| HSP70(Heat Shock protein 70 KDa)protein family, as one of the most conservative and significant heat shock protein families, can be induced in large amount when an organism suffers high temperature and other adverse stimulation and it plays a significant role in the renaturation and decomposion of infraprotein and also has many other physiological functions, such as transporting, folding, assembling and locating the new-born peptide in the organism. The genes of encoded HSP70 can be found easily in many kinds of algae, which range from the primitive microalgae to the macroalgae, and played an extremely important role in the algae’s adaptation to the environment. However, the mechanisum that HSP70 plays in marine algae’s resistance to the adverse environment has not been studied further. Therefore, this experiment uses the protonema of porphyra yezoensis as materials for study. An EST sequence of PyHSP70 has found and cloned according to the database of Japanese porphyra andbased on the EST sequence, the author of this thesis imploried the technique of 3’-RACE,5’-RACE,to clone the intact genes of HSP70. And the full length of this gene sequence is 2221 bp, the predicted ORF of it is 1992 bp, the number of its encoded amino acid is 663, the length of 5′UTR is 119 bp, and the length of 3′UTR is110 bp. The analysis of conserved domains of HSP70 shows that it contains conserved domains of ATPase activity and the prediction of its physicochemical property shows that its molecular weight is 71.7KDa and its isoelectric point is 4.93, which is a kind of stable protein. After the preliminary analysis, the author constructed the Py HSP70 and the expression vector pET-28 a together to build a prokaryotic expression vector and then transformed it into the expression bacteria strain BL21 of Escherichia coli to get fusion protein. And the SDS-PAGE electrophoresis of this fusion protein indicates that the construction of expression vector Pet-28a/PyHSP70 is successful and it also shows that its molecular weight was about 71 KDa,which is approximate to the predicted molecular weight. Finally, the author induced the recombinant protein in large amounts and purified it and then utilized BCA(bicinchoninic acid) technique to determine its protein concentration, which shows the concentration is 1mg/ml. And through ATPase activity detection, it indicates that the amount of ATP decreases with increase of the protein, which shows that protein HSP70 has ATPase bioactivity.The growth curves of E. coli under high temperature stress and normal conditions shows that at high temperature pET-28a/PyHSP70 transformed bacterial strain obviously growed better than that of the control group, which indicates that gene HSP70 can enhance E. coli’s ability to resist high temperature. And high temperature induction of the overexpressed of PyHSP70 gene reveals that the thermal induced genes express greatly compared with those of the control group and it reaches its peak under the thermal induction 3~6h, which indicates this kind of genes is closely related to the heat resistance of plants.Through the cloning of gene PyHSP70, the analysis of its gene expression and the determination of the activity of recombinant protein HSP70,it shows that PyHSP70 has the ability to enhance plant’s heat resistance. Therefore, this experiment provides the basis for further research of the algae resistance mechanisum. |
PDF Full Text Request