【作者】 王亚南；

【导师】 王锡锋；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2008， 博士

【摘要】 为了防止北京近郊甜椒上新出现病害-辣椒轻斑驳病毒(PMMoV)病害的蔓延和危害,本研究开发了一种地高辛标记的cDNA探针利用核酸斑点杂交技术对PMMoV进行检测研究。通过对探针特异性和灵敏度的测试,证明了该探针能够特异检测PMMoV,稀释限点对应的植物组织材料为0.8μg,与RT-PCR及DAS-ELISA两种方法比较,比DAS-ELISA方法灵敏(39.06μg),没有RT-PCR灵敏(0.008μg)。但是综合考虑地高辛标记的cDNA探针是大量田间样品常规检测较好的方法。采取北京郊区和河北保定地区的类似PMMoV症状的93种田间样品和18种商品种子样品进行了检测,将检测结果用更加灵敏的RT-PCR方法进行了验证,两种方法结果基本一致,表明了地高辛标记的cDNA探针在实际应用中检测结果可靠。通过对田间样品的检测,初步揭示了2006年北京附近地区PMMoV病害发生情况,对病害的防治有重要的指导意义。地高辛标记探针的开发为今后PMMoV田间样品的常规检测提供了方便,快捷,灵敏,安全的检测方法,为PMMoV病害的防治奠定了基础。利用分步构建策略构建了含有PMMoV-CN全长基因组序列的侵染性cDNA克隆pMQC。构建过程中在PMMoV-CN基因组序列的5’端引入了T7启动子和提高转录效率的两个G,3’端引入单一酶切位点SmaI,采用pMD18-T simple载体作为基础载体,将PMMoV-CN全基因组序列分为三段逐步构建到pMD18-T simple载体上,得到含有病毒全长基因组序列的重组载体之后,利用SmaI酶切位点将载体线性化,以线性化载体为模板进行体外转录。将体外转录产物接种矮牵牛,苋色藜和昆诺藜,通过症状观察以及分子生物学方法(包括RT-PCR,Western blot和地高辛标记的cDNA探针)验证了pMQC的侵染性。成功构建PMMoV-CN的侵染性cDNA克隆,为今后PMMoV基因组功能及致病机制的研究奠定了基础。将侵染性克隆pMQC的CP置换为TMV-U1和ToMV-S1的CP得到了两个杂合侵染性克隆pM-T-CP和pM-To-CP,将两个杂合侵染性克隆体外转录产物接种矮牵牛和苋色藜观察症状,以TMV-U1(ToMV-S1)和PMMoV-CN为参照,结果采用分子生物学方法(RT-PCR,核酸斑点杂交和Western blot)进行验证。最终结果为:在矮牵牛上,pM-To-CP没有侵染性;PMMoV-CN CP置换为TMV CP之后使PMMoV-CN由局部侵染变为系统侵染,说明TMV CP可能在pM-T-CP系统侵染矮牵牛中起主要作用,CP可能作为主要因子协助该病毒在矮牵牛中进行长距离运动,TMV-U1和PMMoV-CN CP之间的差异是导致两种病毒在矮牵牛上症状差异的决定因子。在苋色藜上,pM-To-CP依然没有侵染性;PMMoV-CN CP置换为TMV CP并没有改变PMMoV-CN在苋色藜上的斑驳症状,说明PMMoV-CN CP可能在PMMoV-CN侵染苋色藜产生斑驳症状过程中没有起到重要作用,或者PMMoV-CN CP在苋色藜上产生斑驳症状的作用被TMV-U1 CP弥补了,而且说明了TMV-U1在苋色藜上产生局部枯斑症状所需要的TMV中的无毒基因可能不是它的CP。TMV-U1 CP与PMMoV-CN CP之间的差异不是两种病毒在苋色藜上症状差异的决定因子。为了进一步确定PMMoV-CN CP序列中的哪一部分序列与TMV-U1序列的差异导致它们在矮牵牛上症状为局部侵染和系统侵染的差异,本研究构建了两个杂合侵染性克隆,pM-T-CP1、pM-T-CP2,它们分别对应着PMMoV-CN CP的1-117、1-237位核苷酸序列置换为TMV-U1 CP对应序列。体外转录之后接种矮牵牛植株,观察症状及通过分子生物学方法验证,证明了两种杂合病毒对矮牵牛为局部侵染。说明PMMoV-CN CP的1-117和1-237位的核苷酸与TMV-U1对应序列的差异并没有导致两种病毒在矮牵牛上的症状差异。为了进一步确定TMV-U1CP序列中哪一部分序列在症状改变中起主要作用,今后的研究将对PMMoV CP其它部分的序列进行置换以及采用定点突变等方法来揭示两种病毒症状差异的原因。更多还原

