| Grapevine is an agriculturally and economically important fruit crop grown all over the world, which also is a significant source of functional compounds, including resveratrol and other derivatives. Resveratrol can play an important role in plant disease resistance, herbivore attack or a variety of abiotic stress. Grape plants are rich in resveratrol. As such, it is one of the most extensively studied natural products. The biosynthesis of resveratrol in grapevine can be affected by biotic and abiotic stresses such as powdery mildew infection, UV radiation treatment and hormone treatment and the ultraviolet radiation has become one of the effective ways of accumulation of resveratrol in grapevine. However, the accumulation of resveratrol in grape and regulatory mechanisms has not been fully studied. Some Chinese wild grape accessions have the character of high resveratrol content. Therefore, to explore the mechanism of its regulation can be used for optimizing grape varieties. In this study, the content of resveratrol in different genotype grapevines were analyzed. We determined that Tonghua-3 had the highest berry resveratrol content of seven grape accessions tested, making it an ideal research material to investigate gene expression responses to UV-C radiation. Then we chose 4 h and 24 h samples after UV-C treatment and using RNA-seq to find important genes which may play a significant role in regulatory mechanisms. And the possible reasons for the formation and accumulation of resveratrol were discussed. More over, this study further cloned two stilbene synthase Genes and their promoters, obtained the transgenic tomato plants and the related promoters activity was studied. The results of this study are as follows:1. Berries from a total of 7 grape accessions, corresponding to five wild species, were analyzed for resveratrol content. The highest levels of resveratrol were found in V. amurensis Tonghua-3(2.19 ?g/g FW) at maturity stage. But resveratrol were not found in V. davidii Tangwei of three fruit development periods. According to the different content of resveratrol,two of these development periods(green and mature) were subsequently targeted to investigate gene expression, using RNA-Seq analysis. We identified 1,861 and 2,234 genes that exhibited changes in transcript abundance at Tonghua-3 and Tangwei in fruit development period, respectively and most of these differential expressed genes were down-regulated. A total of 20 and 14 biochemical pathways were significantly influenced inTonghua-3 and Tangwei fruit development period, respectively. 136 differential expressed transcription factors were found from the analysis. Hormone pathway analysis showed that JA and Eth pathways as well as their related regulatory genes were up-regulated but SA pathway was down-regulated during the development of the two materials. In the phenylalanine pathway, the number of up-regulated genes in Tonghua-3 was higher than that of Tangwei,and the expression of resveratrol/hydroxycinnamic acid O-glucosyltransferase genes wrer higher than that of Tonghua-3. The results show that many ways of grapes during fruit development will change and the mechanism of production and accumulation of resveratrol in different grape genotypes is a complex process. There are a couple of supposed theoris such as the regulation of different hormones and transcription factor or the regulation of the related upstream/downstream genes.2. HPLC was used to analyze the content of resveratrol compounds in Tonghua-3 grape berries at six time points following UV-C treatment. Two of these time points(4 and 24 hours)were subsequently targeted to investigate UV-C induced gene expression responses, using RNA-Seq analysis. We identified 881 and 1,058 genes that exhibited changes in transcript abundance at 4 h and 24 h post-UV-C treatment, respectively. A total of 51 and 76 biochemical pathways were significantly influenced by UV-C treatment(p value < 0.05) at 4h and 24 h post-treatment, respectively. These pathways encompassed the biosynthesis or degradation of diverse metabolites including hormones(JA, Eth and SA), sugars and polysaccharides, amino acids and secondary metabolites. There may be a competitive and/or inhibitory relationship between STS and CHS in response to UV-C exposure. The possible reasons for the formation of resveratrol induced by UV treatment were analyzed. Total results showed that resveratrol could be controlled by multiple genes.3. Based on the data obtained, we cloned STS gene promoter sequences from Tonghua-3and also analyzed the responsiveness of Va STS36 promoter to UV-C irradiation in transgenic tobacco(Nicotiana benthamiana) leaves. After cloned and sequenced, the length was 1570 bp and Genbank accession number is KU376402. Moreover, cis-elements predicted showed that the promoter sequence contained several cis-regulatory elements associated with light response. The GUS activity test showed that the promoter displayed promoter activity after UV-C irradiation.4. The expression pattern of grape stilbene synthase gene family using semi-q RT-PCR analysis in different tissue and organs showed that these genes were expressed differentially.The expression patterns of grape STS genes in fruit development period of Tonghua-3 showed that the majority of STS genes existing a crescendo expression profiles in fruit development period. We cloned Va STS19 and Vd STS19 which may relate o the content of resveratrol, theORF sequence length was 1179 bp, encoding 392 amino acid. The vector harboring Va STS19 and Vd STS19 were successfully transformed to tomato by Agrobacterium-mediated method.Resveratrol contents of transformed plants and wild type were tested using HPLC. The results showed that resveratrol could be detected in the transgenic tomato plants of two fruit development stages. The results indicate that the expression of stilbene synthase gene and the production of resveratrol may be related to the regulation of the upstream region.5. We cloned the promoter of Va STS19 and Vd STS19 and the length of sequence is1461 bp and 1457 bp, respectively. A sequence alignment showed that the Va STS19 promoter was demonstrated 48% homology with the VPst S promoter cloned from Chinese wild V.pseudoreticulata ‘Baihe35-1'. After fused these promoters and 5' deletions to GUS, these promoters and fragments displayed promoter activity response to hormone treatment. To response to Me JA, The highest activity region of PVa STS19 induced by methyl jasmine was1384 bp and PVd STS19 was 1194bp; To response to SA, The highest activity region of PVa STS19 induced by salicylic acid was 1197 bp and PVd STS19 was 868 bp. The results indicate that different types of cis-elements which located in the promoter of stilbene synthase are response to different treatments and this may lead to the different expression patterns. |
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