【作者】 洪鹏翔；

【导师】 胡方平；

【作者基本信息】 福建农林大学 ， 植物病理学， 2007， 硕士

【摘要】 根据GenBank上登录的Bacillus subtilis subsp．subtilis str．168(简称BS168)序列(NC000964)，设计出包含两种不同限制性内切酶位点的1对扩增rpsD启动子序列的特异引物RpsF／RpsR，用PCR方法从BS168的总DNA中扩增出rpsD启动子基因，命名为rpsP。并根据已经在GenBank上登录的Bt vip3A(a)基因序列，设计出包含两种不同的限制性内切酶位点的1对特异引物vip-F／vip-R，以含有对鳞翅目夜蛾科害虫具有广谱高效杀虫活性基因vip3A(a)的苏云金芽孢杆菌(B．thuringiensis，Bt)菌株Bt-WB7的总DNA为模板，用PCR方法扩增出vip3A(a)基因。再将rpsP和vip3A(a)基因的PCR产物分别用NdeⅠ单酶切，之后将两个单酶切产物用T4连接酶连接。以连接产物为模板，用RpsF／vip-R为引物，扩增启动子-vip基因序列，将PCR产物直接连接到pMD-18T克隆载体上转入大肠杆菌(Escherichia coli)JM109。挑取阳性克隆，提取重组质粒pMD-p-vip进行酶切与PCR验证后进行测序。将测序验证正确的启动子-vip基因，用EcoRⅠ和XbaⅠ双酶切从pMD-p-vip重组质粒上切下，回收目的片段与用相同酶切的表达载体Pgfp4412相连，得到重组原核表达质粒pVipgfp。将重组原核表达质粒pVipgfp转化解淀粉链芽孢杆菌(Bacillusamyloliquefaciens)受体菌株TB2中，挑取转化子用PCR检测重组质粒pVipgfp导入情况，最后通过生物测定筛选得到能高效表达Bt vip3A(a)基因的TB2-16工程菌。研究表明TB2-16工程菌株培养液对斜纹夜蛾幼虫的具有很高的毒杀作用；抑菌实验表明TB2-16工程菌株对大豆炭疽病菌、柑橘炭疽病菌、黄瓜枯萎病菌、西瓜枯萎病菌、番茄早疫病菌5种病原菌的抑制效果与TB2无明显差异，而且菌落形态基本上保持不变；内生和对植物生长影响研究表明TB2-16具有良好的内生和促生特性。更多还原

【Abstract】 Abstract: A pair of primers was designed according to the sequence of the strain BS168 (Bacillus subtilis subsp. subtilis str.168, GenBank accession number NC000964) , then to PCR to obtain the promoter of rpsD, designated as rpsP,using a modle of the chromosome DNA of BS 168．Another pair of primers was designed according to the vegetative insecticidal protein (VIPs) gene (GenBank accession number AF442384) , then to PCR to obtain vip3A(a) gene using a modle of total DNA of the strain WB7( Bacillus thuringiensis )which had high insecticidal activity. The rpsP and vip3A(a) sequences were cut by the endoenzyme Nde I and purified by the DNA pulification kit respectively, then the two genes were ligated by T4 DNA Ligas, and to ampify using the ligated genes as a modle to obtain the p-vip gene with RpsF/vip-R as primers. The p-vip gene was cloned into the strain JM109(Escherichia coli) using pMD-18T vector. Then the recombinant plasmid, pMD-p-vip, was extracted and further amplified and analyzed with restrictionendoenzymes. After that the p-vip gene was cut down from the recombinant plasmidpMD-p-vip and ligated with expression vector Pgfp4412 to yield the recombinant plasmid pVipgfp.The recombinant plasmid pVipgfp was transferred into the strain TB2(Bacillus amyloliquefaciens), then transformants were verified by PCR. Therefore, 6 engineering bacterial strains of B. amyloliquefaciens with Bt insecticidal gene were obtained. Bioassays showed that the engineering strain, TB2-16, had high toxicity against the second instar larva of Spodoptera litura. The inhibiting activities of TB2-16 to Colletotrichum truncatum, C.gloeosprioides , Fusarium oxyporum f.sp. cucumerium, F. oxysporum f.sp. niveum, Alternaria solani were as the same as that of the TB2 strain. The endophytic and plant growth promoting tests showed that TB2-16 had no difference from that of TB2．更多还原