【作者】 刘茜；

【导师】 孙丽英；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2019， 硕士

【摘要】 类病毒是一类共价闭合的环状单链RNA,不编码任何蛋白质,其基因组大小在246-434 nt范围内。类病毒自身的稳定性强,不容易被脱氧核糖核酸酶降解。它是迄今发现最小的能进行自我复制的遗传因子。在自然界中,可引起十分严重的植物病害。国际病毒分类委员会将目前已经鉴定的44种类病毒分为两个科,七个属。本实验中,我们选取了具有不同属代表性的7种类病毒,它们分别是:马铃薯纺锤形块茎类病毒(Potato spindle tuber viroid)、柑橘裂皮类病毒(Citrus exocortis viroid)、血苋类病毒1号(Iresine viroid 1)、苹果锈果类病毒(Apple scar skin viroid)、啤酒花矮化类病毒(Hop stunt viroid)、桃潜花叶类病毒(Peach latent mosaic viroid)、鳄梨日斑类病毒(Avocado sunblotch viroid)。本研究所选用的丝状真菌为板栗疫病病原菌,该菌是属于子囊菌亚门、核菌纲、球壳菌目的寄生内座壳(Cryphenctria.Parasitica)。本课题主要研究类病毒能否侵染栗疫病菌并在栗疫病菌中进行复制。此外,研究类病毒的侵染对宿主真菌的生长,发育和致病性的影响。本实验为研究类病毒的宿主范围,发病机制和潜在的生物控制用途提供了新的研究思路和科学依据。本实验首先构建七种类病毒的侵染性克隆,将类病毒的体外转录物通过机械摩擦侵染烟草检验其活性;并通过转化的方法侵染酵母菌株,验证类病毒是否能在酵母菌株中进行复制。通过原生质体转化的方法将类病毒体外转录物人工导入C.Parasitica原生质体细胞中,细胞再生后,在PDA培养基上筛选转化子,提取栗疫病菌ssRNA,设计特异性引物,利用RT-PCR技术对类病毒的侵染情况进行检测。观察稳定侵染的类病毒对寄主真菌的生长、发育及致病力的影响。主要的研究结果如下:1.构建七种类病毒侵染性克隆:本实验利用体外转录的方法,构建了ASBVd HSVd IRVd PLMVd ASSVd IRVd CSVd七种类病毒的侵染性克隆质粒,获得体外转录类病毒RNA后,摩擦接种烟草检测其活性,并验证其是否能侵染酵母菌株AH109。2.检测七种类病毒侵染丝状真菌的能力:将七种供试类病毒导入C.Parasitica原生质细胞后,利用RT-PCR技术检测发现:ASBVd、HSVd、IRVd能够在C.Parasitica中进行复制,其中类病毒ASBVd的复制相较其他两种类病毒更为稳定;ASBVd侵染的菌株经过继代培养后提取ssRNA进行RT-PCR检测,实验结果表明,ASBVd的复制量无明显减少,其他两种类病毒的复制量随继代次数的增加而逐步降低。3.分析ASBVd侵染对C.Parasitica的影响:类病毒侵染后,对栗疫病菌表型产生一定影响,菌株的生长速率及色素积累量等发生变化。4.分析ASBVd在真菌寄主中的传播情况:ASBVd侵染C.Parasitica后,与真菌病毒相似,可通过寄主菌丝融合进行传播。经RT-PCR检测证实,类病毒可从带毒菌株传播至无毒菌株;将ASBVd侵染的C.Parasitica置于光照下培养至菌株产孢后,进行单孢分离培养,提取真菌ssRNA并利用RT-PCR技术检测发现,类病毒可通过孢子进行传播。5.分析ASBVd侵染真菌寄主后的遗传变异情况:类病毒侵染栗疫病菌后,将菌株继代培养,提取第8代菌株ssRNA进行检测并测序发现,随菌株继代培养后,类病毒序列发生了突变。6.对比、分析ASBVd野生型与突变株系的侵染活力及对寄主真菌的影响:ASBVd突变株系侵染栗疫病菌后,菌株气生菌丝的量增加,生长力无明显变化,提取真菌ssRNA进行检测,结果表明,ASBVd的复制量与野生型中相比有所降低,侵染活力有所下降。更多还原

