【作者】 周琴；

【导师】 喻子牛； 孙明；

【作者基本信息】 华中农业大学 ， 微生物学， 2004， 博士

【摘要】 本文围绕绿色荧光蛋白基因在苏云金芽胞杆菌中的表达及应用，从以下几个方面进行了研究，取得结果如下： 1．绿色荧光蛋白基因gfp在苏云金芽胞杆菌中表达的初探 将携带蜡状芽胞杆菌特异启动子的gfpmut3α基因克隆到穿梭载体pHT304后，电转化至苏云金芽胞杆菌BMB171，CryB，IPS78-11，4Q7，HD-80-21中。通过荧光显微镜和Bio-assay Reader对含重组质粒pGFP-304的5个受体菌分别进行荧光检测及定量分析。结果表明，gfpmut3α基因仅在苏云金芽胞杆菌无晶体突变株BMB171，CryB，IPS78-11，4Q7中表达并可检测得到菌体发光，并建立了一套适合于苏云金芽胞杆菌的gfp标记与检测方法。 2．利用绿色荧光蛋白基因gfp研究芽胞杆菌的启动子活性 绿色荧光蛋白基因gfp分别标记苏云金芽胞杆菌的cry3A启动子P_cry3A，BtⅠ-BtⅡ启动子P_BtⅠ-BtⅡ和来自蜡状芽胞杆菌特异启动子P_44-12以研究其表达差异。其中，P_cry3A和P_BtⅠ-BtⅡ分别与gfp构成融合基因，以调控gfp在苏云金芽胞杆菌中的表达。将重组质粒pGFP-304(含P_44-12)、pGFPExpA(含P_cry3A-gfp融合基因)和pGFPExpB(含P_BtⅠ-BtⅡ-gfp融合基因)分别导入大肠杆菌和苏云金芽胞杆菌后发现，P_44-12和P_BtⅠ-BtⅡ在大肠杆菌与苏云金芽胞杆菌中均可表达gfp，其中P_BtⅠ-BtⅡ在大肠杆菌中具有极强的启动基因表达的能力。而P_cry3A不能启动gfp在大肠杆菌中表达，在苏云金芽胞杆菌中启动的gfp表达的荧光强度也较弱。 3．携带不同启动子苏云金芽胞杆菌标记重组菌株的微量热学变化 生物活性检测器对含重组质粒pGFP-304，pGFPExpA和pGFPExpB的转化子分别进行荧光检测及微量热检测。结果表明，3种启动子驱动下的gfp基因均可在苏云金芽胞杆菌无晶体突变株BMB171中表达并检测得到不同的发光类型。微量热法检测发现P_44-12和P（BtⅠ-BtⅡ）启动gfp表达的代谢热低于P_cry3A驱动gfp表达的代谢热。 4．苏云金芽胞杆菌标记重组菌株的构建 本研究利用SOE法将构建的绿色荧光蛋白基因gfp和苏云金芽胞杆菌的杀虫晶体蛋白基因cry1Ac10的嵌合基因克隆到穿梭载体pAD4412上获得成重组质粒pBMBZGC10。同时又将苏云金芽胞杆菌启动子P_BtⅠ-BtⅡ替换蜡状芽胞杆菌启动子P₄₄₁₂，经同一穿梭载体pAD4412构建得到了pBMBZGC11。再通过电转化法将它们导入苏云金芽胞杆菌无质粒突变株BMB171和cryB中获得重组菌株BMB171（PBMBZGC10）;CryB（pBMBZGC10）;BMB171（PBMBZGCll）和CryB（P BMBzGcll）。经荧光显微镜观察标记重组菌株仅能在标记重组菌株BMB171（pBMBzoelO）和CryB（pBMBZGClo）中检测到嵌合基因的表达;SDS一PAGE结果也显示了融合蛋白约为1 50 kDa大小的蛋白带型。5.苏云金芽胞杆菌标记重组菌株的荧光检测分析 本研究分别利用Leiea Stereo MZFlll型荧光显微镜、Leiea Di叩lan型荧光显微镜和Zeiss Axioplan型激光共聚焦扫描显微镜，对苏云金芽胞杆菌标记重组菌株的菌落、菌液、菌体分别进行荧光检测。ZeisS荧光显微镜激发光波长为488nm、发射光波长为522nm（oF32）。并用Laser sharp Z000TM专用软件对收集到的图形信号进行捕获和处理。近一步用Bio一assay Reader对标记重组菌株进行荧光强度的检测，选择激发滤光片为485二、发射滤光片为52Onm。用HT Soft 2.0软件分析所得数据。6.帮助蛋白P20对苏云金芽胞杆菌标一记重组菌株影响的研究 将携带有pZ口基因的穿梭载体pBMBZO一2，利用电转化的方法分别导入苏云金芽胞杆菌标记重组菌株BMB 1 71（pBMBZGClo）和CryB（pBMBZGClo）得到BMB 1 71（pBMBZGC 10一20）和CryB（pBMBZGC 10一20）。经普通光学显微镜和荧光显微镜观察尸2口基因对融合基因表达的影响。结果发现重组菌株BMB 171（pBMBZGC 10一20）和CryB（pBMBZGCIO一20）能够检测到很强的绿色荧光。进一步通过荧光强度分析表明，20 kDa帮助蛋白能够增加融合蛋白的表达量。7.苏云金芽胞杆菌标一记重组菌株与杀虫基因水平转移的研究 将重组菌株CryB（pBMBZGC 10）的发酵液按浓度梯度分次进行喷洒小白菜（Brassica夕exinesis）、雍菜（IP000ea啊uatiea）和番茄（勿c叩ersicon esculentum）作为供试植株，利用标一记基因研究的结果表明:cry了Aojo基因没有向供试土壤细菌、真菌和放线菌转移，也未在植物的供试植物根、茎和叶中检测到该基因。更多还原

