The Preliminary Study On The Function Of OsPLC3 Gene In Rice

Phosphoinositide-specific phospholipase C(PI-PLC)belongs to an important class of enzymes involved in signaling related to lipids.They hydrolyze phosphatidylinositol-4,5-bisphosphate(PIP2),a membrane-associated phospholipid,to produce inositol-1,4,5-trisphosphate(IP3)and diacylglycerol(DAG),both of which are second messengers in the signal transduction pathway.IP3 and DAG can release Ca2+ from intracellular stores and activate members of the protein kinase C(PKC)family respectively which play central roles in various cellular signaling networks.Moreover,as a substrate,PIP2 is not only the precursor for some membrane phospholipids but also serves as a membrane anchor for many proteins.Plant PI-PLC family have been implicated in major biological processes of growth and development as well as in response to high salt and hyperosmotic stress.To explore the function of OsPLC3 in rice,we generated OsPLC3-overexpession transgenic lines in rice and Arabidopsis,knock-down transgenic rice and genome edited rice using the powerful CRISPR/Cas9 technology.To analyze the tissue specific expression pattern of OsPLC3,more efforts were made to generate the transgenic rice plants expressing the GUS reporter gene under the control of the OsPLC3 promoter.Through phenotypic observation and the expression profiles of OsPLC3 assay,we have gained preliminary insights into the function of OsPLC3 in rice internode growth and salt tolerance.The main results are described as follows:1.We did the alignment of amino acid sequence of OsPLCl,OsPLC3 and AtPLC2.The results showed that all of them harbor typical conserved X/Y catalytic domain and C2 domain.OsPLCl had the similar but none-conserved EF-hand domain with AtPLC2,and no EF-hand domain occured in OsPLC3.2.Compared with wild-type,accelerated growth happened in Arabidopsis over-expressing OsPLC3,suggesting that OsPLC3 could promote the growth and development of Arabidopsis.However,in rice,the observation results demonstrated that knock-down of OsPLC3 brings about dwarfism(shortened internodes),reduced panicle length and low seed setting rate.What's more,obvious differences were found in plant height between WT and OsPLC3-OE.3.We analyzed tissue specific expression pattern of OsPLC3 by qRT-PCR and histochemical analysis of GUS activity.qRT-PCR results showed that OsPLC3 had a relatively lower expression level in the seedling stage of rice,and reached to the highest level in the heading stage,especially abundant in the node and internode.Histochemical analysis of GUS activity in OsPLC3pro-GUS transgenic plants showed that GUS activity was mainly detected in panicle and stem of rice flowering stage.What's more,to determine the subcellular localization of OsPLC3,we constructed pCAMBIA-EYFP-OsPLC3 plasmid.We observed the YFP fluorescence in rice protoplasts and onion epidermal cells after the transient transformation,suggested the subcellular localization of OsPLC3 was mainly in the cytoplasm and nucleus.4.Samples were harvested from 7-day-old rice seedlings,which were treated with NaCl(200 mM)?ABA(100 ?M)and PEG4000(w/v,15%)in a short time.The qRT-PCR results suggest that OsPLC3 can be significantly induced by NaCl and ABA treatment.But PEG showed little effects on OsPLC3 expression.Under salt stress,the expression levels of salt responsive genes(OsHKT2;1?OsMSR2?OsCDPK7 and OsAREB2)in wild type and OsPLC3-RNAi were also examined by qRT-PCR.Results display that knock-down of OsPLC3 alters their expression on transcription level.We also found that salt stress can inhibit the growth of rice seedling.And compared with wild-type,the inhibitory effect on OsPLC3-RNAi was becoming much more obvious,especially on shoot length.5.To analyze the enzyme activity of OsPLC3,we constructed the related protein expression vector,and then the OsPLC3-His fusion protein was expressed and purified successfully.Finally,we obtained the preliminary determination of OsPLC3 activity.