【作者】 徐章逸；

【导师】 王国平； 洪霓；

【作者基本信息】 华中农业大学 ， 植物病理学， 2006， 博士

【摘要】 葡萄卷叶伴随病毒(Grapevine leafroll-associated virus，GLRaV)-2和GLRaV-3是引起葡萄卷叶病(Grapevine leafroll disease，GLD)发生的两种比较重要的病毒。目前GLD的防治措施主要是使用无病毒苗木和选育抗病品种，病毒的检测是具体实施过程中的一个重要环节，其中，血清学方法具有灵敏、快速、适合大量样品检测等优点而被广泛应用于葡萄病毒检测中。然而，GLRaVs为韧皮部限制性病毒，在寄主植物中含量低，除GLRaV-2外均没有烟草寄主；另外，GLRaVs的复合侵染现象比较普遍，因此，利用提纯病毒粒子的方法制备特异性的抗体比较困难，而用原核表达系统表达的重组病毒蛋白制备抗体则克服了这个问题。借助于基因工程手段，利用病原获得抗病性(Pathogen Derived Resistance，PDR)进行抗病毒育种方法克服了传统育种方法中育种周期过长的局限性而成为一种有效的育种手段，其中，由病毒复制酶(RNA-dependent RNA polymerase，RdRp)基因介导的抗性在某些方面明显优于外壳蛋白(Coat protein，CP)基因介导的抗性已被成功应用到多个病毒的抗病性研究上，而有关由GLRaVs RdRp基因介导的抗性研究目前尚无报道。另外，葡萄遗传转化较困难，迄今为止仅在少数几个基因型中获得了转基因植株，因此，建立一个高效的葡萄遗传转化体系，是保证葡萄抗病毒育种顺利进行的上游工作。 本研究旨在克隆GLRaV-2 RdRp和GLRaV-3 RdRp和CP基因后进行原核表达获得大量的重组蛋白制备抗血清，为国内葡萄无病毒种苗培育和种苗调运的检验提供有效的检测手段；另外，构建GLRaV-2 RdRp基因的植物表达载体，利用农杆菌介导法进行转化烟草研究，以揭示GLRaV-2 RdRp基因介导的抗病机制，并为进一步的利用此策略进行葡萄抗病毒育种提供理论依据和技术策略；最后，分别以“红地球”、“黑奥林”“美鲁特”试管苗的叶片、叶柄和茎段为试材，通过培养基的筛选，建立葡萄有效再生培养体系，以及构建GLRaV-3野生型RdRp基因植物表达载体及转化烟草验证转入的基因的功能后，通过农杆菌介导的遗传转化方法，对以上三个品种进行初步转化，为培育葡萄抗病毒品种建立一个良好的技术平台。本研究主要结果如下： 1．从“二号大宛红”样品中克隆了GLRaV-3(GLRaV-3-EDH)的RdRp和CP基因。序列测定结果表明，GLRaV-3-EDH RdRp基因全长为1618bp(GenBank登记号为AY495340)，推导其编码538个aa，CP基因全长942bp(GenBank登记号为DQ119574)，推导其编码313个aa；GenBank Blast(nr)检索结果表明，该RdRp基因与已报道的美国分离物GLRaV-3-NY1 RdRp基因核苷酸与氨基酸序列相似性均在99％以上，氨基酸变异主要发生在N端；该CP基因序列与已报道的3个GLRaV-3分离物CP基因核苷酸序列相似性均在99％以上，与巴西的一个分离物相似性为93％；CP氨基酸序列多重比对结果表明，5个GLRaV-3分离物序列中共有20个位点发生变异，其中，GLRaV-3-EDH CP中没有变异位点。 2．从“品丽珠”样品中克隆了GLRaV-2(GLRaV-3-CF)的RdRp基因。序列测更多还原

【Abstract】 Grapevine leafroll associated virus-2 and -3 are two important agents of grapevine leafroll disease. At present, selection of virus-free seedling and virus-resistant grapevine are two main means controlling GLD, in which serology is a widely used tool for routine detection of grapevine viruses due to its sensitivity, quickness and addaption for the detection of amount of samples. However, production of high quality virus-specific antisera to GLRaVs may not be easy, due to the frequency of complex infections in the field and the low yield of virus particles from naturally infected grapevine tissues following purification. Nevertheless, production of virus-specific antibodies using recombinant proteins from cloned viral genes expressed in Escherichia coli can overcome these difficulties. The application of molecular technique using viral genes can overcome the limitation of long breeding age using conventional breeding methods, and was proved to an efficient measure for genetic improvement of grapevine. The RNA-dependent RNA polymerase (RdRp)-mediated resistance provided an atternative measure for virus-resistant breeding with many merits. However, there is no research of RdRp-mediated resistance in GLRaVs. In addition, the grapevine genetic transformation was very difficult, and up to now, only a small number of transgenetic plants were obtained from grapevine cultivars. So establishment of an effective genetic transform system will favour the success of virus-resistant grapevine breeding.The main purpose of the present research is first to supply a practicable virus-detection tool for virus-free grapevine breeding and transporation in China through cloning the RdRp gene of GLRaV-2 and GLRaV-3, and the coat protein (CP) gene of GLRaV-3 and production of virus-specific antibodies using recombinant proteins from cloned viral genes expressed in Escherichia coli; Secondly our researches is to discern the mechanism of RdRp-mediated resistance and supply a new mean for virus-resistant breeding, the wild and deleted GDD motif RdRp genes of GLRaV-2 were transformed into Nicotiana benthamiana via Agrobecterium-mediated transformation; Thirdly, the present research is to establish a effective regeneration system using the leaves, petioles and stems from "RedGlobe", "Black Olympia" and "Meilute" as experimental materials. In addition, the wild RdRp gene of GLRaV-2 was transformed into tobacco to testify the function of transformed gene, and then it was transformed into grapevine via Agrobecterium-mediated transformation. The main results obtained are as followings:1. The RdRp and CP gene of GLRaV-3 (GLRaV-3-EDH) were cloned from "Dawanhong No 2". Sequence analysis showed that the complete RdRp gene consists of 1618 nucleotides (Accession number AY495340) and encodes a polypeptide consisting of 538 amino acids and that the complete CP gene consists of 942 nucleotides (Accession number DQ119574) and encodes a polypeptide consisting of 313 amino acids. Comparison of the RdRp gene sequences with those of the American isolate NY1 of更多还原