【作者】 丁学知；

【导师】 高必达；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2006， 博士

【摘要】 研究和发展现代植物保护生物技术，确保食品安全，保护生态环境和维护人类健康具有重要意义。近年来，利用苏云金芽孢杆菌（Bacillus thuringiensis，Bt）取代有残留杀虫化学农药控制植物害虫，实施生态农业计划，已成为国际上发展的新趋势。但在对提高Bt杀虫毒力、挖掘新的cry杀虫功能基因和研究其蛋白质组学上有待深入进行。本文利用微生物技术，分子生物学技术和蛋白质组学方法，选育出Bt4.0718新菌株，系统研究了cry基因，发现了新的杀虫基因，利用烟草几丁质酶基因tchi B与Bt4.0718菌株的cry1Ac基因重组，获得高效杀虫工程菌。首次对Bt的杀虫功能蛋白质组进行了研究。Bt作为微生物杀虫剂在生产上成功推广和应用，很大程度上取决于高毒力菌株的选育。本研究从湖南不同生态区域采集的858份土样中，分离出30份Bt菌株。从中筛选一株具有典型Bt菌落特征和较高杀虫毒力的701^2c为出发菌株，经紫外线和两次亚硝基胍（NTG+LiCl）的交替复合诱变，发现菌体的伴孢晶体的形状、大小、数量和芽孢同伴孢晶体的比例与杀虫毒力密切相关。以此特征为依据进行快速初筛和毒力生测复筛，获得一株高毒力突变杀虫新菌株4.0718。该突变株杀虫毒力与出发菌株相比提高了7.5倍，在6h、12h、和24h内供试小菜蛾三龄幼虫死亡率分别高达20％、97％和100％。此菌株经连续10代培养稳定遗传。这为高毒力杀虫菌株的选育提供了一种简单和快速的方法。在对4.0718进行单因子培养条件试验的基础上，采用正交设计试验和结合杀虫毒力生测，对该菌株进行发酵试验，获得了最佳发酵条件。Bt菌株的杀虫活性与其携带的编码杀虫晶体蛋白（ICPs）的cry基因密切相关。通过PCR扩增的产物电泳检测，4.0718含有cry1和cry类杀虫基因，显示出4.0718菌株cry基因类型丰富。将4.0718菌株质粒上的cry1Ac基因和烟草几丁质酶基因tchiB（去掉信号肽和去信号肽再加肠激酶位点）重组，经双酶切和亚克隆，将带有cry1Ac基因上游启动序列和下游终止序列的重组基因片段克隆至穿梭载体pHT315中，分别构建重组质粒pHUAccB6和pHUAccB7，转入E.coli，电转入Bt无晶体突变株XBU001中，获得重组菌株HAccB6和HAccB7。通过液体双相孢晶分离提取后，将晶体和上清液分别进行SDS-PAGE分析，双价重组基因与对照cry1Ac基因在无晶体突变株中表达量相比较，获得高效表达的双价融合蛋白。经定量分析：双价融合蛋白分别占总蛋白量的62.5％；Cry1Ac蛋白占蛋白总量的42.0％。经原子力显微镜和电子显微镜观察，表达后的融合蛋白呈菱形和椭圆形晶体，其规格约为1.5×3.0μm。经生测分析，两株工程菌HAccB6和HAccB7的重组基因表达的融合晶体蛋白二者在毒力上比较接近，而与对照工程菌HAc5和野生菌4.0718相比较，速杀时间提前36h。工程菌HAccB6对棉铃虫（Heli coverpa armigera）具有高效杀虫活性，其72h LC₅₀值为9.10μg／mL，为对照菌杀虫毒力的4.529倍。结果显示出tchiB基因与cry1Ac基因重组后的表达产物具有显著的杀虫增效作用。首次对Bt杀虫晶体蛋白质进行了研究。用液体双相法和等电点沉淀法纯化晶体蛋白，结合用2-DE分离的蛋白质进行质谱鉴定，确定了伴孢晶体蛋白总蛋白质的提取方法，获得了更清晰、分辨率更高和聚焦效果更好的2-DE图谱，建立了Bt晶体蛋白的双向电泳方法。对分别属于Bt的3个不同亚种的库斯塔克亚种4.0718、武汉亚种140菌株和以色列亚种H14菌株及相关工程菌株HAccB6和HAccB7的晶体蛋白质进行了系统的研究。工程菌HAccB6和HAccB7菌株分别检测到356±9和400±11个，其中两者129个蛋白质斑点相互匹配，平均匹配百分率为30％。等电点沉淀法双向电泳（2-DE）图谱中检测到90±9和193±11个蛋白质斑点数，其中两者的80个蛋白质斑点相互匹配，平均匹配百分率为40％，说明两者在毒力以及杀虫谱上的相似性和差异性。对2-DE胶上大部份蛋白质点进行了质谱分析，鉴定出各菌株的晶体蛋白成份中的7类功能蛋白质，即杀虫毒蛋白（Cry1Ac、Cry2Aa、CryIG，CryH2，Cry1Ab16，Cry2Ac₂）、晶体形成相关结构蛋白（伴侣蛋白dnak）、蛋白质折叠和组装的重要调节者热休克蛋白H_SP60，物质代谢酶类（分支链氨基酸转氨酶、烯醇酶、丙酮酸脱氢酶复合物E3）、蛋白质合成因子（ATP合成酶F1、ATP合成酶β-链、β-内酰胺酶、翻译延长因子Ts、Tu、G）、延长因子EF-Tu和EF-Ts、假想蛋白、转运蛋白（ABC transporter ATP-binding protein）、和蛋白引发因子（Trigger factor）等。从研究的Bt菌株中都鉴定有HSP-60，翻译延长因子Tu这两种蛋白，认为这两种蛋白很可能为晶体形成相关的重要蛋白，且杀虫晶体蛋白在结构与杀虫活性功能上的关系密切。这些表达谱和功能蛋白质组的研究将有助于对杀虫晶体蛋白杀虫机理的理解。特别是在Bt武汉亚种中新发现了Cry2Ac6蛋白。晶体蛋白种类的不同则有助于解释4.0718菌株高效杀虫的生化功能特征。结合利用CPHmodles、Ds ViewerProTrial和Swiss-Pdb Viewer软件，发现新型的Cry2Ac6与Cry2Aa的毒性核心片段空间结构有一定差异。说明与昆虫中肠受体蛋白结合的亲和力不同，引起其在杀虫毒力上的差异。总之，本项研究为苏云金芽孢杆菌高毒力杀虫菌株的选育、发酵条件、cry基因与外源毒素基因重组及亚种间功能蛋白质组学研究提供了一系列的方法和可借鉴的研究思路，鉴定了与杀虫毒力相关的cry基因及其功能蛋白质组，创新性研究了不同原毒素分子结构与杀虫活性之间的内在规律及其与昆虫细胞受体结合机制的信息。更多还原

【Abstract】 Biotechnology of modern plant protect_is very important for insuring food safety and humanhealth. In the past few years, it has been a world-wide trend to use Bt to replace chemicalinsecticide for pest control in plant. However, further studies are needed to increase itsinsecticidal toxicity, seek new cry functional gene and deepen its proteome analysis.This paper employs microbial technology, molecular biotech and methodology ofProteomics. An insecticidal strain 4.0718 of Ballilus thuringiensis