【Abstract】 Pepper mild mottle virus is a new emerging Tobamovirus disease on capsicum plant in the vicinity of Beijing in recent years. In order to control the damage and prevent the disease spreading。A digoxigenin-labelled cDNA probe PM, complementary to the regions between coat protein (CP) gene and 3’untranslated sequence of PMMoV genome, has been developed to detect the virus by dot-blot hybridization method. The specificity and sensitivity has been tested. The detection limit of the method was equivalent to 0.8μg fresh tissues infected by PMMoV, compared with DAS-ELISA and RT-PCR which was 39.06μg and 0.008μg detection limit respectively. The sensitivity of the dot-blot hybridization was higher than that of DAS-ELISA and lower than that of RT-PCR. 111 pepper samples including 93 field samples collected from pepper fields of Beijing and Baoding during 2006 and 18 commercial seed samples had been evaluated by dot-blot hybridization using this probe. The result has been identified by RT-PCR. The high sensitivity and reliability of the molecular hybridization assay described here, would provide an important alternative method for the detection of PMMoV in large-scale.In order to study the virus deeply, the infectious cDNA clone of PMMoV-CN, pMQC has been constructed. The completed genome sequence of the virus was constructed into a suitable vector by three steps. T7 promoter has been introduced into the 5’end point and two G also introduced in order to increase the efficiency of its in vitro transcription. The restricted enzyme site SmaI has been introduced into the 3’end point of the genome sequence in order to linearize the vector in the latter experiment. The pMD18-T simple vector was applied in constructing infectious cDNA clone, which has no any enzyme site, so we can introduce restriction enzyme site in primer by PCR according to the enzyme sites in the genome sequence of PMMoV-CN. The infectious cDNA clone was linearized and in vitro transcribed. The in vitro transcription product was inoculated onto three host plants, Chenopodium Amaranticolor, Chenopodium quinoa and Petunia hybrida. The infectivity of the infectious cDNA clone had been tested by biology method including RT-PCR, western blot and digoxigenin-labelled probe. The infectious cDNA clone of PMMoV-CN will provide an effective method to study the pathogenicity mechanism and gene function of the virus.In order to make sure the pathogenicity related elements Tobamovirus, two recombinant infectious cDNA clones have been constructed, pM-T-CP and pM-To-CP. The CP nucleotide sequence of pMQC was exchanged by that of TMV and ToMV respectively. The pathogenicity of pM-T-CP and pM-To-CP were tested on Petunia hybrida and Chenopodium amaranticolor. The result showed that pM-To-CP had no pathogenicity and pM-T-CP presented systematically symptom on Petunia hybrida, while PMMoV infected Petunia hybrida locally, so we suggested that TMV CP may help the virus move in long-distance in Petunia hybrida plant. The two recombinant infectious cDNA clones were also inoculated onto Chenopodium amaranticolor. pM-To-CP still had no pathogenicity and pM-T-CP infected Chenopodium amaranticolor showing local mottle symptom, which was as same as PMMoV-CN. So we suggested that the CP of PMMoV-CN may not play an important role in the symptom conformation of PMMoV-CN infecting Chenopodium amaranticolor and TMV CP may not be the avr gene in TMV infecting Chenopodium amaranticolor to show local necrotic lesion.In order to determine which sequence difference between TMV-U1 CP and PMMoV-CN CP plays an important role in causing their different symptom on Petunia hybrida, two recombinant infectious cDNA clones, including pM-T-CP1, pM-T-CP2 had been constructed. The two recombinant vectors including TMV CP sequence corresponding to 1-117,1-237 site in the PMMoV genome sequence. The pathogenicity of the two recombinant vectors had been tested by biology method. The result showed that both of them showed locally symptom on Petunia hybrida, which meaned that the difference of 1-117 and 1-237 sequence of CP were not related to the symptom difference between PMMoV-CN and TMV-U1 on Petunia hybrida. So the other sequence of CP will be exchanged to study the related elements causing different symptom between PMMoV and TMV on Petunia hybrida in the future.更多还原