Cloning And Expression Analysis Of Tryptophan Decarboxylase Tdc1Gene In Camptotheca Acuminata

Camptotheca acuminata is a tree native to China, and its secondary metabolite, Camptothecin (CPT), is a monoterpene indole alkaloid that can effectively inhibit the function of DNA topoisomerse I. Tryptophan decarboxylase (TDC), a key enzyme in CPT biosynthesis, can transform tryptophan to tryptamine. Its activity directly influences CPT biosynthesis and some other related metabolic processes. In this study, we cloned tdcl gene from C. acuminate and subcloned it into the prokaryotic expression vector. Then TDC1was successfully expressed in Escherichia coli and purified. Moreover, according to the spontaneous fluorescence characteristics of camptothecin, we established trace detection methods of CPT in C. acuminata seedlings by HPLC with fluorescence detector. The content of CPT and the expression of tdcl were detected at the different development stage of C. acuminata seedlings. The main results were as follows:1. We successfully cloned tdcl gene of camptotheca acuminata by RT-PCR method and compared its sequence with the registered one in NCBI. There was a high homogeneity of nucleotide (98%) and amino acid (97.41%) between them. Furthermore, tdcl we cloned also show a high amino acid sequence homology among other species, especially at pyridoxal phosphate binding sites.2. We constructed the prokaryotic expression vector pET28a-tdcl, determined tdcl expression in E. coil containing recombinant plasmid and the optimum temperature (37℃) for expression. Prokaryotic expression and over-purified of TDC1protein was carried on. The purified TDC1protein can be used as a basis for further research, such as antibody preparation.3. Because of the autofluorescence characteristics of CPT, a HPLC method with fluorescence detector to determine the content of CPT in milligram-samples of Camptotheca acuminata seedlings was established. Through the analysis to the content of CPT and the expression of tdcl during seedling development, we discovered that both the the content of CPT and the expression of tdcl are changing with the seed germination process. The tdc1expression reached a maximum on10th day, two days earlier than the maximum of CPT accumulation, which suggests that TDC1plays an important role in CPT biosynthesis.