was bred, its cry gene wasstudied systematically and a new insecticidal gene was obtained. And for the first time, theinsecticidal crystal proteins from Balillus thruingiensis were researched using proteome analysis.Also, high toxin insecticidal recombinant strain was constructed by combining B. thuringiensiscry gene and tchiB gene from tobacco.The wide application of Bt as a microbial insecticide in agricultural production depends to alarge degree on the breeding of a high toxin strain. In the research, 30 strains of Bacillusthuringiensis were isolated from 858 samples of dry land, vegetable garden and rice soils indifferent ecological regions of Hunan province, and selected as an original from the 30 strainswas strain 701^2c typical of B. thuringiensis.By means of multiple mutagenesis of UV and double NTG+ LiCl and through microscopicexamination of smear dyeing, it was first found that the insecticidal property of B.thuringiensisis closely related with the shape, size and number of the crystal protein and the proportion ofspore and crystal protein. After screenings, one highly toxic mutant strain 4.0718 was obtained.Tests showed that its toxicity to the larval of Helicoverpa armigera was 4.527 times higher thanthat of the control strain and the lethal rate reached 20%, 97% and 100% respectively 6, 12 and24h after treatment. The virulence of 4.0718 did not change after ten-generation transmission.This method to a simple and efficient way of breeding high toxin insecticidal stains. Still, thefermentation conditions of a highly toxic mutant strain 410718 of Bacillus thuringiensis werediscovered by using orthogonal experiment and bioassays of insecticidal activity.Using a PCR strategy, this study identified rapidly Bacillus thuringiensis strain 4.0718 thatharbored the known cry1 and cry2 type genes. These results suggested the novel Bacillusthuringiensis 4. 0718 had plenty of cry -type genes. This was evidence that Bt 4. 0718 hasconsiderable application and research value. To construct a new fusion protein to enhance thetoxicity of crystal proteins, the cry1Ac gene of Bacillus thuringiensis strain 4.0718 wascombined with a tchiB （deleted signal peptide and Enterokinase site sequence）. In this process,the Enterokinase site sequence was inserted into the midst of the two genes. Then the fusion gene fragment carrying the upstream promoter region and the downstream terminator region ofcry1Ac gene were cloned into the shuttle vector pHT315. And after a series of enzymedigestions and subclonings a new expression vector pHUAccB6and pHUAccB7 was obtained.The two vectors was transformed into B. thuringiensis acrystalliferous strain XBU001 withelectroporation to obtain the recombinant strain HAccB7 and HAccB6.Under AFM and SEM,there were some bipy ramidal crystals with a size of 1.5×3.0μm. Bioassay showed that the fusioncrystals from recombinant strain HAccB7 and HAccB6 were high toxic against third-instarlarvae of Plutella xylostella with the LC₅₀ （after 72 h） value of 9.10μg/ml. The fusion proteinswhich have constructed had