【作者】 张雨；

【导师】 唐纪良； 唐东阶；

【作者基本信息】 广西大学 ， 微生物学， 2013， 硕士

【摘要】 十字花科黑腐病菌（Xanthomonas campestris pv. campestris, Xcc）是引起多种重要的十字花科作物（如白菜、甘蓝、花椰菜、萝卜、芥菜、油菜等）黑腐病的病原菌。Xcc能感染模式植物拟南芥,是研究植物病原细菌与寄主植物相互作用机理的模式菌之一。此外,Xcc还是重要的工业原料,黄原胶的产生菌。由于Xcc在农业和工业上的重要性,它的分子生物学研究越来越受到人们重视。目前,已经有3个Xcc菌株（ATCC33913、8004和B100）的全基因组序列被测定。Xcc8004全基因组编码蛋白基因序列占84.79%,基因间隔区序列（Intergenic Regions, IGR）占15.21%。目前,研究IGR的功能已经成为功能基因学研究领域的热点之一。最近的研究也发现,没有基因注释的区域一基因间隔区也有转录活性并且具有重要的功能。本研究关注的问题是：Xcc8004基因组中两个最重要的致病调控基因hrpX和hrpG间隔区是否有转录活性和生物学功能。在XccS004基因组中,hrpX（基因ID号：XC₃076）与hrpG（基因ID号：XC3077）紧密相邻,但转录方向相反（hrpX是负链编码,hrpG是正链编码）；两个基因的起始密码子间相隔823bp。5’-Race结果显示,hrpX和hrpG的转录起始位点分别位于各自的起始密码子前79bp和129bp处。启动子分析结果显示,hrpX转录起始位点前68bp是hrpX的核心启动子区,hrpG转录起始位点前70bp是hrpG的核心启动子区。因此,hrpX和hrpG间功能未知的区域有476bp,我们把它定义为hrpX和hrpG真正的间隔区,命名为IGRhrpXG。我们课题组之前的RNA测序（RNA-seq）结果显示,无论是在基本培养基还是在丰富培养基的对数生长期,均能够检测到来自IGRhrpXG的转录子,预示IGRhrpXG是有转录活性的。为了进一步确证IGRhrpXG的转录活性,我们用RT-PCR方法检测。RT-PCR结果显示,用多对不同的引物均能扩增出来自IGRhrpXG的特异RT-PCR产物,证明IGRhrpXG确实是转录的。我们试图采用链特异RT-PCR方法确定来自IGRhrpXG的转录子数量、大小和转录方向,但没有成功。为了鉴定IGRhrpXG的生物学功能,保留hrpX和hrpG的核心启动子区缺失掉Xcc8004的IGRhrpXG全长476bp片段,构建的缺失突变体8004ΔIGR476hrpX-G致病力显著下降和不能诱导“非寄主植物上”发生过敏性（HR）反应。半定量RT-PCR显示8004⊿IGR476hrpX-G hrpX和hrpG基因转录水平严重降低,说明8004⊿GR476hrpX-G的致病力显著下降和HR反应丧失是由于hrpX和hrpG基因转录水平严重降低造成的。构建了缺失突变体8004⊿IGR476hrpX-G两个反式功能互补菌8004ΔIGR476hrpX-G/pL6-476和8004⊿IGR476hrpX-G/pL6-828,不能补回致病力和HR反应。猜测hrpX和hrpG的核心启动子不足够它们正常转录。构建⊿hrpG的hrpG基因片段（包含hrpG核心启动子区）反式功能互补菌⊿hrpG/pL6-70hrpG及互补菌8004⊿IGR476hrpX-G/pL6-70hrpG,表型分析显示都能补回致病力和HR反应。半定量RT-PCR结果显示8004⊿IGR476hrpX-G/pL6-70hrpG的hrpX和hrpG基因转录水平恢复正常。证明476bp片段的缺失,保留hrpX和hrpG的核心启动子区足够它们正常转录。为了检验476bp片段的作用。将476bp片段置换回到8004ΔIGR476hrpX-G的hrpX-hrpG基因间隔区,构建了互补菌8004ΔIGR476hrpX-G/476,476bp两边多出两个Xba I酶切位点。表型分析结果显示都能补回致病力和HR反应。8004⊿IGR476hrpX-G/476的hrpG的转录水平有一定程度的提高,HrpG浓度增加,足够激活hrpX转录,hrpX转录恢复到正常水平。推测IGRhrpXG是有功能的,可能包含有属于hrpG核心启动子外的上游元件或上游调控序列,通过对hrpG的转录影响,调控hrpX的转录,从而调控hrp基因簇下游基因。调控转录机制还有待进一步研究。更多还原

