目录
摘要Ⅲ
关键词Ⅲ
AbstractⅣ
引言
引言1
1 材料与方法1
实验材料 1
1.1.1菌株、质粒1
1.1.2 本实验所用引物2
1.1.3 主要仪器与试剂3
1.2 实验方法 3
1.2.1 构建荧光报告载体3
1.2.2 芽胞杆菌转化、荧光观察7
2 结果与分析7
2.1 荧光报告载体的构建7
2.1.1 载体pMK3Phag168gfp的构建7
2.1.2 载体pMK3PcspB168gfp的构建 8
2.1.3 载体pMK3PsspB168mcherry的构建8
2.1.4 载体pMK3PtapA168mcherry的构建9
2.1.5 载体pMK3PppsA168mcherry的构建 10
2.1.6 载体pMK3PtapAFZB42mcherry的构建 10
2.1.7 载体pMK3PppsAFZB42mcherry的构建 11
2.2 芽胞杆菌转化结果11
3 讨论12
3.1 荧光蛋白可行性分析12
3.2 芽胞杆菌转化不成功的原因12
3.2.1 缺少接头蛋白 12
3.2.2 OD600控制不够精确 12
参考文献13
致谢14
附录15
图21 载体pMK3Phag168gfp的验证8
图22 载体pMK3PcspB168gfp的验证8
图23 载体pMK3PsspB168mcherry的验证9
图24 载体pMK3PtapA168mcherry的验证9
图25 载体pMK3PppsA168mcherry的验证10
图26 载体pMK3PtapAFZB42mcherry的验证10
图27 载体pMK3PppsAFZB42mcherry的验证11
图28 载体pMK3Phag168g *51今日免费论文网|www.51jrft.com +Q: @351916072@ 
fp在芽胞杆菌OKB105中的表达情况11
表11 本研究所用的质粒及菌株1
表12 引物名称和序列2
表13 左右臂PCR扩增体系5
表14 酶切体系6
表15 T4连接体系6
荧光蛋白基因标记生防芽胞杆菌生物膜和抗菌物质合成相关基因的研究
摘 要
生物膜和抗菌物质是芽胞杆菌重要的生防因子。本研究我们将构建基于pMK3质粒的生物膜和抗菌物质合成相关基因荧光标记表达载体，转入芽胞杆菌模式菌株并获得转化子，为筛选和鉴定促进生防因子表达的诱导物质打下基础。实验中，我们成功将鞭毛蛋白相关基因（hag）、脂肽类化合物合成基因（srfAA、ppsA）、生物膜合成相关基因（tapA）、芽胞形成相关基因（sspB）、冷激蛋白（cspB）的启动子连接上报告基因（gfp和mcherry），构建了基于pMK3质粒的荧光报告载体。将荧光报告载体pMK3Phag168gfp转化枯草芽胞杆菌模式菌株OKB105，在荧光电镜下观察到较强的绿色荧光，表明hag基因启动子能够正常启动gfp基因的表达。
STUDY ON FLUORESCENT PROTEIN GENES MARKING GENES RELATED TO BIOFLIM AND ANTIBACTERIAL SUBSTANCE SYNTHESIS OF BACILLUS SPP.
ABSTRACT
Biofilms and antibacterial substances are important biocontrol factors for Bacillus. In this study, we constructed fluorescent marker expression vectors for genes related to biofilm and antibacterial substance synthesis, which is based on pMK3 plasmid. Then we transformed it into a Bacillus model strain and obtain transformants, and lay the foundation for screening and identification of inducing substances that promote the expression of biocontrol factors. In the experiment, we successfully linked the promoters of flagellinrelated genes (hag), lipopeptide synthesis genes (srfAA, ppsA), biofilm synthesisrelated genes (tapA), spore formationrelated genes (sspB), and cold shock protein (cspB) to the reporter genes (gfp and mcherry) so that we constructed a fluorescent reporter vector based on the pMK3 plasmid. The fluorescent reporter vector pMK3Phag168gfp was transformed into Bacillus subtilis model strain OKB105, and strong green fluorescence was observed under fluorescent electron microscope, indicating that the hag gene promoter can successfully start the expression of gfp gene.

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