【Abstract】 The expression and application of gfp in Bacillus thuringiensis were described in this thesis. The research results are summarized as following:1. Preliminary Study on the Expression of gfp Gene in Bacillus thuringiensisThe gene gfpmut3a with specific promoter of B.cereus were cloned into a shuttle plasmid pHT304 between E.coli. and B.thuringiensis. It was then introduced into recipient strains BMB171, CryB, IPS78-11, 4Q7 and HD80-21 by electroporation. The results shown that green fluorescence could only been detected in acrystalliferous strains.2. Studies on the activites of promoters of Bacillus by using gfp geneThree kinds of promoters from Bacillus: P441-12, PCry3A and PBtI-BtII were chosen to drive the expression of gfp gene and were cloned into shuttle plasmids. Three recombinant plasmids of pGFP-304, pGFPExpA and pGFPExpB obtained were transformed into recipient strains of E.coli DH5a and B. thuringiensis BMB171 by electroporation. The results shown that gfp gene driven by Pcry3A could only express in BMB171. The VbtI-btII was found to show strong green fluorescence not only in BMB171 but also in DH5a. The activities of Pcry3A in recipient strains were much weaker than that of P44-12 and PBtI-BtII.3. Microcalorinetric study on recombinant strains of Bacillus thuringiensis containing diversity of promotersFluorescent intensities of BMB171(pGFP-304), BMB171(pGFPExpA) and BMB171(pGFPExpB) were detected by using bioactivity monitor. The results showed that gfp gene could be expressed by three kinds of promoters. Thermogenic curves and the results of metabolism heat output revealed that the heat output of BMB171(pGFP-304) and BMB171 (pGFPExpB) were less than that of BMB171(pGFPExpA).4. Construction of recombinant strains labeled with gfp gene in Bacillus thuringiensisBy SOE technic, a recombinant plasmid pBMBZGC10 with P4412 was obtained by the ligation of gfp-crylAc 10 fusion gene and vector plasmid pAD4412. Another recombinant plasmid pBMBZGC11 with PBtI-BtII was obtained by replacing promoter.And then they were introduced by gene pulser into acrystalliferous strains BMB171 and CryB to obtain the recombinant strains BMB171(pBMBZGC10) , CryB(pBMBZGC10), BMB171(pBMBZGC11) and CryB(pBMBZGCll). The results shown that green fluorescence could only been detected in recombinant strains BMB171(pBMBZGC10) and CryB(pBMBZGC10) by fluorescent microscopy. The expression of the fusion genes about 150 kDa ~ 160 kDa can be detected by SDS-PAGE.5. Analysis of fluorescent detection in recombinant strains of Bacillus jhuringiensis by microscopyFor all fluorescence microscopy work, Zeiss Axioplan Leica Diaplan and Leica M2FIII fluorescence Stereomicroscopes equipped with filter sets were used. Finally contained the following filters: excitation filter 488nm, and a 522(DF32)nm (only on the Zeiss microscope) emission filter. Furthermore, fluorescence intensity of recombinant strains detection by instrument of Bio-assay Reader. The GFP-long pass filter set was composed of: excitation filter 485nm, emission filter 520nm. All the data and pictures were done using professional soft of HT Soft 2.0 and Laser Sharp 2000?6. Studies on the effects of the 20kDa helper protein (P20) in recombinant strains of Bacillus thuringiensisA shuttle plasmid pBMB20-2 containing p20 gene was introduced into BMB171 (pBMBZGC10) and CryB(pBMBZGC10) by electroporation to obtain recombinant strains BMB171(pBMBZGC10-20) and CryB(pBMBZGC 10-20). The results showed that green fluorescence could be detected in them under the fluorescent microscope. Further study revealed that the 20 kDa helper protein can increase expressing of fusion protein gene by fluorescence intensity analysis.7. Studies on the construction of labeled recombinant strain and its cry 1 Ac 10 gene horizontal transfer of Bacillus thuringiensisA recombinant plasmid pBMBZGClO was obtained by the ligation of gfp-crylAc10 fusion gene and vector plasmid pAD4412. It was then introduced by gene pulser into acrystalliferous strain CryB and a recombinant strain CryB(pBMBZG更多还原