Cloning And Expression Analysis Of Transcription Factor TwWRKY1 In The Tripterygium Wilfordii Hook.f

As a traditional offcinal, the main active ingredients in Tripterygium wilfordii Hook. f., which are terpenes and alkaloids, have a wide range of applications in agriculture and medicine. However, due to the lack of natural resources and low active substance content in this plant, the application in its secondary metabolites is severely restricted. WRKY is a group of important plant transcription factors. Recent research indicate that WRKY is involved in a wide range of plant growth, development, aging, biotic and abiotic stress responses and secondary metabolite synthesis process. In order to clarify the regulation and expression of WRKY induced by MeJA and SA, as well as its relationship with synthesis of secondary metabolites, hairy roots of T. wilfordii was used as material to do the following research. The TwWRKY1 gene was cloned using RACE technology, and 6 genes associated with terpenes synthesis pathway was induced and analysed about their expression.Some key findings have been made as follows:1. From the hairy roots of T. wilfordii we successfully cloned a WRKY transcription factor and named it as TwWRKY1. Sequence analysis showed that the full-length of the gene is 962 bp, the complete open reading frame size is 537 bp and sequence encoding protein is of 178 amino acids; bioinformatics analysis showed TwWRKY1 contains a typical WRKY conserved domain, zinc finger structure is C2H2, which belongs to Class II of WRKY family; phylogenetic analysis proved that the TwWRKY1 has a close parental relationship with Coptis jiponica CjWRKY1.2. Analysis of different TwWRKY1 expression level in different tissues showed that the gene was expressed in roots, stems and leaves and expression in young leaves is the highest which is 5.34 times higher than that in the natural root, and hairy roots is 3.81, stems is 2.5 and adventitious roots is 1.67 times higher. The expression in natural root is the least.3. The expression of TwWRKY1 was regulated by elicitor MeJA, and with the increase in processing time, gene expression increased and then decreased; SA pre-treatment could inhibit gene expression of TwWRKY1, and the inhibition would eliminate 9h later.4. It was found that in the Tripterygium terpene biosynthesis-related gene expression patterns induced by MeJA and SA, MeJA works significantly both on the early and late biosynthesis genes in the metabolic pathway of terpene, while SA works greatly on the early genes, such as HMGS and HMGR, it shows an inhibition first and then induction of the late gene, means MVK and GGPPS.5. The addition of MeJA promotes the biosynthesis of Tripterygium Triptolide, Wilforgine and Wilforine in the hairy roots, which promotes Wilforgine and Wilforine more than Triptolide; while it shows an inhibition of these three secondary metabolism products in the nutrient solution.