利用CRISPR/Cas9基因组编辑技术定向降低水稻落粒性  

Using CRISPR/Cas9-Mediated Targeted Mutagenesis of qSH1 Reduces the Seed Shattering in Rice

作　　者：盛夏冰[1,2] 谭炎宁[2] 孙志忠 余东[1,2] 汪雪峰 袁贵龙[2] 袁定阳 段美娟[1] SHENG XiaBing;TAN YanNing;SUN ZhiZhong;YU Dong;WANG XueFeng;YUAN GuiLong;YUAN DingYang;DUAN MeiJuan(College of Bioscience and Biotechnology,Hunan Agricultural University,Changsha 410128;State Key Laboratory of Hybrid Rice,Hunan Hybrid Rice Research Centre,Changsha 410125)

机构地区：[1]湖南农业大学生物科学技术学院,长沙410128 [2]湖南杂交水稻研究中心杂交水稻国家重点实验室,长沙410125

出　　处：《中国农业科学》2018年第14期2631-2641,共11页Scientia Agricultura Sinica

基　　金：国家自然科学基金面上项目(31771767);抗虫转基因水稻新品种培育(2016ZX08001-001);转基因生物新品种培育重大专项(2014ZX0801001B-002);香港中文大学合作项目培育超高产杂交稻组合(TK1711793);湖南省自然科学基金青年基金(2017JJ3166)

摘　　要：【目的】易落粒既不利于稻谷收获,也不适宜于水稻机械化生产。利用现阶段前沿的分子育种手段——CRISPR/Cas9技术对水稻落粒性主效基因qSH1进行定点编辑,并调查分析易落粒性状的改良效果,为创制稳产和适合机械化生产的水稻新种质奠定材料基础和探索新途径。【方法】以qSH1为靶标基因,根据CRISPR/Cas9技术原理设计靶标位点。将所设计的靶点序列在水稻参考基因组中比对分析以排除非特异性靶位点,最终筛选出qSH1-T1和qSH1-T5靶标位点。化学合成靶位点寡核苷酸序列,退火后分别与pYLg RNA-U3、pYLg RNA-U6a载体连接构建U3-qSH1T5-g RNA、U6a-qSH1T1-g RNA表达盒,最后将2个g RNA表达盒同时连接至pYLCRISPR/Cas9表达载体中,构建pYLCRISPR/Cas9-qSH1-T51表达载体。利用农杆菌介导转化易落粒的籼稻品种HR1128,以潮霉素抗性为筛选标记筛选获得T0代转基因阳性植株。利用靶位点扩增测序法判断T0代转基因植株在预期靶标位点是否发生突变,并进一步分析突变类型及基因型。将靶点序列在水稻参考基因组中比对,选择与靶点序列匹配度大于或等于15 bp且3′端具有NGG的位点作为潜在脱靶位点进行脱靶效应评估。利用潮霉素基因及靶点检测进一步筛选无T-DNA成分的qsh1突变植株并进一步构建qsh1突变系,并分析qsh1突变系的落粒性、qSH1的表达量及预测编码氨基酸序列。【结果】pYLCRISPR/Cas9-qSH1-T51载体成功地实现了对qSH1靶标位点的定点编辑。在T0代转基因阳性植株中获得7个突变单株,其中qSH1-T1和qSH1-T5靶点的突变频率分别为54.55%和63.64%,突变基因型包括纯合突变、杂合突变、双等位突变和嵌合突变,突变类型包括碱基插入、碱基缺失及碱基突变。通过对T1代植株进行潮霉素基因筛选、靶位点扩增测序,结果表明,在T1代植株中Cas9载体骨架和qSH1突变位点都发生了分离,获得2种不含T-DNA成分的qsh1纯�【Objective】Seed shattering is not good for seeds harvest,nor suitable for mechanized production of rice. In this study, using the latest molecular breeding method-CRISPR/Cas9 system to site-directed edit of the rice seed shattering gene qSH1 and evaluated the improvement effect, in order to lay materials foundation and explore new method for creating stable-yielding rice germplasm suitable for mechanized seed production. 【Method】The sg RNA was designed based on the principle of CRISPR/Cas9 technology. After excluding non-specific target sites by analysis of sequence alignment in the rice genome database, the target sites of qSH1-T1 and qSH1-T5 were selected. The oligonucleotides of sg RNA were chemically synthesized, and then inserted into plasmid pYLg RNA-U3, pYLg RNA-U6 a for constructing the U3-qSH1 T5-g RNA and U6 a-qSH1 T1-g RNA expression-boxes respectively. And the pYLCRISPR/cas9-qsh1-t51 expression vector was constructed by linking expression-boxes to plasmid pYLCRISPR/Cas9. The vector of pYLCRISPR/cas9-qsh1-t51 was introduced into the callus induced from mature seeds of an indica rice variety HR1128 by the Agrobacterium-mediated transformation. At T0 generation, the positive transgenic plants were selected by hygromycin-resistant, and the target site for each plant were detected via a test of PCR combined Sanger-sequencing for confirming whether it was mutated. Besides blasting sg RNA sequence with rice genome in NCBI（https：//blast.ncbi.nlm.nih.gov）, Gramene（http：//ensembl.gramene.org）, highly identical sites with more 15 matching bases and NGG site at 3′region were selected to assess off-target efficiency and specificity of sg RNA. By using hpt gene and target site PCR amplification, mark-free qsh1 mutants were selected and qsh1 mutation lines were constructed by qsh1 mutants self-cross, and the level of rice seed shattering, the expression level of qSH1 and amino acid sequence of gene production of qsh1 lines were analyzed. 【 Result 】 The pYLCRISPR/cas9-qsh1-t51 expression vec

关 键 词：CRISPR/Cas9 基因编辑 水稻 qSH1 落粒性 

分 类 号：Q943.2[生物学—植物学] S511[农业科学—作物学]