| DsbA protein(Disulfide bond formation protein A) was found in Escherichia.coli firstly,the main function of the protein is to oxidize mercaptomethyl to form disulfide bond on the nascent chain. FrnE is a DsbA-like protein containing a C-X-X-C motif., which was presumed to be a thiol oxidoreductase involved in polyketide biosynthesis.One CpDsbA-FrnE gene was cloned by randomly cloning and sequencing, based on the cDNA library construction from Chimonanthus Praecox flower, named CpDsbA-FrnE.The gene has a full-lengths of1200bp, with opening reading frame(ORF) of654bp, encoding a predicted protein of217amino acids. The predicted protein molecular weight is24.6kD and the isoelectric point is5.81.Realtime fluorescence quantitative PCR (qRT-PCR) was used to study the gene expression of CpDsbA-FrnE. The CpDsbA-FrnE gene exhibited different transcription levels in different tissues and had a significantly higher expression in flowers than other tissues. In the bloomed flower, the expression of CpDsbA-FrnE has the maximum at the outer petal, the CpDsbA-FrnE gene in the flower buds gradually increased in the early stages of flower development from sprouting stage to display-petal stage and then decreased at initiating bloom. We also studied the gene expression of the CpDsbA-FrnE under PEG6000and high temperature (42℃)treatment.The results showed that the CpDsbA-FrnE genes were general up-regulated by the CuSO4and high temperature treatment, suggested that the CpDsbA-FrnE is a gene related to drougt and high temperature stress resistens.The Prokaryotic expression vector pET32a-FrnE was constructed and transformed into the expression strain E.coli transetta.A fairly good soluble expression can be gained under the conditions of20℃、0.5mM IPTG and14-16h induced by optimizing the induced conditions. The purified recombinant protein were obtained by the His-Bind protein Purification Kit. The results of the PEG6000and NaCl stress test in vitro showed that the transetta strain containing the plasmid (pET32a-FrnE) has more tolerance than the strain transetta just containing the vector plasmid (pET32a),suggested that the expression of the CpDsbA-FrnE in the transetta strain can improve the ability of E.coli to withstand stress.The plant over-expression vector of CpDsbA-FrnE named pC2301g-FrnE. was consrructed and transformed into tobacco plants using the leaf disc transformation procedure mediated by gene gun.9strain,27transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS (β-glucuronidase)histochemical staining and PCR analysisThe leaves from transgenic tobaccos and wild-type tobaccos were treated with5%PEG20000, After4d observed, part of the wild-type tobacco leaves begin to change to yellow,and no significant changes in transgenic tobacco leaves. Observe the change of the blades under5%NaC1treatment after4d,the result showed that some part of wild-type tobacco leaves becoming yellow and transgenic-tobacco leaves just begin to wilt. Remove the transgenic-tobacco and wild-type tobacco seedlings into MS medium containing50mmol/L NaCl for6days. Soluble sugars content Determine results showed that the soluble sugar content of transgenic-tobacco was higher than the wild-type. |
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