The Preliminary Study On The Function Of OsPLC1 Gene In Rice

Phospholipase C(PLC)is a class of important hydrolytic enzymes.Based on the specificity of substrates,PLC is mainly categorized into two groups:PI-PLC(phosphoinositide-specific PLC)and NPC(non-specific PLC).The roles of PLCs and their products,IP3 and DAG,in mediating signal transduction of animal cells are indispensable.However,in plant cells,the mechanisms on PLC are poorly understood.In this study,combining with the strategies of bioinformatics,genetic and molecular biology,we identified and analyzed the OsPLC1 gene in rice.The preliminary results are summarized as follows.(1)Generated and analyzed the heterologous overexpression transgenic lines of Arabidopsis carrying plasmid pBA-OsPLC1.The stable inheritance of T3 generation lines were obtained.Results indicated that,no significant difference showed in the pBA-OsPLC1 transgenic Arabidopsis plants compared with Col on germination,vegetative and reproductive growth.Meanwhile,the OsPLC1 overexpression transgenic lines in the background of AtPLC2+/-have been constructed.The positive OsPLC1 overexpression lines in the background of plc2-/-were in screening.(2)Generated the transgenic lines of OsPLC 1-OE,OsPLC1-RNAi,OsPLC1-CRISPR/Cas9 by using the method of agrobacterium-mediated transformation.The stable inheritance of T3 generation lines were obtained.(3)We characterized and analyzed phenotype of transgenic lines.The results showed that overexpression of OsPLC1 leads to the abnormal formation of microspores in the process of anther development,which cannot develop normal mature pollen.What's more,the vigor of mature pollen is much lower than that of Nip.(4)By observing and analyzing the RNAi lines,it was found that the seed germination of RNAi lines lags behind than Nip,and this germination lag will increase with the application of exogenous ABA.When all strains grew to 7 days in size,the length of the shoots of the RNAi lines was mostly distributed within the order of 7 cm,and the ratio of R:NAi lines to 100 seedlings was significantly lower than that of wild type in the order of shoot length greater than 12 cm.When we continue to cultivate the Nip and RNAi lines with seedling length greater than 12 cm to about 60 days,there is no obvious difference between them.However,in long-term light conditions,RNAi strains relative to the Nip will be ahead of flowering.(5)Prokaryotic expression vectors of OsPLC1 were constructed,pGEX-4T1-OsPLC1 and pET28-OsPLC1.And we renatured from the inclusion body to get the active GST-tag and His-tag protein.OsPLC1 was identified to hydrolyze PIP2 and PI in vitro.(6)In identifying phospholipase C activity of OsPLCl,a simple set of methods for detecting phospholipase C activity of one protein was established based on the different chemical properties of the phospholipase C substrate and the product.PIP2 or PI and DAG are fat-soluble substances,insoluble in water,while the other product IP3 can be dissolved in water.Thus,the aqueous phase was separated by an organic one(chloroform:methanol = 2:1)to determine-the phospholipase C activity by dephosphorylation of the alkaline phosphatase and malachite green phosphorus.(7)Yeast Two-Hybrid experiments and in vitro Pull-down demonstrated that the C2 domain of OsPLCl interacted with OsMEK6,OsMPK1,OsMPK5.(8)ABA and IAA treatment of rice Nip were carried out,and then the expression of OsPLC1 was detected.It was found that ABA treatment resulted in the increase of OsPLC1 gene expression,while IAA treatment increased the expression of OsPLC1 at 12 hours instantaneously.