【作者】 刘全兰；

【导师】 夏立秋；

【作者基本信息】 湖南师范大学 ， 微生物学， 2002， 硕士

【摘要】 苏云金杆菌（Bacillus thuringiensis，简称Bt）4.0718菌株（CCTCC NO．200016）对小菜蛾（Plutella xylostella）和棉铃虫（Helicoverpa armigera）等鳞翅目昆虫具较高毒性，本研究分离并提纯了其杀虫晶体蛋白基因；运用PCR方法分析4.0718菌株的基因型，并进行了克隆。 实验结果如下： 采用改进的Triton X-100温和裂解法进行趴4.0718菌株的质粒DNA的抽取，取得较理想效果。用0.6％TritonX-100和1 mol／L NaCl的混合液处理菌体，90min的裂解时间效果为好。提取的质粒DNA受到的损伤小，并且酶的抑制剂含量也较少。在0.5％琼脂糖凝胶电泳中，质粒DNA分离相当清晰。Bt 4.0718菌株含有4个大小不等的质粒，依据其分子量大小顺序依次编号为P₁、P₂、P₃和P₄。 采用几种不同的方法回收4.0718菌株的质粒DNA，主要有制备档板挖孔的方法、液氮冻融法和低熔点琼脂糖法。实验中对这三种回收方法进行了不同程度的改进，使它们皆可高质高效的回收Bt 4.0718菌株质粒DNA，基本不受DNA分子长度和量的限制。 目前研究表明，苏云金杆菌cry1、cry2和cry3类基因编码的杀虫晶体蛋白（Insecticidal Crystal Proteins，简称ICPs或Cry蛋白）对鳞翅目昆虫有毒性，并且我室的Bt 4.0718菌株对鳞翅目昆虫有较强毒性。对ee;lba:Ik中eryl一、cryZ一和Cry3一型基因的高度保守区进行同源序列比较，设计了3对通用引物cryl〔U;:l（d）/U，飞一（r）]、eryZ[UnZ（d）/UllZ（r）]和ery3〔U，飞3（d）/U;飞3（，·）]。并根据eryl一和cryZ一型基因的高变区设计特异引物:55、!，11/s3t川l（e ryl人a，ery1Ab，ery1Ac，cry1B，ery1Ca，℃ry1Cb）和SsunZ/s3unZ（cryZAa，cryZ人b，cryz人e）。PCR扩增结果表明质粒P4没有杀虫晶体蛋白基因，质粒P、、p，和p3在用eryl[Unl（d）/Unl（r）]和cryZ【UnZ（d）/UnZ（r）」两对引物扩增时分别产生了277bp和689bp的产物，分别对应cryz一和cryZ一型基因;通用引物cry3[U，13（d）/U，13（r）]没有扩增出DNA片段，说明质粒中不存有cry3型基因。为了对4.07 18菌株的亚类基因型进行精细分析、必须进一步的PCR鉴定。质粒P，用特异引物扩增出1641、1623、1219bp和1471bp的DN八片段，分别对应于cryzAc、cry1Cb、cryZAc和新的Cry基因（暂定名为ery4.5基因）。质粒PZ扩增出的PeR产物大小是1 6 35、1 6 23和1 2 1 gbp，分别对应于ery1Aa、ery1Cb和cryZ八c基因。质粒P3扩增出1623、一557和1219bp的产物，分别对应于ery1Cb、erylAb和eryZAe基因。PCR扩增的数据表明，4.0718菌株含有多个与Cfy家族有关的杀虫基因。即含有cry1Aa、cryl^b、cry1Ac、cry1Cb、cryZ入c和新的cry基因 （。ry4.5基因）等基因型。像Bt 4.0718菌株这样基因类型含量丰富的Bt，国内外报道尚少，这证明Bt 4.0718菌株有重要的研究价值和应用价值。 实验中采用山lljll/Pstl双酶切质粒DNA，寻找携带新基因的片段。发现新基因cry4.5在4.skb酶切片段上，另外还有一5.okb片段携带cry基因。分别回收两片段，将它们与双功能载体nllT3叫进行重组，转化感受态细胞丑coj j Dll sa。通过蓝/白斑显色反应和重组质粒酶切鉴定获得重组子pB t4。更多还原

【Abstract】 The inseclicidal crystal proteins genes were extracted from a novel Bacillus thuringiensis 4.0718 with an improved TritonX-100 gentle lysis, which were analysed by PCR. And the insecticidal ctystal proteins gends were cloned.The results showed as below:The improved TritonX-100 gentle lysis was used to extract plasmids from a novel Bacillus thuringiensis 4.0718. This method was found reproducibly and reliably. When 0.6% TritonX-100 and Imol/L NaCl were usede to extract plasmids ,after 90 lysis time, plasmids were obtained with good qualitiiy. During this course,the damage of plasmids were little and the habitors of enzyme were a few. And plasmid profiles were clear respectively on agarose gel (0.5%), which were named P1,P2, P3 and P4 by their molecular weights.In this study, recovery methods of digging pore , freezing -thawing, low melting gel were used to recover plasmids respectively. In order to keep the good qualities of plasmids and recover plasmids effectively, these methods were improved on a different level. Finally, these methods could recover these plasmids effectively, in spite of their lengths and contents.The main focus in studies of Bacillus thuringiensis 4.0718 strain is the production of insecticidal crystal inclusions during sporulation. These crystals were composed of proteins called 5 -endotoxins or Cry proteins.Workers have described different Cry proteins that exhibit toxicactivity against larvae of very different insects (lepidopteramm, dipterans, and coleopterous), as well as other organisms, including platyhelminths, nematodes,and protozoans. The proteins encoded by cryl-, cry2- and cry3- type genes were toxic to lepidopterans, and the insecticidal crystal inclusions within the novel Bt 4.0718 were found toxic to lepidoptcrans . So, three pairs of universal primers cry1[Unl(d)/Unl(r)], cry2[Un2(d) /Un2(r)] and cry3[Un3(d)/Un3(r)]were designed to probe the most conserved regions of all known cry1-, cry2- and cry3-type genes. The PCR-amplified fragments showed p4 had no any cry-type gene , and the others only have cryl and cry2-type genes. From this , we could identify Bt 4.0718 harbored cryl- and cry2-type genes. On this basis, nine specific primers [S5unl/S3unl( crylAa, crylAb, cryl Ac, cry IB, crylCa, crylCb) and S5un2/S3un2 (crylAa, cry2Ab, cry2Ac)] were designed to amplify P1, P2 and P3. The products of PCR showed that PI had four cry-type genes(cry 1 Ac, crylCb , cry2Ac and a novel cry4.5), P2 had there cry-type genes(cry 1 Ab, crylCb and cry2Ac), P3 had there cry-type genes (crylAa, crylCb and cry2Ac). These results suggested the novel Bacillus thwingiensis 4.0718 had plenty of cry-type genes. There were few reports about genes as many as Bt 4.0718. This gave evidence of application and research values on Bt 4.0718.Besides analysis of the cry-type genes of plasmids within Bt 4.0718, these plasmids were digested with restriction endonucleases BamHI/Pst1. And it was found the novel cry4.5 in the 4.5 kb fragment and the 5.0 kb fragment also harbored the cry gene.These two fragments were recovered from the agarose gel, then were ligated with cohesive ends of expression vector pHT304, transformed into competence cells E.coli DH 5 a. The cloned strains haboring the fragments of 4.5 kb and 5.0 kb were obtained by blue/white selection and digested with restriction endonucleasesBamHI/Pst1. And finally, the recombinant plasmid pBt4 was identified.更多还原