more toxicity than Cry1Ac crystal proteins. The study will enhancethe toxicity of B. thuringiensis Cry toxins protein and make a ground for construction the fusiongenes of B.thuringiensis cry gene and other foreign toxin genes and the recombinant strain withhightoxicity.Bt insecticidal protein has been separated, using two modified methods that two-phaseliquid partition and isoelectric sediment point. The extracted insecticidal crystal proteins werespearateed by 2DE, and submitted to MS identified.We systemly studied the ICPs proteome.Comparatively from three Bt kurstaki 4.0718、wuhanensis 140, israelensis H14 and twoengnerring strains Although pH3-10 and pH4-7gels were used by us, most of the spots were inthe gel pH range 4 to 7.The ICPs are usually high molecular weight, hydrophobic proteins. Thereare 129 matched spots between HAc5and HAccB5 maps （the matching rate was 10%）, usingisoelectric point methods 90±9 spots in HAc5, 193±11spots in HAccB5 strain.There are 80matched spots between gels of HAc5and HAccB5 （the matching rate was 10%）, Thecharacteristics of the 2-DE maps of ICPs from these three strains and two engineering strainpartially explain such differences.After digestion in gel with TPCK-trypsin, Cry1Ac amid the differentially expressedproteins were identified by MALDI-TOF-MS and searched in the SWISS-PROT database withMASCOT in two strains, it is found that the function of some of the different proteins, such astoxins protein （Cry1Ac、Cry2Aa、CryIG, CryH2, Cry1Ab16, Cry2Ac,）, proteinsynthesis,transporter （ATPsynthase F1, gamma subunit, ATP synthase F1, beta subunit,translation elongation factor Ts, translation elongation factor G）, metabolism （branched-chainamino acid aminotransferase, Enolase, pyruvate dehydrogenase complex E3 component,pyruvate dehydrogenase complex e1 component, beta subunit, fructose-bisphosphate aldolase,classⅱ, putative respiratory nitrate reductase delta chain, Negative regulator of geneticcompetence clpC/mecB）, protein folding（chaperone protein dnak）, hypothetical protein tlr2278,synthase beta chain, TEM-7 beta-lactamase, unknown hypothetical. we found two commonproteins. The heat shock proteins （Hsp60） and Translation Elongation Factor Tu, which help with protein refolding and prevent protein degradation, an exogenous insecticidal toxity gene hada significance effect on the expression sperectra of ICPs and on the virulence of the Bt strains aswell.In summary, it is a very usefull reference thought for us to screen, optimize fermentableconditions, fuse between cry gene and other gene, and analyse function protein for hight toxicitystrains from B.thuringiensis. Thus, the characteristic proteins of Cry, as identified in theirinsecticidal crystal proteomes, could also be used as new screening markers of the high toxicity.Cry and function proteomic has been identified the first systemic investigation B. thuringiensisstrain 4.0718, this bring forth new ideas that domain of different protoxin is close relationshipbetween insecticidal toxicity, recombinant mechanism and insect recepter.更多还原