| Vitis vinifera L. is an important commercial fruit and cultivated worldwide for its high ecological and social values. However, grape fungal diseases such as Elsinoe ampelina(de Bary) Shear cause significant yield and quality losses. Substantial evidence has demonstrated that utilizing grapes’ own resistance to breed more resistant grapes is the most effective and economic option to reduce losses caused by fungal diseases. The Chinese wild Vitis has been well known for their desirable resistant germplasms, among which Vitis quinquangularis ‘Shang-24’ accession exhibits synchronous multi-fungal disease defense. However, the relevant molecular resistance mechanism of V. quinquangularis ‘Shang-24’ accession remains still unclear. In this study, disease resistance-related genes were selected from suppression subtractive hybridization libraries of V. quinquangularis ‘Shang-24’ accession infected by pathogens and their expression profiles were analyzed; then RNA-seq was used to compare the changes and differences at the transcription level of resistant V. quinquangularis ‘Shang-24’ accession and susceptible V. vinifera Red Globe, and the expression profile of grapevine in response to E. ampelina was constructed. In addition, JASMONATE ZIM-DOMAIN(JAZ) and basic helix-loop-helix(bHLH) transcription factor families(JAZ and bHLH) related to grape resistance to E. ampelina, were studied through bioinformatic and expression analysis. The main results are described as followings:1. Two SSH libraries of V. quinquangularis ‘Shang-24’ accession infected by E. ampelina and Erysiphe necator were analyzed. 183 putative E. ampelina- resistant unigenes and 87 putative E. necator- resistant unigenes were selected from the two libraries. Twenty putative E. ampelina- resistant genes and 13 putative E. necator- resistant genes, including eight genes involved in metabolism, two transcription factors, two genes involved in signal transduction, one gene involved in transport, six genes with defense-related function, four genes involved in ubiquitination pathway and four genes with unknown function. The results showed that the expression of most candidate genes was up-regulated in resistant grapevines infected by the two pathogens, yet almost unchanged in susceptible grapevines, indicating that these candidate genes may play important roles in Vitis resistance to pathogens.2. Grape leaves of resistant V. quinquangularis ‘Shang-24’ accession and susceptible V. vinifera Red Globe at 6 h, 12 h, 24 h, 48 h and 72 h after inoculation with E. ampelina and with sterile water as control were tested by RNA-seq. 346 Mb reads of high quality were obtained from total 40 samples and the mapping rates of each sample were around 80%. 3414 differential expression genes were identified through calculated differential expression of each gene by RPKM(reads per kilobase of exon model per million mapped reads), among which 2246 genes were from the resistant grape ‘Shang-24’ accession and 2013 genes were from susceptible grape Red Globe. In total, up-regulated genes were more than down-regulated ones. These differential expression genes were functionally classified based on biological process of Gene Ontology(GO). The GO classification of the resistant ‘Shang-24’ accession was quite similar to the susceptible Red Globe: most relevant genes were involved in responses to stress, cellular processes and metabolic processes. The enrichment analysis of metabolic pathways indicated that the differential expression genes and significantly enriched metabolic pathways were unexpectedly similar between the resistant and susceptible grapevines induced by E. ampelina; however, the time points at which the genes expressed differentially and the metabolic pathways enriched were different: in resistant grape ‘Shang-24’ accession, the time points were 6 hours and 24 hours post inoculation and these were earlier than in Red Globe.(48 hours and 72 hours post inoculation).3. The semi-quantitative-RT-PCR analysis of expression patterns of 11 JAZ genes identified from the grape genome before in roots, stems, leaves, tendrils, flowers, pericarps and ovules showed that these genes were expressed differentially in different organs and tissues. The expression profiles of these 11 JAZ genes in grapevines induced by different hormone treatments, salinity and drought stresses, and pathogens infection showed that most JAZ genes could respond to hormone treatments and significantly up-regulated in response to MeJA and ABA, but few JAZ genes responded to SA and ET. Most JAZ genes participated in responses to high salinity(NaCl) and drought stress, yet few JAZ genes participated in responses to pathogens infection. ORFs of three grape JAZ genes(VqJAZ4, VqJAZ7, and VqJAZ9) were cloned from V. quinquangularis ‘Shang-24’accession: the ORF of VqJAZ4 was 861 bp at length and showed 99.19 % similarity to VvJAZ4, encoded 286 amino acid residue; the ORF of VqJAZ7 was 417 bp at length and showed 95.8 % similar to VvJAZ7 nucleotide sequence, encoded 138 amino acid residue; the ORF of VqJAZ9 was 807 bp at length and showed 99.13 % similar to VvJAZ7 nucleotide sequence, encoded 268 amino acid residue.4. 126 bHLH transcription factors were identified in the grape geneome. The phylogenetic tree of four species(grape, Arabidopsis, rice and poplar) classified 126 bHLH genes into 24 subfamilies. The bHLH genes belonging to the same subfamilies always had similar proteins and extron/intron structures. Syntenic analysis indicated that tandem and segmental duplication events have been major contributors to the expansion of the grape bHLH family. Many grape bHLH genes and Arabidopsis homologous bHLH genes were in corresponding syntneic blocks, which implied that these bHLH genes in grapevines and Arabidopsis might possess a common ancestor. Real time-RT-PCR were used to analyzed the expression profiles of 26 gene members of bHLH Ⅲ and Ⅳ subfamily in six diverse grapevines tissues and in grape leaves under different hormone treatments, salinity stress, drought stress, and pathogens infection. The results showed that these genes showed different expression patterns in different tissues. Most bHLH genes could respond to ABA and MeJA treatments, some bHLH genes responded to high salinity(NaCl) and drought stress, but few bHLH genes participated in responses to pathogens infection. |
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