【作者】 肖杨；

【导师】 程萍；

【作者基本信息】 中山大学 ， 微生物学， 2010， 硕士

【摘要】 石斛兰软腐病的致病菌是胡萝卜软腐欧文氏菌,且其致病机理与自身的群感效应系统有关。苏云金芽孢杆菌(Bacillus thuringiensis,Bt)的aiiA基因编码的AiiA蛋白通过降解致病菌群感效应系统中的信号分子来扰乱其群感效应从而达到防治石斛兰软腐病的效果;抗菌肽具有广谱抗性,对于细菌、真菌、病毒、原核生物及癌细胞都有一定毒性,甚至可以直接杀死某些微生物,所以克隆自棉铃虫(Helicoverpa armigera)的hacD基因其编码的抗菌肽也可以作为防治石斛兰软腐病的策略之一。本论文拟通过对aiiA基因与抗菌肽hacD基因的成熟片段进行原核表达,并分析这两种蛋白的活性,然后将aiiA基因与抗菌肽hacD基因的成熟片段融合后,应用农杆菌介导的方法将此融合基因转入石斛兰,以期获得抗软腐病的石斛兰植株。本研究通过PCR方法克隆了aiiA基因(来自Bacillus thuringiensis)和抗菌肽hacD基因的成熟肽片段(来自Helicoverpa armigera),将这两个基因融合,hacD基因融合到aiiA基因的下游端,并在两个基因的融合处引入Factor Xa的酶切位点,将融合基因克隆到原核表达载体pQE-30上并转化大肠杆菌Ml5,经IPTG诱导,高效表达出了大小为34 kDa的融合蛋白。融合蛋白经非变性纯化、脱盐浓缩后,测定了其生物活性。对融合蛋白进行了活性分析,结果显示只有经Factor Xa酶切释放出来抗菌肽能够抑制标准抗菌肽活性测试菌E.coli K12D31的生长;对AiiA的活性进行了测定,结果表明融合蛋白经酶切后释放出的AiiA蛋白及未经酶切的融合蛋白均保持了降解AHLs分子的能力;除此之外,胡萝卜侵染实验发现,经融合蛋白处理过的胡萝卜软腐欧文氏菌能够减缓软腐病症。将aiiA基因和抗菌肽hacD基因的成熟肽片段融合后,克隆进包含有花椰菜花叶病毒35S(Cauliflower Mosaic Virus 35S promoter, CaMV 35S)启动子、胭脂碱合成酶基因的终止子(NOS终止子)的双元转化载体pCAMBIA1301(含有潮霉素选择标记基因)中,构建成高效植物表达载体pC-AH。随后,利用冻融法将pC-AH导入农杆菌EHA105从而获得工程菌,并将此工程菌转化石斛兰。经过17个月的选择培养,获得13株具潮霉素抗性的转基因苗。经PCR,Southern blot分子生物学方法分析,结果证明其中3株抗性苗的基因组中已整合了融合基因aiiA-hacD。更多还原

【Abstract】 Dendrobium is one of the most popular ornamental flowers in the world. However, they’re prone to be infected by the soft rot disease, which tends to result in a lot of loss. The pathogenic bacteria called Erwinia carotovora. are responsible for the soft rot disease on Dendrobium and their expression of virulence factors is coordinated by AHL-mediated quorum-sensing system. The AiiA protein encoded by the aiiA gene from Bacillus thuringiensis (Bt) has been reported to inactivate bacterial virulence through degrades signal molecules of quorum-sensing system. Due to this, it will be one of possible strateges to promote resistance to soft rot; Antibacterial peptides were toxic against a broad- spectrum of bacteria, fungi, viruses, protozoa and cancer cells, they even could directly kill bacteria. So, it will also be one of possible strateges to promote resistance to soft rot; In this study, prokaryotic expression vector of fused aiiA-hacD gene was constructed and expressed in E.coli. Den.Tungku.Anis was transformed with Agrobacterium tumefaciens carrying the fusion gene to expect of acquiring plants that will be more resistant to Erwinia carotovora. infection than the wild-type plants.The region of hacD gene from cotton budworm by PCR amplification (Helicoverpa armigera ), which only codes mature antibacterial peptides, were fused with down-stream sequence of the aiiA gene from Bacillus thuringiensis and a factor Xa recognizing site was designed between them. The resulting fusion gene, aiiA-hacD, was cloned to expression vector pQE-30 (Qiagen) and effectively expressed in E.coli M15. The 34-kDa recombinant AiiA-HacD protein was purified and analyzed its biological activity. The bioassay of AiiA-HacD fusion proteins suggested that only HacD released from the fusion protein can inhibit the reproduction of E.coli K12D31. Both the fusion protein and the production of fusion protein digested by factor Xa protease showed AHL-degrading activity. Besides, the production of fusion protein digested by factor Xa protease could increases the ability to inhibit soft rot caused by Erwinia carotovora.The fusion gene, aiiA-hacD, was cloned into the binary vector pCAMBIA1301 to obtain the recombinant plasmid, pC-AH. The fused gene was driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter and stopped by the nopaline synthase terminator(NOS). Plant expression vector pC-AH were transformed into the Agrobacterium strain EHA105 to create the engineering strain contains aiiA-hacD gene. Then, Dendrobium was transformed by EHA105 equipped with pC-AH. After 17months of selection culture, 13 hygromycin-resistant plantlets were acquired. There were 3 transgenic plantlets containing the fused gene after kanamycin screening, PCR detection and Southern blot analysis.更多还原