【作者】 江晶；

【导师】 檀根甲； 江彤；

【作者基本信息】 安徽农业大学 ， 植物病理学， 2008， 硕士

【摘要】 rDNA核苷酸序列相似性最常用于真菌的分类和鉴定,其中ITS序列相对变异较大,已被用于多种炭疽菌的分类和鉴定。为进一步明确苹果炭疽菌分类地位,提高其检测效率和对苹果炭疽病进行早期诊断,本研究对苹果炭疽菌进行分子鉴定,并设计特异性引物用于苹果炭疽病的快速诊断。分子鉴定确定苹果炭疽菌应属于胶孢炭疽菌(Colletotrichum gloeosporioides)。本研究还在物理诱变筛选苹果炭疽菌低毒性菌株的基础上,运用RAPD和ISSR分子标记技术初步研究了苹果炭疽菌低毒性菌株的分子变异。本研究首先利用引物CgF/CgR克隆了苹果炭疽菌rDNA片段并测序,rDNA核苷酸序列全长3156 bp。截取苹果炭疽菌rDNA的ITS序列,与其它炭疽菌ITS序列进行核苷酸序列比对,发现苹果炭疽菌与胶孢炭疽菌的ITS序列相似性高达99.8%。构建苹果炭疽菌与其它炭疽菌ITS序列的系统关系树,发现苹果炭疽菌与胶孢炭疽菌单独聚成一个分支,因此,可以明确苹果炭疽菌应属于胶孢炭疽菌。进一步的序列比对发现,苹果炭疽菌的18S rDNA3’端比其它胶孢炭疽菌多出一段379 bp的序列,根据这一特有片段设计引物CgF1与通用引物ITS4配对,结果仅能从苹果炭疽菌中扩增出1232 bp的特异性条带。用苹果炭疽菌接种离体苹果,以接种发病的病组织总DNA为模板,利用引物CgF1/ITS4进行PCR扩增,同样可以扩增出1232 bp的特异性条带,而健康苹果组织DNA中未能扩增出任何条带,表明该方法可用于苹果炭疽菌的鉴定和快速检测。利用引物CgF/CgR同样克隆了通过离子注入和磁场照射方法筛选出的苹果炭疽菌低毒菌株rDNA全序列,将诱变的低毒菌株rDNA克隆、测序,序列全长3156bp。序列比较发现,苹果炭疽菌诱变菌株rDNA和未诱变菌株序列只有1-2个核苷酸差异,相似性高达99.8%,几乎没有分子变异。进一步利用RAPD和ISSR分子标记技术对苹果炭疽菌正常菌株和低毒菌株进行多态性扩增。实验筛选了30条RAPD随机引物,绝大多数随机引物不能扩增出丰富的多态性条带,只有随机引物C-9扩增条带较为丰富,但扩增的多态性条带完全相同,不能表现出苹果炭疽菌及其系列诱变株之间序列多态性差异。20条ISSR引物几乎均能扩增出多条多态性片段,但扩增出的苹果炭疽菌及其系列诱变株多态性条带高度一致,菌株间仍未能发现分子标记的差异性。初步研究未能发现苹果炭疽菌低毒菌株的分子变异。更多还原

【Abstract】 The similarity of rDNA nucleotide sequence is most extensively used in the classification and identification of fungi.The ITS sequence of rDNA has been used for the classification and identification of many Colletotrichum pathogens for its relative big variation.To determine the classification status of pathogen causing apple anthracnose more clearly,and to improve its detection efficiency and enable the early diagnosis of apple anthracnose,the research work on the molecular identification of apple anthracnose pathogens was carried out.The specific primers were also designed to be used for the rapid diagnosis of apple anthracnose.It was confirmed that apple anthracnose pathogen should belong to C.gloeosporioides through molecular identification.RAPD and ISSR molecular markers methods were utilized to analysis molecular diversity of low toxic strains of apple anthracnose pathogen on the basis of physical mutation screening low toxic strains of C.gloeosporioides.The rDNA sequence of apple anthracnose pathogen was determined.Sequence comparing of ITS of Colletotrichum was conducted and a phylogenetic tree based on alignment of their ITS nucleotide sequences was constructed.The results showed that Cg-1 and Cg-2 shared the highest sequence similarity(99.8%) with C.gloeosporioides and they could be grouped into one branch with C.gloeosporioides.It was confirmed that apple anthracnose pathogen should belong to C.gloeosporioides.We found that there is a 379 bp sequences residue in 3’ ends of 18S rDNA of apple anthracnose pathogen comparing with the other C.gloeosporioides.Specific primer CgF1 was designed according to the 379 bp sequence,and only a 1232 bp specific band could be amplified using primer pair CgF1 and ITS4.Apple fruits were inoculated with apple anthracnose pathogen.Total DNA was extracted from the diseased tissue,and an identical 1232 bp band was amplified.It was suggested that the method could be applied for the detection and rapid identification of apple anthracnose pathogen.Primers CgF/CgR were utilized to clone the rDNA sequence of low toxic strain of apple anthracnose pathogen which was screened by the ion implantation and magnetic field exposure methods.The rDNA sequence of the low toxic strain was cloned and sequenced.The full-length sequence of the rDNA is 3156 bp.Comparison with the original pathogen strain,the mutation strain has only 1-2 nucleotides different.They shared the sequence similarity as high as 99.8%,and the mutation strain had hardly any molecular variation. RAPD and ISSR molecular markers methods were utilized to amplify DNA polymorphism of the normal strain of apple anthracnose pathogen and its low toxic strains.Thirty RAPD random primers were screened,and most random primers can not amplify abundant patterns except for the primer C-9.But C-9 can only amplify the identical polymorphism patterns between the normal strain and its low toxic strains. Almost all the 20 ISSR primers can amplified multiple polymorphism patterns,but the polymorphism patterns amplified from the normal strain and its low toxic strains were identical completely.The differences of the molecular markers between the strains still could not be found.The molecular diversity also could not be found in the low toxic strains during the preliminary research work.更多还原