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从国内分离的对鞘翅目叶甲科害虫高毒力的Bt菌株中克隆了 3.0kbcry3Aa基因大片段 ,完成了该片段的亚克隆和全序列测定。该基因编码区为 1932bps,编码的蛋白质由 6 4 4个氨基酸残基组成 ,分子量 73.1ku ,等电点为 pH5 .16 5 ,为弱酸性蛋白。该蛋白中Ser、Leu、Thr含量最高 ,分别为 8.2 2 %、8.0 7%和 7.76 %。通过穿梭载体将该基因导入Bt无晶体突变株中 ,获得工程菌Biot2 0 5。cry3Aa7基因在其中能正常表达 ,并形成扁方形晶体。工程菌Biot2 0 5对榆蓝叶甲 (Pyrrhaltaaenescens)校正死亡率为 86 .2 1%。该基因序列已在EMBL、GenBank中登记 ,并被国际Bt δ 内毒素基因命名委员会正式命名为cry3Aa7。
A large fragment of 3.0kbcry3Aa gene was cloned from Bt strains isolated from China and highly virulent to Coleoptera chinensis. The subcloning and full sequencing of this fragment were completed. The coding region of the gene was 1932bps, encoding a protein consisting of 644 amino acid residues with a molecular weight of 73.1ku and an isoelectric point of pH5.165, which is a weakly acidic protein. The contents of Ser, Leu and Thr in this protein were the highest, 8.22%, 8.07% and 7.76% respectively. The gene was introduced into Bt crystal-free mutant by shuttle vector to obtain engineering bacterium Biot2 05. The cry3Aa7 gene can be expressed normally and forms a flat square crystal. The corrected mortality of engineered bacteria Biot2 05 against Pyrrhaltaaenescens was 86.21%. The gene sequence has been registered in EMBL, GenBank, and was officially named cry3Aa7 by the international Bt δ endotoxin gene nomenclature committee.