苏云金芽孢杆菌cry1Ab13基因的克隆及表达研究

The Study on Cloning and Expression of Bt cry1Ab13 Gene

BtC0 0 5是我国自行分离的对多种害虫具有毒杀作用的苏云金芽孢杆菌 ,经PCR RFLP系统鉴定 ,它含有cry1Ab基因。Southernblot结果显示 :PstI酶切C0 0 5质粒所得的 8 5kb长的DNA片段为cry1Ab基因的阳性杂交带。以pUCP1 9为载体 ,克隆了该片段并证明其含有cry1Ab基因。对其进行亚克隆和测序 ,结果表明该基因编码区为 3 4 6 8bp ,其编码的蛋白含1 1 5 5个氨基酸 ,分子量为 1 3 0 6kD ,等电点为pH4 845。该基因已在GenBank基因库中注册 ,Accessionnumber为AF2 5 4 6 4 0 ,并为国际Bt杀虫晶体蛋白基因命名委员会正式命名为cry1Ab1 3。将cry1Ab1 3基因在Bt无晶体突变株cryB- 中表达 ,蛋白质电泳结果表明在 1 3 0kD处有表达带 ,并证明CryAb对小菜蛾有较高的杀虫活性。

B.thuringiensis strain C005 with high insecticidal activity to several kinds of pests,screened from China,was identified that it contained cry1Ab gene by PCR-RFLP.Southern blotting showed that a 8.5kb positive band of plasmid DNA digested with PstI contained cry1Ab gene.The gene was cloned from Bt C005 and the results of sequence analysis showed that cry1Ab13 gene contained a 3468bp open reading frame,encoding a 130.6kD protein composing 1155 amino acids.The IE point of Cry1Ab13 protein was pH4.845.The cry1Ab gene has been registered in GenBank (Accession number is AF254640) and named as cry1Ab13 as a novel gene by International Nomenclature Committee of Bt δ-endotoxin genes.SDS-PAGE analysis indicated that 130kD protein of Cry1Ab13 was expressed in a Bt acrystalliferous mutant cryB - and bioassay results proved that the transformant BiotI81 containing cry1Ab13 gene had high toxicity to Plutella xylostella.