| Grape is the most economically important worldwide fruit. Plant disease is the mainreason leads to production reduction. Chemical control methods increase the production costs,and reduce the quality of grapes. Using the disease resistance germplasm resources tocultivate resistant varieties are effective ways to solve this problem. Chinese wild vitis havestrong resistance on the main diseases of grape. It is great significance to take advantage ofChinese Wild grapes for disease-resistant breeding. This research not only aims at the Chinesewild vitis' low regeneration rate, but also clone and characterizes the disease resistance relatedgenes such as, VpGLOX, VpPR17and VpHsf1. We aimed at better protection and utilization ofChinese wild vitis, further revealing the mechanism of disease resistance of Chinese wild vitis.The novel finding were as follow:1. This research focus on some factors which influence the tissue culture of stem apexof Vitis.pseudoreticulata Baihe-35-1in three stages: initially culture, propagation, rooting,especially the salt concentration, types and concentration of growth regulators, sugar,antioxidants and the material. It show that,the suitable medium as follow: MS+0.2mg.L-1IBA+0.5～1mg.L-1BA for initially culture, MS+1.0mg.L-1BA+0.1mg.L-1NAA for propagation,1/2MS+Ager6g.L-1+IBA0.2mg.L-1+VC1g.L-1for rooting。2. We study the organogenesis using the leaves and others from Baihe-35-1as explants.It showed that: the regeneration rates from leaves are higher than that from the petioles andstems. The callus appears from10days after inoculation, and shoot appears in40days, mostof the shoot are caespitose shoots. MS was suitable basic medium for regeneration ofBaihe-35-1, TDZ was suitable cytokinins for regeneration of Baihe-35-1, NAA was suitableauxin for it, the appropriate concentration was2.0㎎.L-1and0.1㎎.L-1 respectively. Theregeneration rates much higher after the liquid culture, It fromed embryogenic callus at first,then Leaf-like structures, shoot appeared at last.3. We study the somatic embryogensis from10lines of8species of Chinese wild vitisand2Vitis vinifera varieties, And got somatic embryos from two lines 'Baihe-35-1' and'Guanxi-2' belongs to V.pseudoreticulata W.T.Wang,'Ningqiang-6'(V. davidii) and 'Merlot', 'White Riesling'(Vitis vinifera L) among them. The embryoid induction rate using thestamens as are higher than the pistil. MSC is better than other3type medium, There is nodirect relationship between the callus induction rate and the somatic embryos induction rate.Itwas suitable for somatic embryogensis from Chinese Wild grapes using the NN69as the basicmedium and Phytagel.4. Based on the preliminary studies, we analysis the function of glyoxal oxidase relatedgenes(VpGLOX). qRT-PCR are used to analyse the expression of VpGLOX inBaihe-35-1(powdery mildew resistant) and Carignane (susceptible strains), The expression ofVpGLOX in the two kinds of genotypes are both induced by the grape powdery mildew,Genesexpression after inoculation in Baihe-35-1changes greater than in susceptible genotype,Theexpression vector and interference vector of the gene are constructed,4over expressiontransgenic tobacco are obtained through tobacco genetic transformation, glyoxal oxidase andH2O2in the transgenic tobacco was increased and transgenic tobacco added the resistance toPhytophtora parasitica var. nicotianae Tucker. The VpGLOX in transgenic tobacco wasinterfered with agro-infiltration,, the glyoxal oxidase in the plants after interferencedecreased and but no significant changes with H2O2.5. Grape pathogenesis-related protein VpPR17(GenBank accession no. JQ248075) areobtained from Baihe-35-1leaves cDNA by homology cloning methods, and some of thefeatures of the gene encoding acid sequence are analyzed by bioinformatics methods. Thegene has681bp,encoding226amino acids, isoelectric point (PI):6.30,molecular weightof25.41kDa,contain signal peptide. The similarity of VpPR17encoded protein sequence andpotato, tobacco pathogenesis-related proteins27are70%and71%, respectively, Thesubcellular localization shows that VpPR17expresses in the cytoplasm and some shows morein the organelles. qRT-PCR analysis shows that expression of VpPR17in Baihe-35-1arenot induced by powdery mildew inoculation,but it changes obviously after powdery mildewinoculation in Carignane.VpPR17are transferred to prokaryotic expression vectorpGEX-6T-1,then into E.coli BL21, recombinant protein in the position of about50kDa afterIPTG induction shows a specific band, it shows that VpPR17gene expressed in the E.coliBL21, the protein crude extracts have antibacterial activity. VpPR17are transferred toBaihe-35-1leaves by agro-infiltration, and then inoculated with downy mildew, the myceliumon the transgenic leaves at3,5,9d after inoculation was significantly less than that ofcontrol, indicating that VpPR17was related to grape disease resistance.6. An heat shock transcription factor(Hsf), designated as VpHsf1(GenBank accession no.GU393313), was isolated from Chinese wild Vitis pseudoreticulata for the first time using therapid amplification of cDNA ends (RACE). Its full-length cDNA is1428bp, encoding an Hsf protein of306amino acids with a calculated molecular mass of33.96kDa. Multiplesequence alignment and phylogenetic analyses showed that VpHsf1was a novel member ofthe Hsf class B2family. Nuclear localization of the protein of VpHsf1was detected in onionepidermal cells. VpHsf1expression was induced by heat and drought,as well as pathogenErysiphe necator. After the infection of E. Necator, VpHsf11was also induced in twograpevine genotypes, and the induction of VpHsf1kept in a low level in E. necator-resistantgrapevine genotypes different from susceptible ones. VpHsf1overexpression in tobaccoreduced the plant's basal thermotolerance, increased its acquired thermotolerance, andenhanced its susceptibility to osmotic stress and pathogen Phytophtora parasitica var.nicotianae Tucker. Taken together, the results indicated that grapevine VpHsf1is involved inbiotic and abiotic stresses. |
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