【Abstract】 Viroid is an important class of pathogens that can cause many serious plant diseases.It is also the simplest self-replicating infectious agent currently known in nature.The genome of the viroid is small circμLar RNA with the size is generally in the range of 200-400 nt and does not encode proteins.Viroids are divided into two families,Pospiviroidae and Avsunviroidae according to replication strategy.To date,ICTV classifies 44 types of known viroids into 7 genera.In this study,we tested seven representative viroids: Citrus exocortis viroid(CSVd),Iresine viroid 1(IrVd-1),Potato spindle tuber viroid(PSTVd),Apple scar skin viroid(ASSVd),Avocado Sunblotch Viroid(ASBVd),Hop stunt viroid(HSVd),Peach Latent Mosaic Viroid(PLMVd).for their ability to replicate in filamentous plant pathogenic fungi,Cryphonectria parasitica as well as genetic mutation of viroid in fungi.In addition,the effects of viroid infection on the growth,development and pathogenicity of the host fungi were observed.This topic provides new research ideas and scientific basis for understanding the host range,pathogenesis and potential biological control use of viroids.By constructing infectious clones of seven viroids,in-vitro transcripts of viroid were first inocμLated into tobacco and infected yeast strains to detect their activities and then the viroids were artificially introduced into C.parasitica protoplasts by protoplast transfection.After the cells wereregenerated,the transfectants were cultured on the PDA medium and the fungal single-stranded RNA wasextracted and used for the RT-PCR detection of viroids infection.The effects of stable infection of viroids on the growth,development and pathogenicity of host fungi were also observed.The results are listed as follows:1.Construction of seven types of viroid infectious cDNA clones: Plasmids of seven kinds of viroids,ASBVd,HSVd,IRVd,PLMVd,ASSVd,IRVd and CSVd were prepared by oligonucleotide synthesis method.After obtaining in-vitro transcripts,they were tested in yeast strain AH109 for the infectivity.2.Investigation of the ability of seven kinds of viroids to infect filamentous fungi:After introducing seven type of viroids into C.parasitica protoplasts,RT-PCR showed that ASBVd,HSVd,and IRVd coμLd replicate in C.parasitica.The replication of ASBVd was more stable than the other two.Total RNA of ASBVd-infected fungal strains were extracted after subcμLture and the results show that the replication of ASBVd did not significantly decrease,while the replication of the other two viroids gradually decreased.3.Analysis of the effect of ASBVd infection on C.parasitica: Infection of ASBVdinhibited the growth of C.parasitica on PDA medium and the laboratory test for fungal pathogenicity found that ASBVd also has an effect on C.parasitica pathogenicity.4.Analysis of the spread of ASBVd in fungal hosts: Similar to fungal viruses,viroid coμLd be vertically and horizontally transmitted.ASBVd was transmitted by host hyphal fusion as confirmed by RT-PCR.C.parasitica infected with ASBVd was cultured under light.After sporμLation,the spores were isolated,cultured,and the single-stranded RNA of the fungus was extracted and used for RT-PCR detection,The result showed that viroid was vertically transmitted through spores.5.Analysis of the genetic variation of ASBVd in the fungal host: After the viroids infects the filamentous fungus,the infected strain was subcultured and the RNA of the 8th generation strain was extracted for RT-PCR detection.Sequence analysis showed that viroid sequence become mutated after subculture.6.Comparison of ASBVd wild-type and mutant strains and their effects on host fungi:After the ASBVd mutant strain infected the fungus,the amount of fungal aerial hyphae increased although the growth rate did not change significantly.Viroid RNA detection showed that the replication of ASBVd decreased compared to that of wild type.更多还原