【作者】 张春燕；

【导师】 王志伟； 陈怀谷；

【作者基本信息】 南京农业大学 ， 微生物学， 2014， 硕士

【摘要】 丝核菌属(Rhizoctonia)真菌类群繁多、分布范围广泛,大部分类群为植物病原菌,可引发多种严重植物病害。其中,禾谷丝核茵(Rhizoctonia cerealis)可以导致小麦纹枯病,是该病的主要病原菌。茵丝融合群(anastomosis group)是丝核茵最经典的分类方法。目前为止,多核丝核茵共分为14个融合群,双核丝核茵共分为21个融合群。其中禾谷丝核菌是双核丝核茵,属于AG-D融合群。目前对该菌的基因组序列还无报道,对其系统发育学研究也需进一步深入。因此本研究利用实时定量PCR绝对定量的方法对禾谷丝核茵标准菌株R0301的基因组大小进行了预测,并利用翻译延伸因子(translation elongation factor, tef)基因序列对禾谷丝核茵和其他丝核菌融合群进行了系统发育分析,为今后研究禾谷丝核茵的基因组奠定基础。首先用真菌tefA基因通用引物对分离自江苏南京的强致病力禾谷丝核菌R0301菌株的基因组进行了扩增,对克隆测序得到的序列进行Blast分析,结果表明该序列确为禾谷丝核菌的tefA基因序列。应用Southern杂交方法证明了tefA基因在禾谷丝核菌基因组中是单拷贝。利用实时定量PCR绝对定量的方法对已进行全基因组测序的多核立枯丝核茵AG-1-IA标准菌株GD118、AG-1-IB标准菌株7／3／14进行基因组大小的预测。以绝对定量的标准品制作标准曲线,PCR扩增标准菌株的tefA基因进行定量,依据Wilhelm提出的公式计算得到两株标准菌株的基因组大小。该方法预测的结果与基因组测序拼接的基因组大小较接近。结果表明实时定量PCR方法适用于多种真菌进行基因组大小的预测,也可以用于丝核菌的基因组大小预测。实时定量PCR技术可作为一种快速、简便的方法来预测丝核茵基因组大小。本研究利用实时定量PCR技术对禾谷丝核菌R0301菌株进行了基因组大小的预测,首次预测了禾谷丝核茵的基因组大小位于32.2-36.6 Mb。本研究运用实时荧光定量PCR技术首次预测了禾谷丝核茵的基因组大小,对于物种分类、鉴定、系统发育和其致病机制等方面有重要意义,也为全基因组的测序和拼接奠定了一定的基础。从山东、河南、河北和安徽等地小麦田采集分离的18株禾谷丝核菌株及其他融合群的11株标准菌株的tefA基因序列进行了克隆,并挑选阳性克隆进行测序,将这些菌株的tefA基因序列进行了遗传进化分析。克隆测序和PCR产物测序得到的序列一致,且不同克隆问的序列不存在多态性。系统发育树显示,tefA可以很好地区分丝核菌的融合群和融合亚群,且同一融合群及同一菌株之间没有异质性,可作为丝核茵分类鉴定的分子标记。扩增丝核茵同一融合群不同菌株的tefB基因序列,将PCR纯化产物进行克隆测序,并对得到的序列进行了系统进化分析。系统发育分析显示,tefB可以很好地区分丝核茵与其他伞菌纲真菌,但是同一融合群间的菌株具有遗传多样性,序列保守性不及tefA基因。最后,依据tefA基因序列和翻译的氧基酸序列,分析了该基因的结构特征,并按照氨基酸序列预测了二级和三级结构,tefA的二级结构是以卷曲和a-螺旋为主要的结构元件,没有β撕叠和p-转角结构；其目标蛋白序列与模型序列的相似度很高,可认为目标蛋白的三维结构与模型三维结构基榭目似。本研究通过Southern杂交技术确定了禾谷丝核茵体内tefA基因为单拷贝,应用实时定量PCR技术预测了禾谷丝核茵的基因组大小。利用矽基因对不同融合群的丝核茵系统发育学分析结果表明tefA可以很好地区分丝核茵的融合群和融合亚群,且同一融合群及同一菌株之间没有异质性,tefB可以很好地区分丝核茵与其他伞茵纲真茵,但是同一融合群间的茵株具有遗传多样性。研究结果为今后探索tef基因作为丝核茵分类鉴定的分子标记奠定了重要基础。更多还原

