论文部分内容阅读
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pUC57-hA4-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△BtR-175.4龄起蚕经口添食黑胸败血芽孢杆菌,结果△BtR-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△BtR-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△BtR-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 moii 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 pUC57-hA4-Cas9 and T-U6-BtR-175 into silkworm eggs,a BtR-175 knockout mutant named △BtR-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 △BtR-175 mutant and wild-type silkworm were 34.4％+5.8％ and 57.8％±8.4％,respectively,suggesting that △BtR-175 significantly improved the disease resistance against B.bombysepticus.An investigation of economic traits showed no significant difference in cocoon weight between △BtR-175 and wild-type silkworm.Therefore,this mutant could be further developed into breeding material with resistance against bacterial infection.