Identification And Functional Analysis Of WRKY Transcription Factors Regulating Terpenoid Indole Alkaloid Biosynthesis In Catharanthus Roseus

Catharanthus roseus (C roseus) is an important medicinal plant, as well as a model plant for plant secondary metabolism research. Therefore, the research on regulatory mechanism underlying terpenoid indole alkaloid (TIAs) biosynthesis and accumulation in C. roseus has important significance and broad application prospects. WRKY transcription factors (TFs) are one of the largest families of TFs in the regulation of secondary metabolism and stress reponse in plants. However, The types and functions of CrWRKY TFs in C. roseus involved in the regulation of TIAs biosynthesis are poorly understood. In this dissertation, we systematically analyzed the type, function and expression of CrWRKY TFs in C. roseus genome database and chrictarized CrWRKYl promoter which regulates TIAs biosynthesis in C. roseus using molecular biology, biochemistry and bioinformatics technology. This study not only enriches our knowledge about CrWRKY, but also provides a framework for functional identification of the CrWRKYs and understanding the regulation network of terpenoid indole alkaloid biosynthesis in C. roseus.1. A high efficient transformation system of C. roseus mediated by Agrobacterium rhizogens R1000was established using the young leaves as explants. GUS reporter gene histochemical staining and PCR amplification showed that the transgenic system mediated by Agrobacterium rhizogenes R1000can give82±2.49%of hairy root inductivity and100%of the positive transformation. Hairy root lines obtained from this transgenic method are large quantity, high quality, genetic stability and short time needed. It was superior to the present genetic transformation technology in C. roseus. Thus, this transgenic system is a valuable approach for future efforts in regulation mechanism and characterization of related gene in TIAs biosynthesis and anticancer alkaloids metabolic engineering.2.47CrWRKY TFs were identified from26009single-copy proteins of C. roseus by bioinformatics. These CrWRKY TFs were classified into three groups:group1(G1), group2(G2) and group3(G3) based on the number of WRKY domains and the pattern of their zinc finger motif. The G1contained11members of the CrWRKY. The G2consisting of31members were further divided into G2-a, G2-b, G2-c, G2-d and G2-e5subgroups. The5CrWRKY proteins of G3all belong to the G3-a subgroup. There had the conserved WRKY domain motif and atypical zinc finger structure among CrWRKY members of different groups. The zinc-finger structure of G1and G2is C2H2with the sequence of C-X4-C-X22-H-X1-H in G1N-terminus (GIN), C-X4-C-X23-H-X1-H in G1C and G2-c, C-X5-C-X23-H-X1-H in G2-a, G2-b, G2-d and G2-e. The zinc finger structure of G3is C2HC with the sequence of C-X7-C-X23-H-X1-C.3. Digital gene expression (DGE) and quantitative real-time PCR (qRT-PCR) showed that CrWRKY genes expressed in a tissue/organ specific manner. According to the expression pattern,47CrWRKY genes were classified into3groups. CrWRKY genes were involved in the regulation of TIAs biosynthesis and affected by Methy Jasmonate (MeJA) and yeast extract (YE) signals. A group of candidate CrWRKY TFs involed in the regulation of TIAs biosynthesis was identified.4. On the basis of the isolation and characterization of CrWRKY1gene, a524bp CrWRKY1promoter was isolated using a PCR-based genome walking method and functionally characterized by multiple methods of molecular biology. Sequence analysis revealed that CrWRKY1promoter contains several potential eukaryotic regulatory elements, such as basic helix-loop-helix (bHLH) binding sequence E-box, MYB binding element, DOF recognition core sequence, wound response element(WRE) and activation sequence element (ASF-1). The transcription start site (TSS) was determined by5’-RACE and located113bp upstream of the initiation codon ATG of the CrWRKY1gene.5’-or3’-end deletions analysis revealed that the sequence between-230bp to-93bp relative to TSS was critical for promoter activity. Mutation analysis showed that two overlapping ASF-1elements and a CT-rich motif contribute significantly to the promoter activity. The CrWRKY1gene promoter containing various cis-elements with compact structure indicated that the expression of CrWRKY1gene was regulated by various TFs.5. Transgenic analysis showed that CrWRKY1promoter can drive the expression of β-glucuronidase (GUS) reporter gene in protoplasts and transgenic hairy roots in C.roseus and tobacco seedlings. The GUS expression in transgenic hairy roots and tobacco seedlings is similar to the protoplast transient assay. CrWRKY1promoter was weakly induced in response to methyl jasmonate (MeJA) treatment and the·promoter region between-140bp to-93bp contains a putative MeJA-responsive element. These results indicated that CrWRKY1TFs was involed in the TIAs biosynthesis in response to JA.6. A potential binding TFs gene SI FA was identified by yeast one hybrid (Y1H) and another two potential genes MYB44and DOF were cloned according to the cis-element in the CrWRKY1promoter. These results laied a foundation for verification of the interaction between CrWRKY1promoter and this TFs. CrWRKYl promoter could be used as a tool to isolate novel TFs involved in regulation of TIAs pathway...