摘要
本研究主要围绕构建高效表达的GFP解离载体标记苏云金芽胞杆菌而开展的研究工作，主要研究结果如下：
1．利用小质粒复制区构建解离载体
利用Tn4430的解离区和来源于库斯塔克亚种大小为2062bp的小质粒复制区ori2062构建解离载体pBMB1125。将该载体电转化到苏云金芽胞杆菌中，解离载体pBMB1125在编码解离酶的帮助质粒pBMB1200作用下发生解离，消除抗性基因DNA片段，只留下目的基因以及苏云金芽胞杆菌质粒复制区的重组质粒，将解离后的菌株在无抗性培养基培养传代40代后仍可检测到解离后的重组质粒。
2．采用不同启动子、不同质粒复制区构建GFP表达解离载体
分别将带有芽胞依赖型启动子BtⅠ-BtⅡ、非芽胞依赖型启动子pro3A和蜡状芽胞杆菌启动子proB.c的gfp片段插入到以不同质粒复制区构建的解离载体pBMB1205，pBMB1206R以及pBMB1125上，得到9株以不同启动子、不同质粒复制区的表达GFP解离载体。将得到的9种GFP解离载体电转化到苏云金芽胞杆菌无晶体突变株BMB171中，荧光显微镜镜检显示9株含GFP的苏云金芽胞杆菌重组菌在波长为300nm-510nm的激发光下可发射绿色荧光。
3．建杀虫晶体基因与gfp基因的融合基因
PCR扩增全长gfp片段融合于去掉终止结构的杀虫晶体蛋白基因的C-端，构建融合基因。首先PCR扩增去掉终止结构的pro3A+cryⅠAc10片段，用BamHⅠ、SphⅠ酶切PCR产物插入到解离载体pBMB1205上得到中间载体pBMB1205Ac。同时扩增全长gfp片段，将扩增的gfp片段用SphⅠ消化插入到pUC19上得到pUC19G，再利用SphⅠ、BglⅡ双酶切消化pUC19G，回收两端酶切位点分别为SphⅠ、BglⅡ的gfp片段，将得到的gfp片段插入到pBMB1205Ac上最终得到重组的融合基因载体pBMB0474。将pBMB0474电转化到BMB171中得到苏云金芽胞杆菌重组菌，SDS-PAGE结果显示重组菌可以表达大小约150kDa-160kDa的GFP融合蛋白。
4．不同苏云金芽胞杆菌基因工程菌的微量热变化
利用生物活性检测器LKB-2277研究杀虫晶体蛋白含量不同的两株菌YBT-833、YBT-833-2-1的热动力学变化，发现菌体合成杀虫晶体蛋白的过程是一个耗能的过程，杀虫晶体蛋白产量高的菌株向外释放的代谢热少，反之亦然。研究含质粒拷贝数目分别为4、15的GFP菌株BMB304GFP、BMB315GFP的热动力学变化，发现质粒拷贝数目高的菌株向外释放的代谢热少，反之亦然。研究含不同质粒复制区构建的GFP解离载体的菌株BMB0452B、BMB0462B、BMB0472B的微量热变化，证明含小质粒复制区ori2062构建的
解离载体携带外源基因的菌株的代谢热少于以or汀草、oril哪口两种质粒复制区构建的GFP
解离载体。
研究还发现含不同启动子驱动GFP的菌株向外释放的代谢热也不同，含芽胞依赖型
启动子Bll-BtIJ的菌株BMB0462B的代谢热少于含非芽胞依赖型的启动子pro3A菌株
BMB0463B.含蜡状芽胞杆菌启动子proB.。的菌株BMB0461B的代谢热界于两者之间。根
据蛋白表达量同菌株代谢热的关系从9种GFP表达解离载体中筛选出一种高效表达GFP的
载体pBMB0472用以标记苏云金芽胞杆菌。
5.肺基因标记苏云金芽胞杆菌野生菌
将GFP解离载体pBMB0472以及融合蛋白载体pBMB0474分别电转化到拟步行甲亚
种YBT一1765中，得到标记的野生型苏云金芽胞杆菌YBT1765一72B、YBT1765一74。PCR扩
增结果显示均可从两株标记基因工程菌中得到肺片段，且还可以从YBTI 765一74中扩增出
犯T一1765中没有的c尽]A cjo基因。SDS一队GE结果显示标记野生菌YBT1765一72B可以表
达大小为27kDa的GFP，但YBT1765一74中检测不到融合蛋白的表达。荧光显微镜观察结
果表明YBT1765一72B在激发光下可以发射绿色荧光。
The dissertation mainly concerns the construction of resolution vector with green fluorescent protein. The research results are summarized as following.