【Abstract】 The genus Rhizoctonia consists of a wide variety of fungi and is distributed worldwide, among which most of them are phytopathogen, which is responsible for many serious plant diseases. Rhizoctonia cerealis is the causal pathogen of sharp eyespot in wheat.The most classical classifiation method of Rhizoctonia is anastomosis group method based on hyphal fusion interaction. Currently, multinucleate Rhizoctonia is divided into 14 anastomosis groups (AG-1 to AG-13 and AG-BI), The binucleate Rhizoctonia is divided into 21 anastomosis groups (AG-A-AG-U). The binucleate species Rhizoctonia cerealis belongs to the AG-D anastomosis group. The genome information of Rhizoctonia cerealis has not been reported yet, and the phylogenetic analysis of it needs further research. Accordingly, our research estimated the genome size of Rhizoctonia cerealis R0301, made a phylogenetic analysis using amplified translation elongation factor gene of Rhizoctonia cerealis and other anastomosis group Rhizoctonia, which lay the foundation for the analysis of genomy of Rhizoctonia cerealis.Our study amplified the genome of Rhizoctonia cerealis R0301 virulent strain isolated from Nanjing Jiangsu province, using fungal translation elongation factor universal primers, made a Blast analysis of the obtained sequence, identified the sequence as tefA gene of Rhizoctonia cerealis. Southern blot analysis indicated tefA was a single copy in the genome of Rhizoctonia cerealis.We used the quantitative real-time PCR method to calculate the genome size of the sequenced mutinucleat Rhizoctonia solani AG-1-IA strain GD118, AG-1-IB strain 7/3/14. Using the real-time quantitative PCR amplified the tefA gene of standard strains, then quantified the gene based on absolute quantification standard calibration curve, calculated their genome size according to the formula proposed by Wilhelm. The estimated results by this method are almost in accordance with the genome size established by genome sequencing. The results show that real-time PCR method is applicable to a variety of fungal genome size estimation, which can also be used to estimate the genome size of Rhizoctonia.We estimated the genome size of Rhizoctonia cerealis R0301 via the quantitative real-time PCR method and reported the genome size of R. cerealis R0301 was between 32.2-36.6 Mb firstly. Quantitative real-time PCR was a fast, highly accurate and reliable method for the genome size estimation of Rhizoctonia. Our research first reported the genome size of Rhizoctonia cerealis via the quantitative real-time PCR method, which is of great importance for the species classification, identification, phylogenetic analysis and its pathogenic mechanism, but also the foundation for whole genome sequencing and assembly.We cloned and sequenced the partial translation elongation factor 1 gene (tefA) of R. cerealis strain R0301 and other anastomosis group strains isolated from Shandong、HenanN Hebei and Anhui wheat field,and made a phylogenetic analysis of these strains. The cloned sequence is in accordance with the sequence obtained by PCR product, and there is no polymorphism between different clones. The phylogenetic tree shows that tefA gene can well distinguish Rhizoctonia anastomosis group and subgroup and there is no heterogeneity between the same anatomosis group and the same strain, which can be used for Rhizoctonia classification and identification. We also cloned and sequenced the partial translation elongation factor 2 gene (tefB) of R. cerealis strain R0301 and other anastomosis group strains and made a phylogenetic analysis of these strains. The phylogenetic analysis showed that, tefB can well distinguish Rhizoctonia and other AG aric classes, but the same anastomosis group have the genetic diversity, and tefA is more conservative than tefB gene.Finally, we analyzed the structure of the gene and the amino acid sequences translated based on the tefA gene sequence and translated amino acid sequence, from the secondary structure and tertiary structure, we can see that the main secondary structure of tefA is a-helix and crimped elements, no P-sheet and P-turn structure were found; There is a high similarity between the target protein sequence and the model sequence, It can be considered that the three-dimensional structure of the target protein and the three-dimensional structure of the model are similar.Our study indicated tefA gene was a single copy in the genome through Southern blot analysis, estimated the genome size of Rhizoctonia cerealis by real-time quatitative PCR. The phylogenetic analysis of different anastomosis group of Rhizoctonia shows that tefA gene can well distinguish Rhizoctonia anastomosis group and subgroup and there is no heterogeneity between the same anatomosis group and the same strain, te/B can well distinguish Rhizoctonia and other AG aric classes, but the same anastomosis group have the genetic diversity. Our research laid an important foundation for exploring tef gene as molecular markers for Rhizoctonia classification and identification.更多还原