【摘要】 Bt25是中国自行分离的对小菜蛾(Plutellaxylotella)具有高毒力的苏云金芽孢杆菌(Bacillusthuringiensis),经PCR-RFLP鉴定含有cry1Aa基因。以全长基因PCR产物的粘端定向克隆的方法,设计1对特异引物,以Bt25质粒DNA为模板扩增cry1Aa全长基因。序列测定结果表明, 该基因编码区为3 552 bp,编码1 183个氨基酸,分子量为133.7 kD,pI 4.755。该基因序列已在GenBank注册,登记号为AY197341,并获得正式命名cry1Aa14。在氨基酸序列918~1180间,和已知的11种Cry1Aa存在22~23个氨基酸的差异(其中1094~1097的4个氨基酸无对应序列),而这段区域和Cry1Ab氨基酸序列的对应区域无差异。cry1Aa14全长基因插入Bt表达载体,获得了重组表达质粒pBYB1,转化Bt无晶体突变株HD73cry-,经过抗性筛选、DNA酶切分析和PCR检测,证实转化成功。SDS-PAGE分析表明,该基因在上述受体中能正常表达133 kD蛋白。杀虫生物测定结果表明,cry1Aa14表达产物对小菜蛾幼虫具有显著的毒杀作用,与cry1Aa12进行比较,毒力无明显差异。这种单基因菌株的发现及其基因的获得,为害虫抗性研究和高效工程菌的构建提供了重要实验材料。更多还原

【Abstract】 A Bacillus thuringiensis strain Bt25 with high insecticidal activity against several primary lepidopteran pests in China was identified by PCR-RFLP system, and subsequently a novel cry1Aa gene was cloned from it by PCR techniques. The results of sequencing and structural analysis showed that the Cry1Aa14 gene named by International Nomenclature Committee of Bt δ- endotoxin was 3552 bp and the open reading frame encoded a 133.7 kD protein with 1183 amino acids. The isoelectric point of Cry1Aa14 was pI 4.755. Between 918 and 1180 aa, 22~23 amino acids of Cry1Aa14 were different from 11 of all 13 Cry1Aa protein except Cry1Aa9 and Cry1Aa13, and especially four amino acids were additional, but they could be found in Cry1Ab protein. Recombinant plasmid, pBYB, with full length cry1Aa14 gene was constructed and transformed into a Bt acrystalliferous mutant strain HD73 cry-. Result of SDS- PAGE analysis indicated that the expression of the protein was strong in the receptor. Bioassay showed that the toxic protein appeared high insecticidal activity against Plutella xylotella larvea.更多还原