通过CRISPR/Cas9技术靶向编辑SSSⅡb基因改良稻米品质  

Targeted editing of SSSⅡb gene through CRISPR/Cas9 technology to improve rice quality

作　　者：蔡梦颖 张平 宋炜涵 余江峰 郝栖贤 王平 刘世家[1] 王益华[1] 江玲[1] 万建民 CAI Mengying;ZHANG Ping;SONG Weihan;YU Jiangfeng;HAO Qixian;WANG Ping;LIU Shijia;WANG Yihua;JIANG Ling;WAN Jianmin(State Key Laboratory of Corp Genetics and Germplasm Enhancement/Research Center of Jiangsu Plant Gene Engineering,Nanjing Agricultural University,Nanjing 210095,China;Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]南京农业大学作物遗传与种质创新国家重点实验室/江苏省植物基因工程技术研究中心,江苏南京210095 [2]中国农业科学院作物科学研究所,北京100081

出　　处：《南京农业大学学报》2021年第1期18-26,共9页Journal of Nanjing Agricultural University

基　　金：国家转基因生物新品种培育重大专项(2016ZX08001006)。

摘　　要：[目的]本文旨在利用基因编辑技术,降低水稻直链淀粉含量,改善稻米食味品质特性,以培育直链淀粉含量降低的优质育种材料。[方法]利用CRISPR/Cas9基因编辑技术,下调水稻品种‘宁粳4号’淀粉合成基因(SSSⅡb)的表达,对转基因家系进行目标基因的分子鉴定和主要农艺性状、生理生化指标的测定,选出目标育种材料。[结果]对淀粉合酶基因SSSⅡb进行多靶点编辑,得到3个目标基因敲除的转基因家系,分别为SSS2-Q、SSS2-Z和SSS2-H家系。与‘宁粳4号’相比,SSS2-Q家系直链淀粉含量显著下降,支链淀粉的中等链长减少,胶稠度、热浆黏度和冷浆黏度等指标均优于‘宁粳4号’,品质指标达到预期目标;但每穗实粒数和结实率降低,粒长增加。而在SSS2-Z与SSS2-H家系中,品质指标没有实现预期目标。RT-qPCR分析发现,SSS2-Q家系中SSSⅡb基因表达下调,其他淀粉合成相关基因表达也发生变化。[结论]采用CRISPR/Cas9技术,靶向编辑‘宁粳4号’中SSSⅡb基因,降低SSS2-Q家系直链淀粉含量,品质指标得到改善,为优质食味转基因水稻新品种培育提供新种质。[Objectives]This article aims to improve rice eating and cooking quality characteristics and use the gene editing technology to breed rice with reduced amylose content,so as to further obtain high quality breeding materials.[Methods]CRISPR/Cas9 knockout technology is used to down-regulate the expression of the rice variety‘Ningjing 4’starch synthase gene(SSSⅡb).Molecular identification and physiological and biochemical characteristics of the transgenic offspring have been carried out,and we complete the investigation of agronomic traits.[Results]By editing multiple target sites of the starch synthase gene(SSSⅡb),three knockout lines named SSS2-Q,SSS2-Z and SSS2-H were obtained.The amylose content was significantly lower than that of‘Ningjing 4’in SSS2-Q lines with the expected breeding goals of this study.Distribution of branch medium chain lengths of the SSS2-Q lines compared to‘Ningjing 4’reduced.The gel consistency,hot paste viscosity and cool paste viscosity of the SSS2-Q lines were superior to‘Ningjing 4’.In addition,the filled grain of per panicle and the seed setting rate of SSS2-Q decreased,and the grain length increased.SSS2-Z and SSS2-H lines didn’t achieve the expected goals.RT-qPCR analysis revealed that in the SSS2-Q knockout lines the expression of SSSⅡb gene down-regulated and other starch synthesis-related genes showed varying degrees of expression.[Conclusions]The CRISPR/Cas9 knockout technology is used to knockdown SSSⅡb gene in‘Ningjing 4’with reduced amylose content and improve quality index.The study provides material basis for high-quality edible transgenic rice.

关 键 词：水稻 直链淀粉 CRISPR/Cas9基因编辑技术 淀粉合酶 

分 类 号：S511.032[农业科学—作物学]