Font Size: a A A

Cloning And Prokaryotic Expression And Function Analysis Of PyCaM And PyHSP70 Gene From Porphyra Yezoensis

Posted on:2017-11-12Degree:MasterType:Thesis
Country:ChinaCandidate:C Y ZhaoFull Text:PDF
GTID:2310330488968794Subject:Cell biology
Abstract/Summary:PDF Full Text Request
P.yezoensis is one of the most important economic algae in China.It is belongs to the Rhodophyceae,bangiaceae and mainly distributes on the coastal area of Japan,South Korea and China.In the experiment,two contigs were obtained by splicing the EST fragments found in the dbEST database by searching the keywords “CaM gene” and “HSP70 gene”.The sequence analysis was performed using BlastX function in the NCBI website.However,the results pointed to other species.According to the sequence alignment,primers were designed and PCR reactions were performed and then two products were obtained.These two genes were named as PyCaM and PyHSP70 after verification by sequence alignment.Bioinformatics analysis showed that the ORF of PyCaM gene had 453 bp,encoding 150 amino acids with a start codon ATG and a terminator TGA.The protein encoded by the PyCaM gene had a molecular weight of about 16700 Da and the isoelectric point was 4.12.The results of signal peptide prediction showed that PyCaM has no signal peptide sequence.The BLASTx analysis of deduced protein sequence showed that it had high similarities with the sequences of other algae.The highest one was more than 90%.The protein also had EF hand and calcium binding site.The ORF sequence of PyHSP70 had 2007 bp,and encoded 668 amino acids with a start codon ATG and terminator TAG.The deduced protein was with a molecular weight of about 72000 Da and the isoelectric point was 5.17.The result of signal peptide prediction showed that the protein had no signal peptide sequence too.Two recombinant expression vector were constructed by fused the cloned genes into the vector pET28 a and transformed them into BL21 E.coli competent cells.The genes were expressed in the transformed cells by IPTG induction.The result of SDS-PAGE confirmed the molecular weight of two genes were consistent with predicted results.The expressed proteins purified were measured with mass spectrum.The results proved that the two proteins were the target proteins we need.The expression levels of PyHSP70 and PyCaM under high temperature stress were checked by real-time fluorescent quantitative PCR.The results indicated that the expression levels of the two genes in the treatments were significantly higher than those in the control.The expression levels of PyHSP70 and PyCaM reached the highest level after heat stress for1 h or 3h respectively.The assay of ATPase activity of PyHSP70 showed that the ATP contentwas reduced with the increase of the purified PyHSP70 protein.The result indicated that the PyHSP70 had activity of ATPase.The growth of the PyHSP70 transgenic E.coli under heat stress was determined.The results showed that the transgenic E.coli grew obviously better than the control.The over expression of PyHSP70 in the transgenic E.coli can improve the heat resistance of E.coli.Our experiment also demonstrated that the PyCaM and PyHSP70 have a binding effect in vitro by using gel overlay technique.These achievements lay a theoretical foundation for further study of plant heat shock signal transduction pathway.
Keywords/Search Tags:Porphyra yezoensis, CaM, HSP70, prokaryotic expression, proteins interaction
PDF Full Text Request
Related items