【作者】 孟凡宏；

【导师】 王金生；

【作者基本信息】 南京农业大学 ， 植物病理学， 2006， 硕士

【摘要】 大多数革兰氏阴性植物病原细菌都存在hrp基因（hypersensitive response and Pathogenicity），其决定病原菌对感病寄主植物的致病性和非寄主植物的过敏反应。Harpin是一类由革兰氏阴性植物病原细菌hrp基因编码的蛋白质激发子，可以激发烟草过敏性细胞坏死（hypersensitive cell death，HCD），诱导植物抗病、抗虫，促进植物生长。hrfA（HR function）基因是本实验室通过PCR方法，从水稻黄单胞细菌白叶枯致病变种（Xanthomonas oryzae pv．oryzae，Xoo）JxoⅢ菌株中克隆到的。hrfA全长420bp，编码的harpin_Xoo具有其它harpin蛋白所共有的生物活性：激发烟草HR，诱导烟草对多种病原物的抗性。但是harpin_Xoo还显示一些独特性：富含甘氨酸（Gly），同时也含有半胱氨酸（Cys），分子量比其它已报道的harpin都小。本实验室已成功将hrfA转入烟草、水稻、棉花等作物中，都不同程度地提高了转基因植物的抗病能力。本实验室李平通过PCR方法，克隆到hrfA基因的5个缺失突变基因AB、CD、EF、AD和CF，并在Escherichia coli中表达得到缺失多肽片段。Harpin_Xoo N-末端的AB片段仍保持诱导过敏反应和抗病性的能力；中部的CD多肽片段失去了诱导过敏反应的能力，但仍可以诱导抗病性，而且当CD与其它片段组合时，可增强它们的活性。所以推测CD可能在harpin_Xoo分子与植物相互识别的过程中起着增强子的作用。因此，深入研究AB和CD两个多肽片段的诱导抗病性机理将是有趣的理论问题。本研究将hrfA基因及其片段AB，CD分别从pET30a（+）∷hrfA，pET30a（+）∷AB，pET30a（+）∷CD中用BamHⅠ和XbaⅠ双酶切后回收，连接到用这两个酶切过的转基因表达载体pBI121上，构建成重组转基因载体pB-hrf，pB-AB，pB-CD。重组载体转化至大肠杆菌DH5α中，经测序验证后，通过冻融法转化根癌土壤杆菌EHA105。经菌落PCR和酶切检测后，挑选阳性克隆供转化烟草使用。采用叶盘法转化烟草（Nicotiana tobacum cv．Xanthi nc．），用卡那霉素抗性筛选再生植株。通过PCR扩增检测了转化烟草中的靶基因序列及35S启动子序列，对T1代PCR阳性植株进行了PCR-Southern杂交和RT-PCR分析。结果显示外源基因已经整合到转基因烟草基因组中，并且能在转录水平正常表达。从转hrfA基因和AB片段的烟草中提取的可溶性蛋白注射烟草能引起过敏反应，而转CD片段的烟草蛋白则不能。在相同培养条件下，转基因烟草的株高、叶片大小和花期等性状与受体植株无明显差异，生长发育正常，无肉眼可见的HR。用trypan blue对转基因烟草叶片进行染色发现，转基因表达harpin_Xoo及其AB多肽片段能够诱导烟草产生micro-HR，而转基因表达CD多肽片段不能诱导烟草产生micro-HR。抗病性鉴定结果表明，转hrfA基因及其两个缺失片段的烟草对烟草花叶病毒（TMV）都有较好的抗性。更多还原

【Abstract】 Most gram-negative bacterial pathogen possess hrp （hypersensitive response and pathogenicity） gene cluster, which controls the ability of the pathogen to cause disease in susceptible plants and elicit HR in resistant plants. Harpins, a group of proteins encoded by hrp genes of gram-negative bacteria, can elicit hypersensitive cell death in tobacco leaves, induce disease and insect resistance in plants, and promote plant growth. hrfA （HR function） gene was identified by our lab in a Japanese strain of Xanthomonas oryzae pv.oryzae. It is 420 bp in length and encodes harpin_xoo protein, which can also elicit HR in tobacco and induce disease to many pathogens. Harpin_xoo has some specialties: rich in Gly, it also contains Cys; moreover, it is smaller than many other harpins. Our lab has successfully transformed hrfA into tobacco, rice and cotton, and disease resistance of these transgenic plants have been enhanced. Li Ping has cloned five deletion derivatives of hrfA （i.e. AB, CD, EF, AD and CF） by PCR and expressed them in Escherichia coli BL21/pET30a（+）. Fragment AB, which contains N-terminal 59 amino acids, can trigger HR and induce resistance and is the best one with multifunction among deletion derivatives. The middle fragment CD （99 bp） fails to trigger HR but can induce resistance normally. When combined with other fragments, CD can enhance their bioactivity, thus, the fragment CD may act as enhancer in triggering HR. So it would be very interesting to study the resistance mechanisms of AB and CD.In this study, we excised the hrfA gene and its two deletion derivatives, AB and CD, from pET30（a） by digestion with restriction enzymes BamH I and Xbal I, and inserted them into the transformation vector pBI121 between the CaMV35S promoter and theβ-glucuronidase gene uidA. The resulting three recombinant constructs, pB-hrf, pB-AB, pB-CD, were transformed to E. coli DH5a. After verifying correct orientation of genes, these three transgenic consrtucts were transformed into Agribacterium tumefaciens EHA105 by the freeze-thaw method. Transformation was conducted by infection of tobacco leaf discs with recombinant A. tumefaciens, and transgenic tobacco plants were obtained by screening with kanamycin. Genomic integration and expression of the transferred genes were determined by PCR, PCR-Southern blot hybridization and reverse transcriptase PCR. Soluble proteins extracted from plants transformed with hrfA and AB could elicit HR on tobacco leaves, but those from CD-transformed plants could not.Under the same culture conditions, there was no apparent difference between the transgenic and parent plants in heights, size of leaves, and flowering time. The transgenic plants growed normally, without macroscopical HR. When stained with trypan blue, leaves from plants transformed with hrfA and AB showed micro-HR, whereas leaves from CD-transformed plants could not. Disease bioassays showed that, the tobacco plants transformed with deletion derivatives, AB and CD, enhanced resistance to TMV similarly as those transformed with the whole hrfA gene.更多还原