利用HRM技术快速筛选低镉烟草突变体  

Rapid screening of low-cadmium tobacco mutants by HRM technology

作　　者：张超 张吉顺 王仁刚 曾吉凡 王自力 任学良 舒庆尧[2] 武立权[3] 赵海军[2] ZHANG Chao;ZHANG Jishun;WANG Rengang;ZENG Jifan;WANG Zili;REN Xueliang;SHU Qingyao;WU Liquan;ZHAO Haijun(Molecular Genetics Key Laboratory of China Tobacco,Guizhou Academy of Tobacco Science,Guiyang 550081,China;College of Agriculture ＆ Biotechnology,Zhejiang University,Hangzhou 310058,China;School of Agronomy,Anhui Agricultural University,Hefei 230036,China)

机构地区：[1]烟草行业分子遗传重点实验室贵州省烟草科学研究院,贵州贵阳550081 [2]浙江大学农业与生物技术学院,浙江杭州310058 [3]安徽农业大学农学院,安徽合肥230036

出　　处：《中国烟草学报》2018年第3期37-45,共9页Acta Tabacaria Sinica

基　　金：烟草行业重点实验室项目:利用分子标记辅助筛选低镉和低降烟碱烟草种质研究(合同号:110201403019);中国烟草总公司贵州省公司科技项目<贵州烤烟种质创制及新品种选育研究>(中烟黔科[2016]6号)

摘　　要：低镉烟草种质资源对烟草育种及烟叶安全具有重要意义。本研究利用EMS处理贵烟1号获得诱变群体,按株系种植2020个M2株系,每株系种植10株,株系内3株叶片混样提取DNA,并采用HRM技术对样本池筛选烟草镉转运基因Nt HMA4突变体,共获得49份疑似突变材料。DNA测序鉴定发现,其中21个株系发生SNP突变,其中15个为错义突变,其余为同义突变。对突变体与和野生型材料叶片的镉含量分析发现,有8份突变体的镉含量较野生型显著下降(16.19%~42.08%)。利用HRM技术跟踪检测10份种子可正常萌发的错义突变体M3家系,结果表明,HRM技术不仅可快速筛选烟草EMS诱变群体中的突变材料,结合DNA测序也可进一步对突变杂合体后代进行快速、准确的基因分型。Low-cadmium tobacco germplasm resources are essential for tobacco breeding and production safety. In this study, we treated Guiyan No.1 with EMS, and planted 2020 M2 lines including 10 individuals, then extracted bulk DNA from leaves of three individuals. 49 materials with putative mutation in Nt HMA4 were screened using high-resolution melting（HRM） curve analysis. Among them, 21 mutants with SNP mutation were confirmed by DNA sequencing, of which 15 were non-synonymous mutations caused amino acid changed and the rest were synonymous mutations. Subsequently, cadmium content in the leaves of eight mutants decreased significantly from 16.19% to 42.08% compared to wild type. Ten M3 progeny with non-synonymous mutations, which can germinate normally, were detected by HRM. Results showed that HRM technology could not only quickly screen mutants in tobacco EMS mutagenesis, but could also be used for rapid and accurate genotyping of heterozygous mutant offspring combined with DNA sequencing.

关 键 词：镉转运基因 诱变 烟草 低镉 HRM 

分 类 号：S572.03[农业科学—烟草工业]