Biological Function Of Ca²⁺-dependent Nuclease In Rice

Nuclease is a kind of protein that can participate in many important molecular biology and biotechnology and acts on phosphodiester bond.It exists in a wide range of living organisms and is of great significance for the growth and development of physiological organisms and physiological activities.In this study,we used Ca2+-dependent nuclease OsCaN1,OsCaN3,and OsCaN4 gene of rices as the research object,and analyzed its biological functions by expression characteristics,subcellular localization,and enzyme activity and obtaining overexpressed plants.First,we used real-time quantitative PCR to analyze the expression of OsCaNl?OsCaN3?OsCaN4 gene in wild-type rices,and to study its expression characteristics when the plants were subjected to salt stress treatment.The results showed that OsCaN gene were the most expressed in mature leaves,indicating that they may all be genes related to senescence;at the same time they were all induced by salt stress,indicating that OsCaN gene related to stress.To explore the location of OsCaN protein expression,plasmids containing GFP tags were transformed into onion epidermis by gene gun and the subcellular localization of OsCaN-GFP was observed by fluorescence microscopy.The results showed that the OsCaNl-GFP?OsCaN3-GFP?OsCaN4-GFP fusion protein was localized in the cytoplasm.Next,in order to explore the enzyme activity of Ca2+-dependent nuclease in rice,the OsCaN gene was cloned into the pGEX-6p-3 plasmid,GST-OsCaN fusion protein was obtained through induction and purification.for enzyme activity detection.The results showed that OsCaN protein has the ability to degrade circular plasmid,dsDNA,ssDNA,mRNA,rRNA,and tRNA with the help of Ca2+,and the metal chelating agent EDTA could inhibit the enzymatic activity of the nuclease,The results showed that OsCaN gene belongs to the Ca2+-dependent nuclease.In order to better explore the funinction of the Ca2+-dependent nuclease OsCaN gene in rice,we have constructed a gene overexpression vector and successfully obtained transgenic rice plants and seeds through Agrobacterium infestation,this laid the foundation for exploration of the function of gene in rice and its response mechanism under stress.