Study On Gene Clone,Prokaryotic Expression And Functional Analysis Of CrMT From Catharanthus Roseus

Metallothioneins, which widely distribute in eukaryotic and prokaryotic organisms, are a class of cystein-rich and heavy metal-binding proteins with low molecular weights. Recently, many study demonstrated that metallothionein played a role in storing and carting trace element, scavenging of free radicals and detoxifying of heavy metals. The study on plant MTs is just started because of the discovery of plant MTs is relatively later. Many study demonstrated that the plant metallothionein may function in both metal chaperoning and scavenging of reactive oxygen species with their large number of cysteine residues to protect plant from oxidative damage and participated in many physiology processes, such as development of plant, embryogenesis and stress resistance, and was regarded as an important functional gene. But, the function of plant MTs remains unclear. To demonstrate the function(s) of plant MTs, more studies on the cloning and functional analysis of plant MTs genes would be of important scientific value for further researching and developing plant metallothionein.In this paper, the Catharanthus roseus metallothionein (CrMT) gene was first isolated from the cDNA library of Catharanthus roseus. CrMT gene was amplified by PCR and cloned into prokaryotic expression vector pGEX-6P-1, the vector pGEX-6p-1 has a giutathione-S-transferase (GST) gene, which was in favor of the purification of the fusion protein by affinity chromatography subsequently. The recombinant plasmids were successfully introduced into E. coli BL21, SDS-PAGE analysis showed that the fusion protein GST-CrMT was successfully expressed by the induction of IPTG. We optimized the prokaryotic expression conditions through studing the effects of various factors on the protein expression, which included culture temperature, inducing time and the final concentration of inductor IPTG. Finally, we produced high concentration of GST-MT fusion protein and lay the foundation of producing antibody for Western-blotting. The Western-blotting test showed that the fusion protein had immunological reactive activity by using Anti-GST. Through analyzing the metal-ion tolerance of CrMT, concentrations of various metal ions were suitable for comparing metal-ion tolerance of transformed and non-transformed E. coli BL21were 1.0 mM CdCl₂ and 1.6 mM ZnCl₂. The metal-ion (Cd²⁺ and Zn²⁺) tolerance of E. coli cells expressing GST-CrMT fusion protein increased. These results suggest that CrMT functions in E. coli cells.In this paper, we analyzed the Signal Peptide through using SignalP 3.0 Server and applying the CrMT gene as a heterogeneous gene. The CrMT gene and GFP were together cloned into yeast expression vector pYES2, then the plasmid pYES2-CrMT-GFP was transformed into Saccharo cerevisiae INVScl by electric impulse transformation, observing the cell by Confocal Laser Scanning Biological Microscope (Nikon), so, the gene coding CrMT was studied on the localization of CrMT gene encoding protein in yeast. The results demonstrated that the intracellular localization sites was in cytoplasm.The results have important scientific value for further researching on regulation mechanism, structure and function of plant metallothionein expression and will provide important scientific basis for developing the products of plant metallothionein.