bases,insertion behinds deletion of bases,and long fragment deletion of genomic DNA.The major mutation type in this study was 1 bp insertion and 1 bp deletion mutation.In addition,The results found that the positions of the mutation could occur in the upstream of the proto spacer adjacent motif (PAM) region ranging 4～7 bp,and the mutation occurred in the 4 bp upstream of the PAM region was more often.Consequently,3 different mutation types mediated by the CRISPR/Cas9 system were formed in T0 transgenic lines,including homozygous mutation,biallelic mutation and heterozygous mutation.All these mutation types could descend stably into the next generation.Furthermore,protein analysis indicated that frameshift or premature of proteins which caused function loss of TIFY1a or TIFY1b gene happened in T0 transgenic mutants.Collectively,a series of different types of tify1 mutant lines were successfully obtained by using CRISPR/Cas9 technology in this study and could be used to investigate the role of TIFY1 genes in rice adaptation to chilling temperature.Our studies might reveal a novel pathway that controls cold adaptation in rice and will help to broaden the possibilities for genetically engineering cold-tolerant rice cultivars.
展开▼
