Sequencing Of Cdna Library And Functional Analysis Of Transcription Factors Hrf And Nac From Chinese Wild Vitis Pseudoreticulata

Powdery mildew (Erysiphe necator.(schw.) Burr.) causes great losse to the grapeproduction in the world. The most worldwide cultivated varieties are from Vitis vinifera whichwas generally susceptible to E. necator. The modern fruit breeding theory and practice provedthat it is feasible to breed resistant cultivars using wild grape. Chinese wild grape is preciousresource for resistance breeding, among which Vitis pseudoreticulata accession Baihe-35-1showed high resistance to E. necator, but the disease resistant mechanism is not understood.Transcription factor plays an important role in response to pathogen. The grapevine genomecontains more than2000transcription factors, but little is known to the function. Thisresearch aims to understand the types of the defense related gene of Chinese wild Vitispseudoreticulata through large-scale sequencing, and study the expression and function of thetranscription factors (ERF and NAC gene). Mainly results obtained are as follows:1. Large-scale sequencing of the cDNA library from V. pseudoreticulata accessionBaihe-35-1inoculated with E. necator.3500clones were randomly sequenced,1727ESTswere obtained by removal of low quality sequences and cluster analysis. All the ESTs weredeposited at the NCBI GenBank database under the accession numbers GR882788-GR884514. The ESTs were submitted to the public databases (InterProScan and GO) forsimilarity analysis and assigned GO categories,56.9%(983) ESTs could be annotated. For thecategory of molecular function, there contain 'binding'48.2%; for the category of biologicalprocess, there contain 'metabolic process'60.1%and 'cellular process'56.2%; for thecategory of cellular component, there contain 'cell'42.7%and 'cell part'42.7%.2. Four disease resistance related genes (Transcription factor, defense genes, R gene andactive oxygen metabolism) were identified from the annotated ESTs. The semi-RT-PCRanalysis indicated that these disease resistance related genes may be involved in the prosess ofChinese wild Vitis pseudoreticulata Baihe-35-1against powdery mildew. Twenty twofull-length cDNAs related to disease resistance were cloned by rapid-amplification of cDNAends technique. They are VpERF1, VpERF2, VpERF3, VpNAC1, VpWRKY3, VpHsf1,VpMYB1, VpMYB2, VpDRE1, VpDRE2, VpB-Zip1, VpPR1, VpPR1.2, VpPR4, VpPR10.2,VpPR14, VpDRP1, VpMLO1, VpUSP1, VpSGT1, VpTBE1, VpGST1. The GenBank numbers are: GU393310, GU393312, GU393311, GU393316, JF500755, GU393313, GU393314,GU393315, GU393308, GU393309, GU393307, GU269633, GU269634, JN977472,HQ634186, JQ814381, JF500756, JQ814382, GU946975, JQ814383, JQ814369, JQ814380.3. Chinese wild Vitis pseudoreticulata VpERF1, VpERF2and VpERF3belong to the B3,B2and B3subfamily of ERF family, respectively. There is a low similarity (21.9%) betweenthe three ERF proteins, but they possess a conserved AP2/ERF domain with the similarity82.49%. Although three VpERFs proteins have high similarity with V. vinifera ERF proteins,but they exist difference in protein sequence because of the protein fragment deletion andsingle amino acid mutation, indicating the three VpERFs are novel ERF genes.4. VpERF1, VpERF2and VpERF3were induced by E. necator, but the expressionpatterns are different. The maximum induction of VpERF2and VpERF3in E.necator-resistant grapevine was higher than that in E. necator-susceptible grapevine butVpERF1. Under the abiotic stress, VpERF1was induced by drought and heat stresses;VpERF2was induced by drought, cold and heat stresses; VpERF3was induced by cold andheat stresses, but the induction was very low. Moreover, VpERF1and VpERF3were involvedin SA, MeJA and ET signal pathways, VpERF2was involved in MeJA and ET signalpathways.5. VpERF1, VpERF2and VpERF3contain AP2/ERF domain, nuclear localization signaland trans-activation domain. The sub-cellular localization indicated that three ERF geneslocalized to nucleus. Yeast hybrids indicated that VpERF1and VpERF2functioned as atranscriptional activator but VpERF3. Transient transformation in grapevine indicated thatthree ERF genes function as transcriptional activators by binding to the GCC box cis-element.6. Transient transfection in grapevine indicated that VpERF2and VpERF3could hinderthe development of hypha but VpERF1. Overexpression of VpERF2and VpERF3in tobaccoincreased the resistance to bacterial pathogen Ralstonia solanacearum and fungal pathogenPhytophtora parasitica var．nicotianae Tucker, while VpERF1increased the susceptible tothese two pathogens. In addition, VpERF2and VpERF3resulted in the accumulation of fourPR genes expression in transgenic tobacco plants. But, only part of PR gene transcripts wereup-regulated by VpERF17. The1616bp VpERF3promoter was obtained by homologous cloning. Sequenceanalysis indicated that VpERF3promoter contains root specific cis-acting element and guardcell specific cis-acting element. Chemical histological staining in transgenic Arabidopsisindicated that VpERF3specially expressed in root and guard cell, indicating VpERF3is atissue-specific promoter.8. VpNAC1belongs to the TERN subgroup. The sub-cellular localization and trans-activation activity assays indicated that VpNAC1is a nuclear targeting protein andfunctions as a transcriptional activator. After inoculation with E. necator, expression ofVpNAC1showed the pattern with increased first and then declined, but the maximuminduction in E. necator-resistant grapevine was higher than that in E. necator-susceptiblegrapevine. VpNAC1was strongly induced by methyl jasmonate, while induction level ofsalicylic acid and ethylene was low. VpNAC1was induced by drought, cold and heat stresses,especially the induction level of drought treatment was more40fold than control.Over-expression of VpNAC1in tobacco plants enhanced their resistance to Erysiphecichoracearum and Phytophtora parasitica var． nicotianae Tucker, and regualted theexpression of PR genes as a positive regulator.