生防细菌NCD-2中抑菌功能相关基因的定位及克隆

枯草芽孢杆菌NCD-2菌株是一株有效防治棉花黄萎病的细菌,它通过产生抑菌物质达到对大丽轮枝菌的抑制作用。以该菌株作为有效成分的微生物农药已通过国家农药登记。通过原生质体转化法将含有转座子mini-Tn10的质粒pHV1249转入枯草芽孢杆菌NCD-2中,获得转化子。对转化子通过高温诱导转座子转座,获得4 000个NCD-2菌株的突变子。对这些突变子进行对大丽轮枝菌的抑菌作用测定,筛选到2株抑菌作用增强的突变子和4株抑菌作用降低的突变子。采用染色体步移技术对此6个突变子中转座子插入位点基因的侧翼序列进行克隆和测序,结合枯草芽孢杆菌168菌株的全基因组序列对测序结果进行序列同源性和基因定位分析,结果表明:抑菌作用增强的2个突变子中,转座子插入到一个功能未知的基因内部,此基因与枯草芽孢杆菌168菌株中的yvoA基因的同源性为98%;抑菌作用降低的4个突变子中,转座子插入位点对应于枯草芽孢杆菌168菌株中的phoP基因内部,插入位点的侧翼序列与枯草芽孢杆菌168菌株中的phoP基因的同源性达到98%。

Mapping and Cloning Function Genes from Antigonistic Bacterium NCD-2 Against Verticillium dahliae

Bacillus subtilis NCD-2 was an effective bacterial strain to control verticillium wilt of cotton,and could produce antibiotic substances to inhibit the gowth of Verticillium dahliae,the pathogen causing this disease.The wild type of strain NCD-2 was transformed with the plasmid pHV1249 carring transposon mini-Tn10 by protoplast measure and the transformants were screened.Through the transposon mini-Tn10 mediated insertional mutagenesis technique,more than 4 000 mutants were screened.The inhibiting activity of all mutants were evaluated against V.dahliae by inhibiting zone observation,2 mutants with significantly enhanced inhibition activity and 4 mutants with signifcantly decreased inhibition activity were obtained.Using genome walking technique,gene flanking the mini-Tn10 insertion site was cloned from the two mutants with enhanced inhibition activity,respectively.Gene sequences are most similar to the sequence of the function-unknown gene yvoA of B.subtilis model strain 168(similarity 98%).Genes flanking the mini-Tn10 insertion site were cloned from the four mutants with decreased inhibition activity,respectively.Gene sequences are most similar to the sequence of the phoP gene of B.subtilis model strain 168(similarity 98%).