RAPD Analysis Of Jatropha Curcas L. And Expression In E.coli Of Curcin

Seed samples of seven populations of Jatropha curcas L. were collected southern Sichuan and northern Yunnan. RAPD analysis was carried out using seedlings of each sample after optimizing PCR conditions. Due to high sensitivity of RAPD analysis to reaction conditions,main factors affecting the results including composition of the buffering system,concentrations of Taq DNA polymerase,primers and templates,and number of PCR cycle etc.,were examined,and conditions applicable to RAPD analysis of J. curcas were determined. Forty arbitrary primers were used in the analysis of the seven samples,within which five primers produced relatively stable diversity. In total 49 loci were detected by these five primers,including 25 diverse loci(51%). Pairwise genetic distances were calculated and accordingly cluster analysis was carried out and the genetic dendrogram of the seven samples were generated.After getting the cDNA sequence of curcin,four fragments of the gene were cloned through PCR and high-level expression in E. coli was achieved. The four target DNA fragments,i.e. the full-length sequence,the 3'- deletion fragment,the sequence encoding the mature protein and the sequence encoding the conservative domain,were cloned using synthesized primers. Recombinant expression vectors were constructed through directional cloning and then host E. coli were transformed by the vectors. Recombinant proteins were expressed by the hosts after being induced with IPTG.Two expression systems were used,one of which was QIAexpress system,the other Glutathione S-transferase(GST) Gene Fusion System. The former expressesrecombinant proteins with a 6XHis tag at N-terminal. The fore mentioned four fragments were all used for expression in this system and the mature protein and the conservative domain were effectively expressed while the expression of the other two was unobvious. The latter expresses proteins fused with GST at the N-terminal. Fragment encoding the mature protein was used in this system. The expression of mature protein using this system was less effective compared with QIAexpress. So QIAexpress was preferred in producing target proteins,and the purified products were to be used for further studies in activity analysis.