1. Construction of a resolution vector with a small plasmid origional replion
A small plasmid original replion about 2062bp come from B. thuringiensis subsp. kurstaki strain YBT-1520 and the res site of Tn4430 were used to construct a resolution vector. The resolution vector based on Tnpl-mediated site-specific recombination system of B. thuringiensis transposon Tn4430. The gene of or/2062 and other target DNA were inserted into two copy sets of res sites. The res sites have the same direction. With the help of the integrase( Tnpl) the antibiotic resistance genes and other non-5, thuringiensis DNA can be eliminated. When the engineered strain, which had the recombinant plasmid without resistance gene cultured for 40 generations the very recombinant plasmid could still be detected.
2. Construction of green fluorescent protein resolution vector with various promoters and different origional replicons
In this study, three kinds of specific promoter of B.thuringiensis: crySA promoter, Btl-BtH promoter and 5. cereus promoter were chosen to drive the expression of gfp. Three kinds of plasmid original replion of B.thuringiensis subsp. kustaki: ori44, oriW30 and ori2062 were chosen to construct the GFP resolution vector. There were 9 kinds of GFP resolution vector. All of the recombinant plasmids were introduced into crystal negative B. thuringiensis 8MB 171.The fluorescence of the 9 kinds engineered strain can be detected by the fluorescent microscope.
3. Construction of fusion genes with insecticidal protein gene and gfp
The fusion genes was constructed by PCR. The pesticidal crystal protein gene crylAc10 and gfp were chosen to construct the fusion genes. The gfp gene was fused to the C-terminate of crylAclO. The two genes were in an open reading frame. The fragment of pro3A+crylAc10 without terminated structure was inserted into plasmid pBMB1205 when it was digested by BamHI and SphI. The recombinant plasmid pBMB1205Ac was obtained. Recombinant plasmid of pUC19G was constructed when PCR product of gfp was digested by Sphl and inserted into pUC19. The gfp fragment with Sphl and BglII enzyme sites from pUC19G was inserted into pBMB1205Ac. The very plasmid pBMB0474 was constructed. When the recombinant plasmid pBMB0474 was transferred into crystal negative Bt strain BMB171 through
electroporation, the expression of the fusion genes about 150kDa-160kDa can be detected by SDS-PAGE.
4. Microcalorinetric study on B. thuringiensis
By using an LKB-2277 Bioacitivity Monitor, the thermogenic curves of different B thuringiensis strains YBT-833 and YBT-833-2-I, have been determined. The metabolism heat output revealed the heat output was correlated to the yield of the protein, the higher yield protein, the less heat output. A microcalorimetric technique based on the bacterial heat-output was explored to evaluate the effect of various promoters and different plasmid original replicons on the expression of GFP. In this study the heat output also suggested there was obvious relation between the gene copy number and heat output. The heat output rate of different strains with various copy number is BMB304GFP) BMB315GFP. The relation of the heat output rate with original replion is or/1030) or/44) or/2062. The heat output indicated that different promoter had various impact on the expression of GFP. The drive impact was promoter Btl-Btll } promoter B. cereus ) promoter cry3A.
5. Wild-type B.thuringiensis tabled with green fluorescent protein
Plasmid pBMB0472 and pBMB0474 were introduced into wild type B.thuringiensis subsp. tenebrionis YBT-1765 by electropotation. The transformams YBT1765-72B and YBT1765-74. were obtained. SDS-PAGE result suggested GFP about 27kDa was expressed in YBT1765-72. But the fusion protein could not be detected in YBT 1765-74. Fluorescent microscope observation results indicated there was fluorescence in YBT1765-72B. but there was no flu
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