Cloning And Expression Analysis Of Heavy Metal Adaptability Genes From Rhodotorula Mucilaginosa AN5

Glutathione peroxidase and glutathione S-transferase are two important enzymes of glutathione antioxidant system, which can eliminate the H2O2 and organic peroxide produced in physiological and pathological processes. Metallothionein is a class of cystein-rich protein with low molecular weight, which can combine with heavy metals and reduce the toxic effects of heavy metals, also can maintain the homeosistance of heavy metal ions. In this experiment, the heavy metal adaptability genes GPx, GST, MT from Rhodotorula mucilaginosa AN5 were cloned, then analyzed the sequences, constructed the recombinant plasmids, expressed and purified the proteins.(1) Cloning, expression and purification of GPxThe target GPx gene was cloned by PCR amplification, and named RmGPx. Sequence analysis indicated that the RmGPx had an open reading frame(ORF) of 498 bp, encoding a hydrophilic protein of 165 amino acid residues with a theoretical molecular mass of 18.3 k Da and a theoretical pI of 8.37. No signal peptide and transmembrance domain existed in the protein sequences. Two N-glycosylation sites, seven phosphorylation sites and a coiled-coil domain were predicted in RmGPx protein. The RmGPx gene was inserted into prokaryotic expression vector pET-28a(+), and then the recombinant plasmid(pET-28a-RmGPx) was constructed successfully, the GPx recombinant protein was expressed with the induction of IPTG and purified with Ni-NTA affinity chromatography.(2) Cloning, expression and purification of GSTThe target GST gene was cloned by PCR amplification, and named RmGST. Sequence analysis indicated that the RmGST had an open reading frame(ORF) of 843 bp, encoding a hydrophilic protein of 280 amino acid residues with a theoretical molecular mass of 30.4 kDa and a theoretical pI of 5.40. No signal peptide and transmembrance domain existed in the protein sequences and the subcellular location was predicted in cell nucleus. The RmGST had GST_N and GST_C two conserved domains. The blastp search in the NCBI GenBank revealed that it had high sequence similarity to Rhodotorula sp. JG-1b. The pET-28a-RmGST recombinant plasmid was constructed successfully, the GST recombinant protein was expressed and purified.(3) Expression and purification of MTThe target MT gene was amplified by PCR, and named RmMT. The RmMT gene was inserted into prokaryotic expression vector pET-28a(+), and then the recombinant plasmid(pET-28a-RmMT) was constructed successfully. The MT recombinant protein was expressed with the induction of IPTG and purified with Ni-NTA affinity chromatography.These studies obtained the purified protein of GPx, GST and MT, aimed to provide the basis for the study of these proteins’ characteristics and function, and provide a reference for the theoretical study of adaptation mechanisms of Rhodotorula mucilaginosa AN5 to heavy metals.