家蚕类钙粘蛋白BtR-175基因敲除突变体创制和抗性检测  

Creation of Bombyx mori Cadherin BtR-175 Knockout Mutant and Investigation of Its Ｒesistance Against Bacterial Infection

作　　者：程廷才[1] 林平[1] 付剑锋[1] 蒋亮[1] 马三垣[1] 夏庆友[1] 

机构地区：[1]家蚕基因组生物学国家重点实验室西南大学,重庆400715

出　　处：《蚕业科学》2016年第5期814-819,共6页ACTA SERICOLOGICA SINICA

基　　金：国家重点基础研究计划"973"项目(No.2012CB114600);重庆市自然科学基金项目(No.CSTC2014JCYJA80004)

摘　　要：家蚕类钙粘蛋白BtR-175不仅是苏云金芽孢杆菌(Bacillus thuringiensis)晶体毒素蛋白的受体蛋白,也是黑胸败血芽孢杆菌(Bacillus bombysepticus)晶体毒素蛋白的受体分子。为建立对黑胸败血芽孢杆菌具有抵抗能力的家蚕品系,采用CRISPR/Cas9介导的基因组编辑技术,设计引导RNA(gRNA)对家蚕BtR-175基因进行精确定点编辑,对蚕卵显微注射重组质粒pU C57-hA 4-Cas9和T-U6-BtR-175后,筛选获得2个碱基插入导致蛋白质翻译提前终止的BtR-175基因敲除突变体,命名为ΔBtR-175。4龄起蚕经口添食黑胸败血芽孢杆菌,结果ΔBtR-175突变体家蚕的死亡率为34.4%±5.8%,野生型家蚕的死亡率为57.8%±8.4%,表明ΔBtR-175对黑胸败血芽孢杆菌的侵染抵抗性显著提高。饲养试验显示ΔBtR-175突变体和野生型家蚕的茧层量没有显著性差异。该突变体有望进一步培育为家蚕抗性育种素材。Bombyx mori cadherin-like protein（ BtR-175） functions as not only a receptor protein of Bacillus thuringiensis toxin,but also a receptor of Bacillus bombysepticus crystal toxin. To enhance disease resistance of the silkworm to B.bombysepticus,CRISPR/ cas9 technology was used to accurately edit BtR-175 by designing gRNAs. By micro-injecting p UC57-hA 4-Cas9 and T-U6-BtR-175 into silkworm eggs,a BtR-175 knockout mutant named ΔBtR-175 was obtained with two bases inserted which led to premature termination of the target protein. In the 4th instar,by feeding B. bombysepticus,the mortality of ΔBtR-175 mutant and wild-type silkworm were 34. 4% ± 5. 8% and 57. 8% ± 8. 4%,respectively,suggesting that ΔBtR-175 significantly improved the disease resistance against B. bombysepticus. An investigation of economic traits showed no significant difference in cocoon weight between ΔBtR-175 and wild-type silkworm. Therefore,this mutant could be further developed into breeding material with resistance against bacterial infection.

关 键 词：类钙粘蛋白 基因编辑 黑胸败血芽孢杆菌 抗性 家蚕 

分 类 号：S882[农业科学—特种经济动物饲养]