| Powdery mildew, Uncinula necator (Schw.) Burr, is one of the most important fungal diseases of grapevine (Vitis vinifera L.). Most cultivated Vitis vinifera varieties worldwide,although excellent in quality, are generally susceptible to U. necator. The accession Baihe-35-1 of Chinese wild Vitis pseudoreticulata, as a highly resistant wild grape germplasm, was previously reported several times. Grapevine resveratrol, the expression product of stilbene synthase in phenylalanine pathway, can enhance plant disease resistance to various pahtogens, yet also shows significant health-promoting effects including anti-cancer, anti-tumor, prevention of cardiovascular disease and neuroprotective properties. In this study, a powdery mildew-resistant accession of Chinese wild Vitis pseudoreticulata, and two susceptible cultivars of Vitis vinifera Carigane and Thompson Seedless were used as materials. The study mainly focused on the expression pattern of stilbene synthase in grape-powdery mildew interaction, comparison of the stilbene synthase genomic structures, and cloning the upstream regions of sitlbene synthases. Furthermore, the stilbene synthase promoter activities were analyzed using Agrobacterium-mediated transiently transformed tobacco. The resveratrol content in pathogen-inoculated tobacco leaves were detected using HPLC. The main results are described as followings.1.The expression patterns of stilbene synthase between powdery mildew-resistant accession Baihe-35-1 of Chinese wild V. pseudoreticulata (VpSTS) and powdery mildew-susceptible cultivars of V. Carigane and Thompson Seedless (VvcSTS and VvtSTS) were studies. In pathogen-grapevine interactions, VpSTS displayed a unique expression pattern that differed from V. vinifera cvs. Carigane and Thompson Seedless, which demonstrated a suppression expression at early inoculation stages but a enhanced expressing at late stages. A further analysis showed that the VpSTS expression of this specific model involved two types of coordination of stilbene synthase gene expression, whereas two susceptible varieties of V. vinifera were only found in a class of stilbene synthase gene expression. 2. The two full-length genomic stilbene synthase genes were cloned from Chinese wild V. pseudoreticulata accession Baihe-35-1, V. vinifera cvs. Carigane and Thompson Seedless, and Genbank accession nos. are FJ830329,FJ830330, FJ851182, FJ851183, FJ851184 and FJ851185, respectively. Comparative analysis revealed that the nucleotide homology was 94 to 99% among the cloned stilbene synthase from disease-resistant and disease-susceptible genotypes. Structure analysis of stilbene synthase genomic sequence indicated that the cloned stilbene synthases contained two exons and one intron. The single nucleotide polymorphism (SNP) caused the main differences in two exon regions, whereas insertion-deletion (indel) mutations were presented on intron regions. Restriction enzyme digestion analysis showed the 6 cloned stilbene synthase genomic sequences can be divided into two types. The deduced amino acid of the cloned stilbene synthases had 94.5 to 99.5% identities with previously reported stilbene synthases from grapevine plants. The sequence features of"IPNSAGAIAGN"and"GVLFGFGPGLT"in the cloned stilbene synthases revealed that they were distinct from chalcone synthases, which further proved the obtained target sequeces are stilbene synthases.3. Genomic walking method was used to isolate 3 upstream regulatory sequences from disease-resistant and disease-susceptible genotypes, and GenBank accession nos. are FJ605484, GU269272 and GU269273, respectively. Sequence prediction analysis indicated the 3 upstream regions have some cis-regulatory elements associated with biotic and abiotic stress responses. Alignment results revealed that the upstream sequence of stilbene synthase from Chinese wild V. pseudoreticulata accession Baihe-35-1 had 53% and 51.7% identities with the corresponding regions of V. vinifera cvs. Carigane and Thompson Seedless, respectively.4. The 3 upstream regulatory sequences of stilbene synthases were fused to UdiA gene, respectively. The GUS gene expression patterns in infiltrated tobacco with different constructs were analyzed using Real-time PCR technique. The results showed the GUS expression profile driven by VpSTS promoter in response to Alternaria alternate was quite different from GUS expression profiles driven by VvcSTS and VvtSTS, whereas similar GUS gene expression patterns were found in VvcSTS and VvtSTS promoters fusion constructs. Compared to non-inoculated control leaves, the 3 upstream sequences of stilbene synthases possessed pathogen-inducible promoter activities. The different fusion constructs in response to pathogen were analyzed by GUS histochemical staining and fluorescence quantitative methods. The results indicated the highest promoter activity was detected on GUS gene driven by VvcSTS promoter, the activity of VpSTS promoter took second place, and VvcSTS promoter activity was lower. In abiotic stress treatments with injury and MeJA, VpSTS promoter in response to injury regulated GUS expression with negative regulation, while no significant changes in response to MeJA treatment was found. VvcSTS and VvtSTS promoters in response to injury and MeJA treatments presented positive regulation patterns.5. Based on the different expression pattern of VpSTS promoter from VvcSTS and VvtSTS promoters, the 5'deletion analysis of VpSTS promoter were further analyzed. GUS activities of different deletion constructs were tested in disease-susceptible V. vinifera cv. Carigane using Agrobacterium-mediated vacuum method. Also, the heterologous system of tobacco leaves were used to test deleted promoter activities. The results showed that 1772-bp region of VpSTS promoter in response to pathogens had high inducible expression activity in homologous and heterologous systems. To response to SA treatment, 162-bp promoter region presented high inducible activity. For cold treatment, 1129-bp promoter region had highest inducible activity. These results suggested that positive and negative cis-regulatory elements in response to biotic and abiotic stress co-existed in the cloned 1772-bp promoter region of VpSTS, and the 162-bp promoter region regulated with constitutive expression.6. The 3 stilbene synthase promoters were respectively fused with its stilbene synthases to generate fusion expression vectors. Agrobacterium-mediated transiently transformed tobacco system and HPLC analysis were used to evaluate resveratrol content in infiltrated tobacco leaves inoculated with A. alternate or mock. The results indicated that the infiltrated tobacco leaves transformed with PvpSTS::VpSTS G-2 fusion construct after A. alternate inoculation can produced a high-content resveratrol 2.81μg.g-1FW, which was 3.37 times relative to the mock-inoculated leaves. The resveratrol produced in the tobacco leaves transformed with PvvcSTS::VvcSTS G-1 and PvvcSTS::VvcSTS G-2 were 2.29 and 2.28μg.g-1FW, which were respectively1.82 times to the mock-inoculated leaves. The resveratrol produced in the tobacco leaves transformed with PvvtSTS::VvtSTS G-1 and PvvtSTS::VvtSTS G-2 were 1.78 and 1.83μg.g-1FW, which were 1.53 and 1.51 times to the mock-inoculated leaves. However, the resveratrol produced in the tobacco leaves transformed with PvpSTS::VpSTS C-1, PvpSTS::VpSTS C-2 and PvpSTS::VpSTS G-1, were 1.06, 0.84 and 0.67μg.g-1FW, which were respectively1.68,1.31 and 1.1 times to the mock-inoculated leaves. These results suggested that, the expression of stilbene synthase is co-regulated both the gene itself and the upstream promoter.
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