PCR-RFLP筛选DNA文库克隆Bt cry基因的研究

Cloning of Bt cry Genes by Rapid Screening of DNA Libraries with PCR-RFLP

Bt菌株C0 0 2对水稻二化螟、甜菜夜蛾具有高毒力 ,PCR RFLP鉴定含有cry1Aa、cry2Ab、cry1Ca和未知杀虫蛋白基因cryX等 ,其中cry1Ca位于染色体DNA 6～ 9kb的EcoRI片段。染色体和质粒DNA分别经EcoRI完全酶切和Sau3AI部分酶切、电泳回收 6～ 9kb片段。E .coli Bt穿梭载体pHT315分别与目的DNA连接、转化大肠杆菌感受态细胞后获得了相应的DNA文库。约 5 0个转化子合为一个转化子池 ,采用PCR RFLP方法快速检测 ,分别从约 2 0 0 0质粒DNA文库转化子和 4 0 0个染色体文库转化子中筛选获得了cry1Aa、cry2Ab、cry1Ca和未知基因cryX的阳性克隆 ,相应命名为pHT 1Aa、pHT 1Ca、pHT 2Ab和pHT X。限制酶切分析表明 ,含有cry1Aa、cry1Ca和cry2Ab基因的克隆片段均含有相应基因的保守物理图谱。进一步将这些质粒分别导入Bt无晶体突变株CryB-,SDS PAGE分析表明 ,只有cry1Ca表达了约 130ku杀虫晶体蛋白。初步杀虫生测结果显示 ,cry1Ca对甜菜夜蛾具有高毒力 ,7d校正死亡率为 10 0 %。

Bacillus thuringiensis (Bt) strain C002 contains cry1Aa, cry2Ab, cry1Ca insecticidal crystal genes and an unkown gene cryX , among which cry1Ca is located in a 69 kb EcoRⅠ fragment of the chromosomal DNA. The total DNA and the plasmids DNA libraries of C002 were constructed in Bt E. coli shuttle plasmid pHT315 by inserting 69 kb chromosomal and plasmid DNA fragments prepared respectively with EcoRⅠ complete and Sau3AⅠ partial digestion. On the basis of every 50 transformants pooled together from 510 tubes, the pools containing about 2 000 transformants from the plasmids DNA library and 400 transformants from the total DNA library were rapidly screened by PCR RFLP. Clones containing cry1Aa, cryX, cry1Ca, and cry2Ab were isolated and named as pHT 1Aa, pHT X, pHT 1Ca and pHT 2Ab respectively. Restriction analysis indicated that pHT 1Aa, pHT 1Ca and pHT 2Ab had the typical physical map of the homologous cry genes. Furthermore, each plasmid was transferred into Bt acrystalliferous strain CryB by eletroporation. SDS PAGE result showed that transformant of pHT 1Ca expressed 130 ku protein and bioassay result proved its high toxicity against Spodotera exigua 1st instar larvae with 100% corrected motality.