【Abstract】 Xanthomonas campestris pv. campestris（Xcc） is the causal agent of cruciferous plant including cabbage, cauliflower, radish, mustard, and arabidopsis thaliana. Xcc is one of the model bacteria for studying the mechanism of plant pathogenic bacteria-host plants interactions. Xcc is also an important industrial bacteria used for producing the industrial raw material xanthan gum. Up to date, the whole genomes of three Xcc strains,8004, ATCC33913and B100, have been sequenced.In the genome of Xcc8004,84.79%of the sequences are protein-coding sequences and15.21%are locating in intergenic regions （IGRs）. At present, the functional study of IGR has become one of hotspots in the field of functional genomics. Recently, it has been shown that some IGRs are transcribed and have important functions. In this study, we focus on:whether the IGR of hrpX and hrpG, the two key virulence regulatory genes of Xcc8004, is transcribed and has any biological function.In Xcc8004genome, hrpX （gene ID:XC₃076） and hrpG （gene ID: XC₃077） are closely adjacent but with opposite transcriptional orientations: hrpX is encoded on the minus strand and hrpG is encoded on the plus strand of the genome. The spacer between the start codons of the two genes is823bp （base pair）.5’-Race analysis demonstrated that the transcriptional start site of hrpX is located in79bp upstream of its start codon, and the transcriptional start site of hrpG is located in129bp upstream of its start codon. Promoter analyses reveal that the core promoter regions of hrpX and hrpG are68bp and70bp upstream of their transcriptional start sites, respectively. Thus, the476bp region between the hrpX and hrpG promoters is the area without any known function; we define this region as the IGR of hrpX and hrpG, named IGRhrpXG·Our previous RNA deep sequencing results showed that transcripts from IGRhrpXG are detected in RNA samples isolated from Xcc8004grown in the minimal medium MMX and the rich medium NYG, indicating that IGRhrpXG is transcribed. RT-PCR analysis show that specific products are detectable using different IGRhrpXG-specific primer sets, demonstrating that IGRhrpXG is indeed transcribed. We try to clarify the transcriptional orientation of the transcripts from IGRhrpXG by using Strand-Specific RT-PCR, but failed for an unknown reason.To identify the biology function of IGRhrpXG, the deletion mutant of named8004⊿GR476hrpX-G, was constructed. Phenotype analysis showed that,8004⊿IGR476hrpX-G displayed a reduced virulence on host plant and lost hypersensitive reaction （HR） on non-host plant. Semi-quantitative RT-PCR assays showed that the transcriptional level of hrpX and hrpG was significantly reduced compared to that in the wild type strain, suggesting that the reduction in virulence and lost HR of the IGRhrpXG deletion mutant are due to the reduction in hrpX and hrpG transcription.The trans-complementary strains8004⊿IGR476hrpX-G/pL6-476and8004⊿hrpG/pL6-828were constructed, can’t recover pathogenicity and HR response of wild type strain. Guess hrpX and hrpG core promoter is not enough to their normal transcription.The trans-complementary strain8004⊿hrpG/pL6-70hrpG and8004⊿IGR476hrpX-G/pL6-70hrppG were constructed. Phenotypes of the assay results demonstrated that the virulence and hypersensitive reaction of complementary compensated. By means of semiquantitative RT-PCR assays, transcriptional level of hrpX and hrpG recover in two. The core promoter of hrpX and hrpG is enough their normal transcription.In order to explore the function of the476bp sequence, we constructed cis-complementary strain8004⊿IGR476hrpX-G/476,476bp of sequence in which with two extra Xba I enzyme loci. The phenotypes of the assay results shewed that the virulence and hypersensitive reaction werere covered. Transcription level of hrpG in8004⊿IGR476hrpX-G/476increased to a certain degree, as a result, HrpG concentration increased, which enough to activate the hrpX transcription and hrpX transcription returned to normal levels.We harbor the expection that hrpX-hrpG gene spacer in Xcc8004is functional, which might contain upstream components outside the core promoter or upstream regulation sequence of hrpG. The hrpX-hrpG spacer regulates the transcription of hrpX, via regulating transcription of hrpG, thereby regulates the hrp gene cluster downstream. However, the transcription regulation mechanism remains to be further study.更多还原