Function Analysis And Transgenic Tobacco Of Metallothionein HCMT From Halostachys Caspica

Metallothioneins?MTs? are a family of ubiquitous low molecular weight?6–7kD?, metal binding, Cys-rich cytosolic proteins. They are widely distributed in the plant kingdom and can be induced by many abiotic stresses. Their abilities of high free radical scavenging and metal binding made them the hot topic in the study of plants HcMT, a metallothionein from Halostachys caspica, encoded 78 amino acids which included 14 cysteines. The molecular weight and pI of HcMT was 7.70 kD and5.05. Bioinformatics analyses show HcMT belonged to plant Class ? type 2 MTs,had closer genetic relationship with Salicornia brachiata and the similarity of their amino acids sequence attain 93%. To evaluate the metal-binding ability of HcMT, it was expressed in Escherichia coli BL21 as a glutathione S-transferase?GST? fusion protein?GST- HcMT?. The metal-binding ability of GST-HcMT was analyzed and compared with GST alone using atomic absorption spectrometry. These results showed that GST-HcMT has a about 34 kD molecular weight is as expected, and was confirmed by Western blot. Furthermore, GST- HcMT could bind about 25, 10, 4, 2folds of Cu²⁺, Zn²⁺, Pb²⁺ and Cd²⁺ separately than GST alone, and the affinity ability of metal ions for recombinant form of GST-HcMT was as follows: Cd²⁺>Zn²⁺>Pb²⁺.Concurrently, it was found that pGEX-6p-2-HcMT/BL21 could act well under the stress of H₂O₂?NaHCO₃?Na₂CO₃. When it was cloned into the Escherichia coli expression vector pET32 a as a thioredoxin?TrxA? fusion protein?TrxA- HcMT?,TrxA- HcMT could bind Cd²⁺?Co²⁺?Cu²⁺?Fe3+?Mg²⁺?Pb²⁺?Zn²⁺?Hg²⁺ when the irons mixture was added during the inducing, the affinity ability of metal ions for recombinant form of GST-HcMT was as follows: Pb²⁺?Cu²⁺?Zn²⁺?Cd²⁺ when the irons was added separately. Compared with GST-HcMT,TrxA-HcMT can bind more metal irons. Under the stress of Cd²⁺, Cu²⁺, Zn²⁺, pET32a-HcMT/BL21 acted better than pET32a/BL21. The differences between GST-HcMT and TrxA-HcMT may due to the different number of cysteines in their tags.The fermentation conditions for production of recombinant HcMT expressed in E. coli were optimal and produced in a 20 L fermentor. Single factor experiments on the effect of different component in the medium and their concentrations, induce's concentrations shows that the optimized medium contains 4 g sucrose, 24 g Yeast extract, 8 g peptone, 2.31 g KH₂PO₄, 12.54 g K₂HPO₄?3H₂O and the recombinant protein was effective induced by 6 mmol/L lactose for 4 h. Further optimization of the culture conditions were performed in 20 L fermentor, the dry weight of bacterial reaches 2.68 g/L increased by 31% and the bacterial can bind 315.3 mg Cu²⁺ per gram of dry bacterial increased by 69%. The recombinant HcMT with high level production will be useful in various industrial applications.To investigate some other functions of HcMT and evaluate the posibility of HcMT in phytoremediation, HcMT was subcloned into the pCAMBIA1301 vector,several strains of the transgenic tobacco was confirmed by PCR using the genome as the template. Further researches about the metal binding ability and stress tolerance of the transgenic tobacco would be done.