Cloning And Expression Analysis Of Powdery Mildew Responsive Genes In Chinese Wild Vitis Quinquangularis

Powdery mildew (PM), caused by the fungus Uncinula necator (Schw.) Burr, is one ofthe fungal diseases of grapevine which harm Vitis vinifera and can cause severe yield loss.Researches have identified that Chinese wild Vitis quinquangularis is highly resistant topowdery mildew, cloning and expression analysis of powdery mildew resistance relatedgenes in Chinese wild V. quinquangularis is important to reveal the molecular mechanism ofgrape resistance to PM. Based on some EST sequences related to defense in the subtractivesuppression hybridization (SSH) cDNA library we have obtained from young leaves ofChinese wild V. quinquangularis clone ‘shang-24’ inoculated with U. necator, then annotatedfunction and classificated these ESTs using bioinformatic analysis. We isolated the cDNAsequence of SAP, AP, COI1and JAZ2gene. Main conclusions of this paper are as follows:1. Function annotion and classification of264ESTs obtained from the SSH. Geneontology (GO) analysis indicated that188unigenes could be assigned with at least one GOterm in the biological process category, and176unigenes in the molecular function category.Sequence analysis showed that a large number of genes were homologous to those involvedin biotic stress responses. Genes involved in metabolism, photosynthesis, energy, transportand signal transduction were also enriched in the EST collection. Further expression analysisof11unigenes using qRT-PCR revealed that all these genes were induced by U. necatorinfection with different expression patterns. They may be involved in many metabolic process,such as ubiquitin pathyway, active oxygen metabolis, defense response, secondary metabolicand cell metabolic et al.2. Based on the EST sequence of SAP gene from constructed SSH, we isolated thecDNA sequence of the SAP gene using the RT-PCR technology, which was designated asVqSAP. The sequence analysis indicated that its complete ORF was819bp, which encodes272amino acids, with a predicted molecular weight of30.56kD and the isoelectric point of8.78. Multiple Alignment of deduced amino acid sequence of showed that65-74%sequencesimilarity with other SAP from different plants. QRT-PCR was used to detect the expressionpatterns of VqSAP in response to different hormones and pathogen infection in resistant andsusceptible lines. The QRT-PCR showed that the expression of VqSAP was induced by U. nector in resistant material, it may be involved in the programmed cell death. VqSAP was alsoregulated by Eth, MeJA and SA.The different expression of VqSAP was observed in leaves,stems, flowers, pericarps and tendrils. These results revealed that VqSAP was apathogen-responsive gene and it was regulated by certain biotic stress-related hormones,indicating it may play an important role in grape pathogen resistance.3. Based on the EST sequence of AP gene from constructed SSH, We isolate the cDNAsequence of the AP gene using the RT-PCR technology, which was designated as VqAP. Thesequence analysis indicated that its complete ORF was1377bp, which encodes458aminoacids, with a predicted molecular weight of50.48kD and the isoelectric point of8.73. It isatypical aspartic proteases belongs to clan A1of plant aspartic proteases gene family.QRT-PCR analysis showed that the gene involved in defense response at the early period ofpathogen infection and different hormones also inducing its expression indicated it may beregulated some pathogen-related gene.The different expression level of VqAP was observedin leaves, stems, flowers, pericarps and tendrils indicated that it involved in synthesis anddegradation of different cell in plants tissues.4In the subtractive suppression hybridization (SSH) cDNA-library from Uncinulanecator (Schw.) Burr-inoculated young leaves of Chinese wild Vitis quinquangularis clone‘shang-24’, We isolate the cDNA sequence of the COI1gene and JAZ2gene using theRT-PCR technology, which was designated as VqCOI1and VqJAZ2, respectively. Thesequence analysis indicated that the length of VqCOI1was2455bp, which encodes598amino acids, with a predicted molecular weight of67.87kD and the isoelectric point of5.85.Multiple Alignment of deduced amino acid sequence of showed that it has the highestsequence similarity, approximately79%similarity with other COI1in Glycine max（GenBankacc.no: AAZ66745.1）the ORF of VqJAZ2was1167bp, which encodes388amino acids, witha predicted molecular weight of40.26kD and the isoelectric point of9.19. Existing consevedCCT2domain and TIFY domain, VqJAZ2gene can involed in SCFCOI1ubiquitinationpathway and regulate JA-mediated defense response. Semi-Quantitative RT-PCR in leaves,stems, flowers, pericarps and tendrils showed that VqCOI1and VqJAZ2play a role in thedifferent tissues of the plants. After treatment with SA, JA and Eth and post-inoculation withU. necator. The expression pattern showed that they have a interaction in ubiquintationpathway and regulation plant defense response.