Cloning And Prokaryotic Expression Of A CDNA Encoding A Putative Ent-copalyl Diphosphate Synthase From Andrographis Paniculata (Burm. F.) Nees

1. Objective"Chuan-Xin-Lian", a widely used Chinese herbal medicine, is the dried aerial parts of herbaceous plant Andrographis Paniculta (Burm. f.) Nees. The major constituents of "Chuan-Xin-Lian" are diterpene lactones known as andrographolides. These bitter constituents have many biological activities and are believed to contribute to medical functions of "Chuan-Xin-Lian". Andrographolides are formed through a very complex biosynthetic pathway in A Paniculta. According to chemical structures of andrographolides, we proposed that a diterpene synthase, namely ent-copalyl diphosphate synthase (CPS), was the first key enzyme involved in andrographolide biosynthesis, determining their parent skeleton. Geranylgeranyl diphosphate (GGDP) is catalyzed by CPS to form cyclization, followed by some successive modifications to finally obtain andrographolides. Cloning and functional identification of ent-copalyl diphosphate synthase gene from A. Paniculta (Burm. f.) Nees (designated as ApCPS) will not only help to elucidate the biosynthetic pathway of andrographolide, but also lay the foundation for genetic regulation of andrographolide biosynthesis for improvement of the medical efficacy of " Chuan-Xin-Lian " in the future.2 Methods2.1 Cloning of the core fragment of ApCPSDegenerate primers were designed based on the conserved region by comparing the sequences of Andrographis Paniculta partial cps gene (Genbank Accession Number: AJ9731191) and some CPS genes from other plants published in Genbank. Using total RNA isolated from the leaves of A. Paniculta as template, the core fragment of ApCPS was amplified with the degenerate primers by reverse transcription PCR (RT-PCR) and the product was sequenced. 2.2 Cloning of 3'-end and 5'-end of ApCPSUsing rapid amplification of cDNA ends (RACE-PCR), the 3'-end and 5'-end of ApCPS gene were amplified with specific primers that were designed according to the sequence of ApCPS core fragment obtained.2.3 Construction of recombinant cloning plasmids, sequencing and analysis of the assembly sequenceThe 3'RACE and 5'RACE products were purified and subcloned into the vector pMD19-T and transformed into E. coli DH5 a competent cells. Transformants were selected by ampicillin resistance and Blue-White Screening. The positive colonies were further identified by restriction digest of isolated plasmid DNA and sequencing. The full-length cDNA sequence of ApCPS was obtained by assembling the sequences of the core fragment,5'-end and 3'-end. The open reading frame (ORF) finding analysis was performed with DNAMAN software.2.4 Amplification of the full-length cDNA of ApCPSAccording to the coding sequence of ApCPS and the multiple cloning sites of expression vector pGEX-4T-3, specific primers (5'-end primer containing BamH I restriction site and 3'-end primer containing Sma I restriction site) were designed. Using total RNA as template, the full-length cDNA of ApCPS was amplified by RT-PCR with high fidelity DNA polymerase.2.5 Construction of prokaryotic expression plasmidThe full-length cDNA of ApCPS was purified, double-digested with BamHⅠ/Sma I and then inserted into the expression vector pGEX-4T-3 which was digested with the same enzymes, to give recombinant plasmid. E. coli DH5 a competent cells were transformed with the plasmid and plated on LB plates containing ampicillin. The positive colonies were identified by restriction digest of isolated plasmid DNA and sequencing.2.6 Expression of ApCPS as fusion proteinExpression of ApCPS gene was investigated in different E. coli strains (E. coli JM109··E. coli BL21 and E. coli XLI-Blue) carrying prokaryotic expression plasmid under the induction of IPTG. SDS-PAGE was used to analyze the results of protein expression. As negative control, E. coli strains carrying the empty vector pGEX-4T-3 were used. 3 Results3.1 Cloning of the core fragment of ApCPSGel electrophoresis showed that an approximately 420bp fragment was specifically amplified as expected. The fragment was sequenced to generate a 373bp sequence.3.2 Cloning of 3'end and 5'end of ApCPSThe results of gel electrophoresis and sequence alignment demonstrated that the expected plasmids harboring 3'-end and 5'-end of ApCPS, respectively, were successfully constructed. They were named as pMD19-apCPS(3'RACE) and pMD19-apCPS(5'RACE), respectively. The BLASTx searching result showed that the translated protein of 3'-end exhibited 62% identity to Coffea arabica CPS (Accession No.: ACQ99373.1) while that of 5'-end exhibited 55% identity to Scoparia dulcis CPS (Accession No.:AB046689.1).3 3 Amplification of the full-length cDNA of ApCPSGel electrophoresis showed an approximately 2.5kb fragment was specifically amplified as expected. The purified fragment was used to construct prokaryotic expression plasmid.3.4 Construction of prokaryotic expression plasmidThe results of gel electrophoresis and sequence alignment showed that the expected expression plasmid containing the full-length cDNA of ApCPS was successfully constructed. Sequence analysis of the insertion identified an ORF encoding a polypeptide of 833 amino acids, which showed 64% and 60% homology to Scoparia dulcis putative CPS (Accession No.:BAD91286.1) and Scoparia dulcis copalyl diphosphate(Accession No.:BAB03594.1), respectively. The prokaryotic expression plasmid was named as pGEX-apCPS.3.5 Expression of fusion proteinThe results of SDS-PAGE showed there was no expected band corresponding to GST-tagged ApCPS of about 121.4KDa in either E. coli JM109, E. coli BL21 (DE3) pLysS or E. coli XLI-Blue host containing pGEX-apCPS after IPTG induction. But all the negative controls showed the marker protein GST of about 26KDa. 4 ConclusionsApCPS was successfully cloned and a recombinant plasmid harboring the full-length cDNA of ApCPS in expression vector pGEX-4T-3 was constructed, laying the foundation for prokaryotic expression of ApCPS.Overexpression of ApCPS in E. coli was not yet succeeded. No expected fusion protein could be observed in E. coli JM109, E. coli BL21 (DE3) pLysS and E. coli XLI-Blue strains harboring ApCPS. The exact reasons remained unknown. It could be thesignal peptide in 5'-end of the full-length eukaryotic gene or the non-functional sequences, which influence gene transcription and translation in prokaryotes. Thus it is worthwhile to study the expression of ApCPS without the 5'-end signal peptide or just the functional regions. Another possible reason may be the cell toxicity or instability of the target protein. So, it is necessary to optimize further the expression conditions.