基于热处理CRISPR/Cas9马铃薯植株的基因编辑体系优化  

Optimization of Gene Editing System Based on Heat-treated CRISPR/Cas9 Potato Plants

作　　者：熊文汶 刘慧敏[1] 陈凯园 杜鹃 宋波涛[1] 景晟林 XIONG Wenwen;LIU Huimin;CHEN Kaiyuan;DU Juan;SONG Botao;JING Shenglin(Key Laboratory of Horticultural Plant Biology Ministry of Education,Key Laboratory of Potato Biology and Biotechnology,Ministry of Agriculture and Rural Affairs,School of Horticulture and Forestry,Huazhong Agricultural University,Wuhan 430070,China;National Key Laboratory of Crop Genetic Improvement,College of Plant Science&Technology of Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[1]华中农业大学园艺林学学院,园艺植物生物学教育部重点试验室,农业农村部马铃薯生物学与生物技术重点试验室,湖北武汉430070 [2]华中农业大学植物科学技术学院,作物遗传改良全国重点实验室,湖北武汉430070

出　　处：《园艺学报》2023年第12期2591-2600,共10页Acta Horticulturae Sinica

基　　金：国家自然科学基金面上项目(31871683);现代农业产业技术体系建设专项(CARS-09-P07)。

摘　　要：以二倍体马铃薯栽培种AC142和四倍体栽培种E3为试验材料,构建并优化了马铃薯内源启动子驱动的CRISPR/Cas9基因编辑体系。以控制白化表型的八氢番茄红素脱氢酶基因(StPDS)为靶标基因,通过农杆菌介导的遗传转化侵染试管薯,根据转化效率和表型突变频率的分析,筛选出马铃薯内源启动子StU6和StUBI10驱动的HM4载体为最优CRISPR载体。以常温处理为对照,对HM4获得的AC142的37个全绿株系和白绿嵌合株系、E3的4个全绿株系的侧芽进行循环8次的短期热处理(37℃24h+22℃24h)共15d,表型分析和测序检测表明,对敲除株系进行短期热处理能显著提高CRISPR/Cas9的编辑效率。常温下,AC142和E3敲除株系的表型突变频率分别为16.2%(6/37)和0(0/4),循环8次短期热处理之后,表型突变频率分别提高到54.1%(20/37)和75%(3/4)。其中,AC142敲除株系的侧芽能得到10.8%(4/37)白化株系,而E3未能得到白化株系,只能得到75%(3/4)的白绿嵌合株系。4次连续15 d的短期热处理后,2.1%(3/144)的E3敲除株系的侧芽可在常温下保持白化。对短期热处理后的白化植株进行TA克隆,测序结果显示二倍体敲除株系CR-AC142-StPDS-17/21和四倍体敲除株系CR-E3-StPDS-3/4的8~12个单克隆都不存在野生型,说明本研究的敲除体系可在马铃薯中实现对靶标序列的完全诱导突变。该体系可广泛应用于马铃薯突变体的创制,为马铃薯基因功能组学研究和目标性状精准改良提供技术基础。The development of endogenous promoters driven CRISPR/Cas9 gene editing system in diploid potato cultivar AC142 and tetraploid cultivar E3 were reported.The StPDS gene was targeted by Agrobacterium-mediated potato microtuber transformation.Through the analysis of transformation efficiency and phenotypic mutation rate,HM4 driven by potato endogenous promoter StU6 and StUBI10 was selected as the optimal CRISPR vector.The short-term cycle of eight heat treatment(37℃24 h,22℃24 h)was performed on the lateral buds of 37 AC142 all-green lines and chimeric lines and four E3 all-green lines obtained from HM4 for 15 days.Phenotypic analysis and sequencing showed that short-term heat treatment of knockout lines could significantly improve the editing efficiency of CRISPR/Cas9.At room temperature,the phenotypic mutation rates of AC142 and E3 knockout lines were 16.2%(6/37)and 0(0/4),respectively.After one short-term heat treatment,the phenotypic mutation rates were increased to 54.1%(20/37)and 75%(3/4),respectively.Among them,AC142 knockout lines can obtain 10.8%(4/37)lines that maintain the albino phenotype,while E3 can only obtain 75%(3/4)chimeras with white spots.Four consecutive short-term heat treatments could result in 2.1%(3/144)albino mutants at room temperature in E3 knockout lines.TA cloning was performed on the albino lines after short-term heat treatment.The sequencing results showed that 8-12 single clones of the diploid knockout line CR-AC142-StPDS-17,21 and the tetraploid knockout line CR-E3-StPDS-3,four did not contain wild type,indicating that the knockout system in this study could induce mutations in the target sequence.The results provide a technical basis for functional genomics research and precise improvement of target traits in potato.

关 键 词：马铃薯 基因编辑 CRISPR/Cas9 StPDS 热处理 

分 类 号：S532[农业科学—作物学]