TILLING技术应用于家蚕化学诱导突变检测的体系优化  

The Optimization of TILLING Reaction System in Silkworm,Bombyx mori

作　　者：林英[1] 陈冬妹[1] 杨瑜[1] 王艳霞[1] 杨从文[1] 夏庆友[1] 

机构地区：[1]西南大学生物技术学院蚕学与系统生物学研究所,重庆400716

出　　处：《蚕学通讯》2009年第4期1-6,共6页Newsletter of Sericultural Science

基　　金：重庆市自然科学基金(2008BB1246);长江学者和创新团队发展计划(IRT0750);973计划(2005CB121000)

摘　　要：TILLING(Targeting Induced Local Lesions In Genomes)即定向诱导基因组局部突变技术,是近年发展起来的一种高通量检测点突变的反向遗传学方法。本研究从PCR模板量及酶切体系中CELI的酶量两个方面对家蚕TILLING技术的检测体系进行了优化。结果表明:本实验在应用TILLING技术筛选家蚕突变体过程中,用于PCR扩增的家蚕基因组DNA模板最佳用量为20ng;CELI酶切的20μL反应体系中,加入的最适酶量为0.5μL(本实验室从芹菜中自提的CELI酶10倍稀释液)。本研究初步对TILLING技术应用于家蚕化学诱导突变检测体系进行了优化,为TILLING技术在家蚕功能基因组研究中的应用奠定了基础。Targeting induced local lesions in genomes （TILLING） is a recently developed reverse genetics strategy for high throughput identification of point mutations In the present study, the reaction system of TILLING in silkworm was optimized, involving the concentration of genomic DNA for PCR and the amount of CELI enzyme for digestion reaction. The result showed that the optimal amount of genomic DNA for PCR reaction was 20ng and the optimum amount of CEL I, which was diluted in ten--fold solution, was 0. 5μL for every 20 μL digestion reaction system. The optimization of TILLING application for detecting chemical-- induced mutant system in this study should provide convenience with silkworm functional research by TILLING technology.

关 键 词：家蚕 TILLING 优化 功能基因组 点突变 

分 类 号：S882[农业科学—特种经济动物饲养]