Cloning,Prokaryotic Expression Of CaM And HSP70 Genes From Chondrus Crispus And Functional Analysis Of CcHSP70

The environmental changes will impose influences on plant growth,development as well as metabolism.In recent decades,the signaling pathways of algae responding to environmental stresses and the function of corresponding protein have become hot research spots.HSP70(Heat Shock protein 70),as one member of heat shock protein families,can be induced to express when an organism is suffering high temperature,drought,cold,and other adverse stimulation.The protein also functions to recover or depredate the denatured protein.It is obvious that the protein plays some significant roles in the response to environmental stresses and metabolic regulation of the organism.As the greenhouse effect aggravated,high temperature is one of the major abiotic stress encountered by algae in growth and development.However under high temperature stress,plants will quickly initiate the complex regulatory mechanism to respond the thermal stimulation.One of the regulatory mechanisms is known as Ca2+-Calmodulin heat shock signal pathway,which is a key regulatory mechanism in response to heat stress.For exploring the regulation mechanisms of economic algae Chondrus crispus,CaM(designated as CcCaM)and HSP70(designated as CcHSP70)genes were cloned by using RT-PCR technology.The results of bioinformatics analyzed using online software showed that the ORF of CcCaM is 453 bp,encoding 150 amino acids,and the ORF of CcHSP70 is 1971 bp,encoding 656 amino acids.A CaM binding domain,which was composed by 21 amino acid residues,was found in the CcHSP70 protein sequence.The result suggests that there may exist recognition and binding between CcCaM and CcHSP70 to regulate the downstream events of the heat shock signal network.This data provides the basis for further research on heat shock signal pathway in C.crispus.In order to study the function of HSP70 in C.crispus resistance to heat and cold stresses,the thallus of C.crispus was treated at 28? for heat stress and 4? for cold stress and then the relative expression levels of CcHSP70 were measured by Real-Time PCR.The CcHSP70 was transformed into E.coli to be prokaryotic expressed and then purify the target protein.With purified target protein,ATPase activity was checked.The growth of transformed E.coli cells under 45? high temperature stress and 10? cold stress were measured too.The results showed that overexpression of CcHSP70 in transformed E.coli could be induced by high and cold temperature stress.Under continuous high temperature stress,CcHSP70 expression level reached to maximum at 4h post treatment and then maintained at a high level.The differencewas significant compared to the control.Under continued cold stress,CcHSP70 expression level reached highest level at 8h and the difference was significant between treatments and the control.The analysis result of ATPase activity indicated that the CcHSP70 had ATPase activity.Overexpression of CcHSP70 in E.coli conferred the transformed cell resistance to high and cold temperature stress.These results implied that the expression of CcHSP70 was induced by heat and cold stresses,and overexpression of CcHSP70 in E.coli increase the ability of the cell to tolerate the high or cold temperature stress.