| Taxol, which has special anti-cancer efficacy, is one of the secondary metabolites extracted in Taxus chinensis. However, it is hard to fulfil the commercial requirements of taxol due to the low content and complicated biosynthesis steps. To solve this contradiction, large-scale plant-cell-culture technique has been applied to taxol production. And clarification of transcriptional regulatory mechanism would help to improve the industrialization level. WRKY transcription factors are one of the largest families of transcriptional regulators in plants, which involved in many plant processes including plant growth, development, defense and metabolism. In this study, Tc WRKY genes were screened and cloned on the basis of Taxus chinensis transcriptome and genome databases, the regulatory mechanism of taxol biosynthesis was also studied. The main results achieved are as follows:(1) Screening and phylogenetic analysis of TcWRKY.61 TcWRKY genes were screened from Taxus chinensis transcriptome and genome databases. And phylogenetic analysis showed that 16 of these TcWRKY transcription factors belong to group I, only one belongs to group III, the rest of them belong to group II. 44 groupII TcWRKY transcription factors were further subdivided into five subgroups: 8 of group IIa, 10 of group IIb, 14 of group IIc, 5 of group IId, 7 of group IIe.(2) Analysis of TcWRKY expression patterns induced by hormones.Seven representative Tc WRKY genes were selected from group I(TcWRKY26), group IIa(TcWRKY47), IIb(TcWRKY52), IIc(TcWRKY8), IId(TcWRKY44), IIe(TcWRKY41), group III(TcWRKY20) respectively based on the phylogenetic analysis result. After MJ and SA treatment, the expression of TcWRKY44 and TcWRKY47 dramatically increased, and the expression of TcWRKY8, TcWRKY20, TcWRKY41, Tc WRKY44 and TcWRKY47 obviously increased after MJ treatment, but were inhibited by SA treatment. TcWRKY52 had no obvious response to MJ, while significantly inhibited by SA.(3) Expression of taxol-synthesis related genes in Tc WRKY-Overexpression Taxus chinensis cells.Tc WRKY8, TcWRKY20, TcWRKY26, TcWRKY41, TcWRKY44 and TcWRKY47, which significant responsed to MJ, were overexpressed in Taxus chinensis cells respectively. Then expression of taxol-synthesis related gene DBAT, T5 H, and regulatory gene TcERF12, TcERF15 in these cells were analyzed. The results showed that, the expressions of these four genes were apparently inhibited in Tc WRKY44-overexpressing cells, while increased in different degree in TcWRKY20, 26-overexpressing cells. Meanwhile, in TcWRKY8 and Tc WRKY 47-overexpression cells, expressions of DBAT, T5 H and TcERF15 were improved while TcERF12 expression inhibited.(4) Study on the mechanism of interaction between TcWRKY47 transcription factor with DBAT promoter.Considering that Tc WRKY47 gene significantly responded to MJ and SA, also activated several taxol-biosynthesis related genes, the interaction between TcWRKY47 transcription factor and DBAT promoter was studied in Taxus chinensis cells. Three W-boxes of DBAT promoter were mutanted and constructed respectively. When only WA-box or WC-box exists, TcWRKY47 slightly interacts with DBAT promoter, while WB-box exists with no WA-box, combination between TcWRKY47 and DBAT promoter improves significantly. These results showed that TcWRKY47 could combine with WA-box, and an activation effect when combines with WB-box. While TcWRKY47 has no obvious effect with WC-box.In conclusion, our study has screened the TcWRKY47 transcription factor among 61 TcWRKY transcription factors, which could significantly increase the biosynthesis of taxol, its interaction with DBAT promoter has also been explored. These results lay a foundation to study mechanism of TcWRKY transcription factors regulatory effects on taxol biosynthesis, as well as constructing high-content cell lines for production. |
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