烟草TILLING技术体系的建立及NtPhyB基因的筛选  

Establishment of TILLING in Tobacco and its Application to Screen the NtphyB Gene in Mutant Populations

作　　者：宗鹏[1,2] 李凤霞[1] 王倩[1] 刘贯山[1] 龚达平[1] 陈雅琼[1,2] 孙玉合[1] 

机构地区：[1]中国农业科学院烟草研究所,烟草行业基因资源利用重点实验室,青岛266101 [2]中国农业科学院研究生院,北京100081

出　　处：《中国烟草科学》2014年第2期32-36,共5页Chinese Tobacco Science

基　　金：中国烟草总公司烟草基因组计划重大专项项目“烟草突变体库创制、筛选与分析”[110201101009(JY-03)];“烟草突变体筛选与鉴定”[110201201004(JY-04)];“烟草突变体库创建与功能基因组学研究”(110200701022)

摘　　要：定向诱导基因组局部突变技术（Targeting Induced Local Lesions In Genomes，TILLING）是一种新近发展起来的高通量检测点突变的反向遗传学方法。本研究首次将TILLING技术用于EMS处理的烟草突变体筛选中，通过对正反向荧光引物使用量的摸索，确定了10μL PCR体系中IRDye-700标记的正向引物（1μM）适用量为0.32～0.64μL，IRDye-800标记的反向引物（1μM）适用量1.12～1.6μL；对异源双链CELⅠ酶切体系实验表明，10μL PCR产物中加入0.6μL CEL I酶，1.5μL 10× Buffer酶切效果最好。从而建立并优化了适用于普通烟草基因组研究的TILLING技术体系。在此基础上，以NtPhyB为目标基因，在1000份0.6% EMS诱变的M2突变体中进行筛选，共得到13个突变体，该基因突变频率约为1/94 kb。本试验建立的TILLING筛选体系能够快速、高效地筛选任意已知序列基因的突变体，对烟草功能基因组研究具有重要意义。Targeting Induced Local Lesions In Genomes （TILLING） is a newly developed high-throughput reverse genetic method used for detection of point mutations. We used TILLING technology to screen EMS treated tobacco mutant population. The concentration of forward and reverse fluorescent labeled primers was determined：the 10μL PCR mix consisted of 0.32~0.64μL （1μM） fluorescence-labeled forward primer and 1.12~1.6μL （1μM） fluorescence-labeled reverse primer. The 10μL PCR product was used as a substrate for the cellI digestion. The concentration of cellI enzyme and 10× buffer optimized to cut the heteroduplex was 0.6 μL and 1.5 μL respectively. This optimized and established TILLING system was applied to screen the NtPhyB gene in 1000 （0.6% EMS mutagenized） M2 mutant population. The screening procedure provided a total of 13 mutants. The gene mutation frequency of approximately 1/94 kb was obtained. TILLING screening system established in our study is faster and efficient for mutant genes of any known sequence and can play a vital role in tobacco functional genomics research in future.

关 键 词：功能基因组学 烟草 化学诱变 突变体 

分 类 号：S572[农业科学—烟草工业]