摘要
以人工合成的130kd 杀虫蛋白基因的18-base 序列为探针,通过 Southern 分子杂交,验证了在 Bacillus thuringiensis var.israelensis 4Q5菌株75Md 质粒上含有130kd 杀蚊蛋白基因,并且证明了提纯的晶体蛋白具有杀蚊幼虫活性。对该质粒进行 HindⅢ完全酶切,以pUC18为载体,以 E.coli TG1为受体,得到四个与探针有强杂交信号的阳性克隆,其中两个(pFH2,pFH4)含有5.2kb HindⅢ插入片段,包含第一类130kd 杀蚊蛋白基因;另两个(pFH1,pFH3)含有2.3kb HindⅢ插入片段,包含第二类130kd 杀蚊蛋白基因的3′部分.pFH2和 pFH4中,第一类130kd 杀蚊基因的插入方位不同。
The location of 130kd mosquitocidal protein gene of Bti 4Q5 strain on its 75Md plasmid wasconfirmed by southern hybridization using a 18-base oligonucleotide probe.The crystal proteincontaining the component of 130kd toxic protein was purified.The crystal protein exhibiting themosquitocidal activity against larvae of Aedes aegypti was shown by bioassay.The purified 75Mdplasmid DNA of Bti 4Q5 strain was completely digested with HindⅢ restriction enzyme,li-gated with the vector pUC18 and transformed into the recipient cells of E.coli TG1.From Ap^(?)transformants,four clones with HindⅢ restriction fragment inserts highly homologous to the18-base oligonucleotide probe were obtained by in situ hybridization and southern hyb:idization.The 5.2kb HindⅢ restriction fragment insert was obtained in clone pFH2 and clone pFH4,and2.3kb HindⅢ restriction fragment insert in clone pFH1 and pFH3.For pFH2 and pFH4,he5.2kb fragment was inserted in pUC18 in opposite orientation.It contained 130kd mosquit(?)dalprotein gene(typeⅠ)identified by restriction enzyme map analysis.The 2.3kb HindⅢ fragmentinsert in other two clones(pFH1 and pFH3)harbored a part of the type Ⅱ mosquitoc(?)dal proteingene which can be used as a probe for cloning of the typeⅡmosquitocidal protein gene.
出处
《微生物学报》
CAS
CSCD
北大核心
1992年第5期314-319,共6页
Acta Microbiologica Sinica
关键词
苏云金杆菌
以色列种
杀蚊蛋白基因
Bacillus thuringiensis var.israelensis
130kd mosquirocidal protein gene
Molecular cloning
