球形芽孢杆菌2362株杀蚊幼虫蛋白基因的克隆

CLONING OF THE LARVICIDAL PROTEIN GENES OF BACILLUS SPHAERICUS 2362

利用合成的寡核苷酸探针从球形芽孢杆菌2362株基因文库中筛选了四个杂交阳性克隆,它们分别携带质粒 pDC4(7.8kb)、pDC5(7.8kb)、pDC6(8.4kb)和 pDC7(6.4kb)。斑点杂交结果显示,探针与pDC7结合能力明显比与 pDC4和 pDC6结合能力弱。四个杂合质粒 EcoRI 酶切图谱不同,但插入片段中都含有一个3.3kb 片段。Southern 印迹杂交实验进一步揭示该3.3kb 带可为探针识别;pDC24除此之外还有-1.4kb 杂交带,表明该质粒中携有两个与探针同源的序列。在以上四个克隆中,E.coli HB101(pDC4)对淡色库蚊幼虫有显著毒性,因而携有完整的基因51和基因42序列。

Four purpose gene clones were obtained by probing with synthetic oligonucleotide from thegene library of Bacillus sphaericus 2362,carrying the plasmids pDC4(7.8kb),pDCS(7.8kb),pDC6(8.4kb),pDC7(6.4kb)respectively.Dot blot analysis showed that combination of the probe with pDC7 was ob-viously weaker than that with other two plasmids pDC4 and pDC6.Although they had different EcoRI cleav-age patterns,the four hybrid plasmids shared a 3.3kb fragment,which was a hybridization band in the au-toradiogram of Southern blot analysis.However,pDC4 had a more hybridization band of 1.4kb.This resultsuggests that the cloned fragment in this plasmid has two homologous sequences with the labelled probe.Foris obvious tocxicity to the larvae of Culexpipens E.coli HB101(pDC4)must be with complete sequences ofgene 51 and gene 42.