Gene Cloning, Expressing In E.coli Of Two Grapevine Leaf-roll Associated Viruses And Preliminary Establishment Of Grapevine Transformation System

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...