苏云金芽孢杆菌cry1Ia基因的克隆、表达与活性研究

根据cry1Ia类基因的全长序列设计引物，以苏云金芽孢杆菌（Bacillus thuringiensis）菌Btc008的总DNA为模板扩增出片段长为2.1kb的cry1Ia的全长基因，插入大肠杆菌（Escherichia coli）表达载体pET-21b，转化大肠杆菌BL21（DE3）菌株，诱导表达出81kD的蛋白。该蛋白由719个氨基酸组成，推导的分子量为81.2kDa。该蛋白的氨基酸序列不同于已知的12种Cry1Ia蛋白，是一种新的Cry1Ia蛋白，该基因已被国际基因命名委员会正式命名为cry1Ia8。杀虫活性测定结果表明：Cry1Ia8对亚洲玉米螟（Ostrinia furnacalis）、小菜蛾（Plutella xylostella）有很强的杀虫活性，LC50分别为0.268 µg/g、2.227 µg/ml，其杀虫效果与Cry1Ab、Cry1Ac相当。对大豆食心虫（Leguminivora glycinivorella）也有较好的活性，但对鞘翅目叶甲科害虫榆兰叶甲（Pyrrhalta aenescens）没有活性。该基因的获得将为我国抗虫转基因作物和工程菌的研制提供新的基因来源，为筛选延缓昆虫抗性产生的基因组合提供了极为重要的依据。

Cloning, Expression and activity of cry1Ia Genes from Bacillus thuringiensis isolate

A Full-length cry1Ia gene fragment, which obtained by PCR amplification with a pair of primers designed according to cryIa-type gene sequences and DNA from Bacillus thuringiensi Btc008 as template, was introduced into expression vector pET-21b and transformed into E. coli BL21(DE3). Molecular weight of the induced express product was 81kD.The encoded protein was composed of 719 amino acid residues and the predicted MW is 81.2kD. The amino acid sequence of the Cry1Ia was very different from those of 12 known Cry1Ia-type proteins. This gene with accession number AF373207 was designated as cry1Ia8 by International Bt Insecticidal Gene Nomenclature Committee. The bioassay results indicated that the Cry1Ia toxin protein showed distinctly insecticidal activity against Ostrinia furnacalis and Plutella xylostella with LC50 of 0.268 µg/g and 2.227 µg/ml respectively, while it also had insecticidal activity against Leguminivora glycinivorella，but no activity against Pyrrhalta aenescens. The novel cry1Ia8 gene will be new resource for the construction of genetically engineered bacterium and transgenic plant for biocontrol of insect pests. It also is available for screening gene stacks to delay the resistance produce of the pests.