【作者】 段瑞旭；

【导师】 王金生； 宋从凤；

【作者基本信息】 南京农业大学 ， 植物病理学， 2008， 硕士

【摘要】 水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)引起的水稻白叶枯病(Bacterial Leaf Blight of Rice),是世界性的水稻重要病害。如同其它革兰氏阴性植物病原细菌—样拥有hrp基因(hypersensitive response on nonhost plants and pathogenicityon host plants)和无毒基因(avrirulence gene)或毒性基因(virulence gene)。hrp基因决定着植物病原细菌在非寄主植物上激发过敏反应(hypersensitive response,HR)和在感病寄主植物上的致病力(pathogenicity)。毒性基因则是以基因对基因互作的方式决定着寄主专化性。研究水稻白叶枯病菌的无毒基因,将有助于鉴定和克隆水稻的抗病基因,进一步明确无毒基因与抗病基因的互作的分子机制。Xoo基因组中存在着众多的avrBs3/pthA家族基因,为了了解这些基因在PXO99基因组中的存在特点,本实验利用来源于水稻白叶枯病菌菌株JXOⅢ的2.67Kb的无毒基因avrXa3重复区的2.4kb-BamHI片段作探针检测PXO99基因文库,通过菌落原位杂交及Southern杂交,将所得结果进行归类和分析,从87个阳性克隆中鉴定出13个不同的阳性克隆,它们在基因组中单独存在,或2个和2个以上串联排列。这基本上明确了PXO99基因文库所含的avrBs3/pthA家族基因的数量、类型及排列方式,为今后的研究提供了基本信息。在前人研究基础上,本实验对PXO99及其突变体PXO△avr做了进一步的研究,通过对其基因组酶切、southern杂交及作图分析,发现突变体PXO△avr与PXO99相比缺失了三条带,而有两条带信号减弱。对比PXO99基因文库的检测结果,发现有三个克隆P5、P8和P67与突变体PXO△avr缺失的信号带相对应,克隆P5包含了这五条带,克隆P8和P67分别包含了其中的三条与两条带。(丬寽)这三个文库克隆(P5,P8,P67)导入突变体PXO△avr,经苗期接种实验表明:P5互补突变体能使其毒性恹复接近PXO99菌株的致病表型,但并不是全部恹复,P8对表型影响较小,P67也能部分恹复突变体的功能。因此,推测由于随机交换产生的突变体中有5个基因被敲除,文库克隆P5、P8和P67与缺失突变体缺少的条带相关。同时也表明缺失的5个串联基因的综合作用主要表现为毒性功能。avrBs3/pthA家族基因之间的作用比较复杂,受多重因素的影响。经过对PXO99基因文库中无毒基因的研究分析,从中获得2个avrBs3/pthA家族基因克隆,P3.1和P3.4。序列测定结果显示,它们存在与PXO99基因组中,与avrBs3/phA家族成员一样,具有几乎一样的5′端和3′端、1个亮氨酸拉链(leucine zipper,LZ)、3个核定位信号(nuclear localization signal,NLS)和1个酸性转录活化域(acidictranscriptional activation domain,AAD),不同的只是所含102 bp重复单元的重复次数不等:P3.1有12.5个重复,P3.4有15.5个重复。它们分别与本实验室从JXOV中得到的克隆S11及已知的从PXO86中得到的克隆avrXa10舍有相同的重复数,经过比对,它们的可变区及其它区域均存在着差异。为了比较这些具有相同重复数,但序列不完全一致的克隆其功能是否相似或相同,本实验将其导人经过200mM 5-氮胞苷(5-anacytidine)标记的中国菌株OS-198和KS6-6中接种水稻近等基因系进行了致病性测验。结果表明这些具有相同重复数,但序列不完全相同的avrBs3/pthA家族基因,在水稻近等基因系上的致病能力并不完全一致,这也说明,无毒基因重复区中的可变区对于基因的功能是重要的。更多还原

【Abstract】 Xanthomonas oryzae pv.oryzae(Xoo) is one of the most important bacterial pathogens,which caused bacterial blight in rice.This pathogen possesses hrp genes and avirulence gene(avr) or virulence gene(vir) like other gram-negative plant pathogenic bacteria.The hrp genes confer the pathogenic bacteria the ability to trigger hypersensitive response(HR) on non-host plants and to cause diseade on host plants.The avirulence(avr) or virulence(vir) genes determine host specificity via gene-for-gene interactions.Research on avr genes in Xoo can help to identify and clone R genes from rice and to understand the molecule mechanisms between pathogenic bacteria and host plant.There are many avrBs3/pthA genes in Xoo,in order to understand the existance of avirulence genes in Xoo strain PXO99,its genomic library was screened by using a probe of avrXa3 which covers a 2.67kb- avrXa3 gene from Xoo JXOⅢ.87 positive clones were screened out of 1100 library clones.It indicates that there are multiple avrBs3/pthA genesin PXO99 genome.The positive cosmids DNA was digested with BamHI and Southern blot showed that there were 13 different clones in the pathogenic genome.The avrBs3/pthA genes occurred at isolated individual portions or in clusters.This study definitude the quantity、types and arrange manner of avirulence gene in the genome of PXO99.And it may provide some clues for further study.According to the previouse work done in our lab,an avirulence gene mutant PXO△avr was constructed by homologous recombination of avrXa3 from JxoⅢin PXO99.In this study,by comparing genomic DNA Southern blot result of PXO99 and PXO△avr,for mutant PXO△avr,three bands disapeared in size 4.3Kb,3.3Kb,2.9Kb and two bands weakened in size 4.0Kb and 3.6Kb as compared to wild-type.By analyzing the above 87 positive cosmid clones screened from PXO99 genomic library,three cosmid clones P5,P8 and P67 were found to contain five,three and two of the same size bands respectively as that changed in PXO99△avr.P5,P8 and P67 were introduced into PXO△avr, pathogenicity tests in near isogenic lines of rice confirmed that the cosmid P5 could restored the pathogenicity of PXO△avr;cosmid P8 made no change;cosmid P67 partly restored the pathogenicity of PXO△avr.The results showd bands in cosmid P5 were relevant with the changed bands in PXO△avr,but as to what role of each band play in the pathogenicity is still needed to be revealed.By screening the genomic library of PXO99,two avrBs3/pthA genes P3.1 and P3.4 were obtained.The two putative proteins of these genes were similar to other avrBs3/pthA genes,possessing the same 5’-and 3’-terminals,one leucine zipper(LZ),three nuclear localization signals(NLSs) and one acidic transcriptional activation domain(AAD).The repeat numbers of 102bp unit contained in P3.1 and P3.4 were 12.5 and 15.5,which were same as S11 from JXOV and avrXa10 from PXO86 respectively.Alignments of 12-13th residues in 34-amino-acid unit showed that they are different.In order to compare whether these genes have the same function or not,constructed vectors pZW::P3.4、pZW:: avrXa10;pZW::P3.1、pZW::S11 were introduced into Chinese strains OS-198 and KS6-6 which were mutanted with 5-anacytidine.pathogenicity tests in near isogenic lines of rice showed that:although these genes have the same number of repeats,but their function are not complely identical.更多还原