Studies Of Mechanism Of TcWRKY47 Transcription Factor In Regulating Synthesis Of Taxane In Taxus Chinensis

Taxane compounds are characteristic secondary metabolites of Taxus,among which paclitaxel has a significant broad-spectrum anti-tumor effect and is the most widely used antitumor drug in clinical practice.However,its content in plant tissue is extremely low,leading to direct extraction from plants can not meet the market application.There are many ways to produce taxanes,and the use of transgenic technology at transcriptional level is one of the common means.WRKY transcription factor is one of the largest family of transcription factors in plants.It is widely involved in many biological processes such as plant development,senescence and growth.It also plays a significant role in plant defense and secondary metabolites synthesis.In this paper,the simple genomic data of Taxus were screened to obtain the gene sequence containing W-box element in the 5'flanking sequence.The molecular mechanism of the biosynthesis of taxane compounds was investigated by using TcWRKY47 transcription factor as the research object.The main results achieved are as follows:(1)Screening the potential target gene of WRKY transcription factor based on simple genome and transcriptome data of Taxus chinensis.Based on the transcriptome of Taxus and simple genomic data,the 31618 genes were screened with complete 5'flanking flanking sequences,and 5017 5'flanking sequences containing W-box elements and their downstream genes were extracted.Through the functional annotation of 5017genes,the results showed that some of the six genes including taxane synthase T5H,T7H,T10H,T14H,DBAT and BAPT were the key to the synthesis of terpenoids,and partial of the synthesis of alkaloids and the synthesis of flavonoids enzymes are potential target genes for WRKY.In addition,four 5-terminal flanking sequences of TcERFs,5 TcbHLHs,and 16 TcMYBs were also found to contain W-box elements,indicating that WRKY has the effect of regulating the transcription factor and regulating the synthesis of taxane.(2)Expression pattern analysis of TcWRKY47 and Its potential target genes.TcWRKY47 is a typical group IIa WRKY transcription factor,which was found to be a potential regulator of paclitaxel synthesis.After MJ and GA hormone treatment,TcWRKY47 transcription factor and taxane synthase T5H,T7H,T10H,T14H,BAPT and DBAT and other six key enzyme genes consistent with the expression trend.Further quantitative analysis showed that TcWRKY47 overexpression could up-regulate the expression of T5H,T7H,T10H,T14H,BAPT,DBAT and TASY genes in different degrees.Therefore,TcWRKY47 can significantly regulate the synthesis of taxane compounds.(3)Mechanism of TcWRKY47 transcription factor regulates T5H and TcERF12/15 gene expression.The activity of the 5'flanking sequence of the T5H gene and the TcERF12/15 gene in the preliminary work showed that the three sequences had promoter activity in the plant cells.EMSA and yeast single-hybrid experiments show that TcWRKY47 can specifically bind to W-box on the three promoters.In the taxus cells,TcWRKY47 is also capable of binding to T5H and W-box on the TcERF12/15 gene promoter and exerts a different effect:i)When the TcWRKY47 transcription factor binds to the W-box promoter on the T5H gene promoter,it up-regulated the gene expression;ii)and binded to TcERF12 gene promoter on the W-box,it play a negative regulatory role;iii)when it combined with ERF15W_A-box and ERF15W_C-box on the TcERF15 gene promoter,it played a positive regulatory role,while negatively regulating when it binded to ERF15W_B-box.In conclusion,In this paper,based on analyzing simple genome and transcriptome,the potential target gene of TcWRKY47 was annotated,and six promoter regions containing W-box elements of the taxane synthesis of key enzyme genes elected from the potential genes.Furthermore,the mechanism of TcWRKY47 transcription factor regulation of T5H and TcERF12/15 gene expression was further explored,and the mechanism of TcWRKY47 transcription factor at the transcriptional level to regulate the biosynthesis of taxane compounds was clarified to a certain extent.