Studies On PHSP1 Gene Expression In Escherichia Coli And Nicotiana Tobacum

PHSP1, the cDNA for mtHSP70 (mitochondrial heat shock protein,mtHSP70) has been isolated from Pisum sativum. The protein encoded by PHSP1 is a homologue of the mtHSP70 proteins, involving in the translocation of pre-proteins across the inner mitochondrial membrane during the process of mitochondrial protein import.In the study, PHSP1 was sub-cloned into the prokaryotic expression vector pET-28a and pET-His using recombinant DNA techniques. Transforming pET-28a-PHSP1 into BL21(DE3) cells and were grown at 37℃to mid log phase. Protein expression was induced by addition of 0.5mmol/L IPTG (Isopropyl-β-D- thiogalactopyranoside). 4 hours later, bacterial proteins were separated by 12% SDS-PAGE. A new protein with MW approximately 70kDa was observed. With anti-mtHSP70 antibodies, Western blotting hybridization experiment confirmed this new protein as mtHSP70 (PHSP1).With the change of IPTG and culturing conditions, different PHSP1 expression yields were induced, in which maximum PHSP1 expression yields were observed at 0.25mmol/L IPTG, 37℃and 5 hours after induction. E. coli BL21(DE3) cells, transformed by pET-28a-PHSP1, expressed more PHSP1 than ER2566. pET-28a-PHSP1 had better expression than pET-His-PHSP1 in BL21(DE3) cells.Plant expression plasmid pBI121-PHSP1 was constructed for transformation, including a strong, constitutive 35S promoter of Cauliflower Mosaic Virus (CaMV), which can be highly expressed in eukaryotic cell. Tobacco leaf disks were infected and pBI121-PHSP1 was transformed into tobacco cells by the Agrobacterium-mediated transformation techniques with EHA105. Transformants were selected on the basis of resistance to the antibiotic kanamycin. After 30 days'culture, we successfully got transformed seedlings. PCR technique proved that extra PHSP1 gene existed in the seedlings. By Western blotting hybridization, more mtHSP70 had been found in transgenic seedlings. The results indicated that PHSP1 gene has been integrated into the chromosomal DNA of these transgenic plants and normally expressed.NaCl treatments (0, 100, 300 mmol/L NaCl) were applied to the leaves of wild type t and ransgenic tobacco for 0, 6, 24, 48 hours. We found that leaves of wild type tobacco were injured more seriously than transgenic tobacco at 300mmol/L NaCl after 48 hours. Leaf SOD (superoxide dismutase), POD (peroxidase) and CAT (catalase) activities had different changes with the rise of NaCl concentration and extending time. But at same treatment condition, transgenic tobacco leaves always had higher SOD, POD and CAT activities than wild type. We concluded that PHSP1 expression in transgenic tobacco protected or stabilized the structure of three enzymes under NaCl treatment. Therefore, transgenic tobacco had higher resistance to NaCl stress.