Cloning And Functional Analysis Of NB-ARC Encoding Putative Disease Resistant Genes To Powdery Mildew From Chinese Wild Vitis

Grape is the most economically important fruits of worldwide, powdery mildew(PM) caused by(Uncinula necator [Schw.] Burr.), is one of the most important fungal diseases of grapevine(Vitis vinifera), cause great loss to grape industry. Chinese wild Vitis pseudoreticulata W. T. Wang ‘Baihe-35-1’ and Vitis quinquangularis ‘Shang-24’ both possess a high resistance to U. necator. Identification and function analysis of relative disease resistance gene to powdery mildew from Chines wild Vitis will be great importance to grape resistance breeding. This study cloning and function analysis of VpCN and VpTNL1 from Chinese wild V. pseudoreticulata ‘Baihe-35-1’, ecotopic expression in Arabidopsis to verify the disease resistance function; Cloning the promoters and fused to GUS, transient transformation to grapevine leaves via Arobacterium to verify the promoter activity. Further more, VqTNL2 and its promoter sequence were cloned from V. quinquangularis ‘Shang-24’ and sequences were analyzed, cloned RPM1 genes from Chinese wild Vitis, analysis their expression pattern and bioinformatics. The main results are described as followings:1. In early studies, a global gene transcriptome was obtained from Chinese V. pseudoreticulata ‘Baihe-35-1’ after inoculation with U. necator, one of sequence was induced after inoculation PM. After cloned and sequenced, the ORF was 1,773 bp, encoding 590 amino acid, designed as VpCN. Bioinformatics analysis showed that the deduced amino acid contained a RX CC like domain in N terminal and an NB-ARC in C terminal. Ecotopic expression of VpCN in Arabidopsis displayed three morphological phenotype, normal wild-type phenotype, dwarf phenotype and fused rounded rosette leaves with slightly curled edges. The normal transgenic Arabidopsis showed enhanced resistance to Arabidopsis Golovinomyces cichoracearum and a virulent bacterial pathogen Pseudomonas syringae pv.tomato DC3000. Moreover, the promoter sequence of VpCN was isolated and fused to GUS(β-glucuronidase), promoter GUS fusion assays revealed that powdery mildew infection could induced the promoter activity in V. vinifera cv. Red globe leaves,-400 bp may the minimal segment of promoter sequence for respond to PM infection. Furthermore, deleted promoter analysis showed that TC rich repeats elements play an important role in response to E.necator infection.2. In early studies, a global gene transcriptome was obtained from Chinese V. pseudoreticulata ‘Baihe-35-1’ after inoculation with U. necator, one of sequence was induced after inoculation. After cloned and sequenced, the full length sequence was 3,903 bp, ORF 2,622 bp, encoding 873 amino acid, the 3’UTR was 1,281 bp, designed as VpTNL1. Bioinformatics analysis showed that the deduced amino acid contained a TIR and NB-ARC domain in N terminal, 4 LRR domains in C terminal. Ecotopic expression of VpTNL1 in Arabidopsis displayed two morphological phenotype, normal wild phenotype and dwarf phenotype. The normal wild phenotype Arabidopsis exhibited enhanced disease resistance to G.cichoracearum and a virulent bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Moreover, isolated the promoter and cis-elements predicted showed that the promoter sequence contained several cis-regulatory elements associated with fungal and abiotic response. After fused the promoter to GUS, the promoter displayed promoter activity in V. vinifera cvs. Red Globe leaves, deleted promoter analysis showed TCA elements may involved in response to SA treatment and TC rich repeats may involved in PM infection.3. In early studies, a global gene transcriptome was obtained from Chinese V. quinquangularis‘Shang-24’ after inoculation with U. necator, one of sequence was decrease after inoculation PM, expression pattern analysis performed in roots, stems, leaves, flowers and pericarps, displayed different expression patterns. After cloned and sequenced, the full length of the sequence is 3,835 bp, ORF is 3783 bp, encoding 1,260 amino acid, designed as VqTNL2. Bioinformatics analysis showed that the deduced amino acid contained a TIR and NB-ARC domain in N terminal, 6 LRR domains in C terminal. After isolated its promoter sequence and bioinformatics analysis showed that the promoter regions contain some cis-regulatory elements associated with biotic and abiotic stress responses. After generate the transient expression vector fused to GUS, transformed into grapevine leaves via Agrobacterium, the promoter displayed promoter activity in grapevine leaves.4. Isolated VpRPM1,VpRPM1-4 and VpRPM1-5 from Chinese V. pseudoreticulata ‘Baihe-35-1’, the ORF is 2794, 465 and 2556 bp, respectively. Isolated VqRPM1-2 and VqRPM1-3 from Chinese V. quinquangularis‘Shang-24’, the ORF is 2805 and 2727 bp. These five genes encoding NB-ARC domain. Analysis these five genes expression pattern after inoculation with U. necator showed that these five genes displayed significant decrease after inoculation. After analysis the RPM1 genes in V. vinifera “Pinot Noir”, 17 RPM1 genes were identified from V. vinifera “Pinot Noir” genome. Synthetic analysis indicated that tandem duplication and large fragments chromosome replication were main amplification ways of RPM1 genes.