摘要：
为扩充鳞翅目害虫杀虫基因资源,本研究从苏云金芽胞杆菌BN23-5中克隆得到一个新的cry基因,并对其进行鉴定和分析.该基因为一个完整的cry1D基因,全长3501 bp,编码1166个氨基酸残基.该氨基酸序列是一个新的Cry氨基酸序列,与Cry1Db1的同源性最高,为86％,命名为Cry1Dd1(登录号为KJ728844).将该基因插入穿梭表达载体pSTK中,转入BT无晶体突变株-HD73中进行表达.结果表明,cry1Dd1基因能在BT无晶体突变株中表达,并形成菱形伴孢晶体.SDS-PAGE验证其分子量为132.2 kD,与预测的大小相符.生物活性测定表明,Cry1Dd1晶体蛋白对小菜蛾的幼虫具有杀虫活性,LC50为13.1μg/mL;能明显抑制甜菜夜蛾幼虫的生长;但对棉铃虫幼虫没有杀虫活性.对cry1Dd1基因序列进行分析,cry1Dd1包含8个block保守区域,这和目前其他的cry基因相似;Cry1Dd1蛋白的活性区域为N端的37～593位氨基酸残基.%A novel holotype cry gene (cry1Dd1) was cloned from a Bt strain BN23-5, which enriches novel lepidopteran insecticidal Bt genes. In this study, the full length of the cry1Dd1 gene was cloned. The full-length cry1D gene was 3501 bp, encoding a polypeptide of 1166 amino acid residues. Its amino acid sequence was 86％ identical to that of Cry1Db1, and the gene was named as cry1Dd1 by International Nomenclature Commitee of B. thuringiensis endotoxin with a Gen Bank number of KJ728844. The cry1Dd1 gene was inserted into a shuttle vector (p STK) and expressed in an acrystalliferous mutant B. thuringiensis HD73-. In this transformant, cry1Dd1 was expressed and diamond-shaped parasporal crystals were formed. SDS-PAGE analysis determined a molecular mass of approximately 130 kD of Cry1Dd1 protein. The Cry1Dd1 protein exhibited high larvicidal activity against the diamondback moth (Plutella xylostella), with an LC50 of 13.1 μg/mL, and obviously inhibited growth of Spodoptera exigua larvae, but showed no insecticidal activity against Helicoverpa armigera larvae. Sequence analysis of the gene indicated that the cry1Dd1 gene retained eight conserved regions commonly found in the existing cry genes, and the function domain of Cry1Dd1 protein was the 37 to 593 amino acid residues at the N-terminal.
