Expression, purification, and subcellular localization of phospholipase C in Dunaliella salina  

作　　者：CONG Yuting WANG Yuan YUE Jinrong XING Zhenyu GAO Xiangnan CHAI Xiaojie 

机构地区：[1]Key Laboratory of Hydrobiology in Liaoning Province,College of Fisheries and Life Science, Dalian Ocean University

出　　处：《Journal of Oceanology and Limnology》2019年第4期1363-1371,共9页海洋湖沼学报（英文）

基　　金：Supported by the National Natural Science Foundation of China(No.31472260);the Key Laboratory of Hydrobiology in Liaoning Province,College of Fisheries and Life Science,Dalian Ocean University

摘　　要：Plants possess effective mechanisms to respond quickly to the external environment. Rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs after a stimulus. The PLC in Dunaliella salina plays important roles in growth and stress responses. However, the molecular basis of PLC action in D. salina remains little understood. To gain insight into the potential biological functions of this enzyme, we cloned a phospholipase C gene from D. salina in a previous study, named DsPLC (GenBank No. KF573428). Here, we present the prokaryotic expression, purification, and characterization of the DsPLC gene. The entire coding region of DsPLC was inserted into an expression vector pET32a, and the DsPLC gene was successfully expressed in Escherichia coli. The DsPLC protein was purified and identified using a polyclonal antibody and western blotting. Expressing DsPLC fused with a green fluorescent protein (GFP) in onion showed that DsPLC-GFP was localized to the intracellular membrane. Quantitative real-time PCR analysis revealed that the relative expression of the DsPLC gene was induced significantly by 3.0-mol/L NaCl at 4 h. Our results support the importance of PLC enzymes in plant defense signaling. This study provides a basis for further functional studies of the DsPLC gene and for additional analysis of the potential roles of PLC enzymes in response to abiotic stress.Plants possess effective mechanisms to respond quickly to the external environment.Rapid activation of phosphatidylinositol-specific phospholipase C(PLC)enzymes occurs after a stimulus.The PLC in Dunaliella salina play s important roles in growth and stress responses.However,the molecular basis of PLC action in D.salina remains little understood.To gain insight into the potential biological functions of this enzyme,we cloned a phospholipase C gene from D.salina in a previous study,named DsPLC(GenBank No.KF573428).Here,we present the prokaryotic expression,purification,and characterization of the DsPLC gene.The entire coding region of DsPLC was inserted into an expression vector pET32 a,and the DsPLC gene was successfully expressed in Escherichia coli.The DsPLC protein was purified and identified using a polyclonal antibody and western blotting.Expressing DsPLC fused with a green fluorescent protein(GFP)in onion showed that DsPLC-GFP was localized to the intracellular membrane.Quantitative real-time PCR analysis revealed that the relative expression of the DsPLC gene was induced significantly by 3.0-mol/L NaCl at 4 h.Our results support the importance of PLC enzymes in plant defense signaling.This study provides a basis for further functional studies of the DsPLC gene and for additional analysis of the potential roles of PLC enzymes in response to abiotic stress.

关 键 词：DUNALIELLA SALINA DsPLC gene PROKARYOTIC EXPRESSION SUBCELLULAR localization salt stress 

分 类 号：P[天文地球]