苏云金芽孢杆菌CrylAb13基因的克隆及表达研究

BtC005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crylAb基因。Southern blot结果显示；PstI酶切C005质粒所得的8.5kb长的DNA片段为crylAb基因的阳性杂交带。以pUCP19为载体，克隆了该片段并证明其含有crylAb基因，对其进行亚克隆和测序，结果表明该基因编码区为3468bp，其编码的蛋白含1155个氨基酸，分子量为130.6kD，等电点为pH4.845。该基因已在GenBank基因库中注册，Accession number为AF254640，并为国际Bt杀虫晶体蛋白基因命名委员会正式命名为crylAb13。将crylAb13基因在Bt无晶体突变株cryB^-中表达，蛋白质电泳结果表明在130kD处有表达带，并证明GryAb对小菜蛾有较高的杀虫活性。

The Study on Cloning and Expression of Bt crylAb13 Gene

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 8.5 kb 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 3468 bp open reading frame, encoding a 130.6 kD protein composing 1155 amino acids. The IE point of Cry1Ab13 protein was pH 4.845. 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 delta-endotoxin genes. SDS-PAGE analysis indicated that 130 kD 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.