Characterization And Expression Analysis Of OsIPCS Family Genes In Rice (Oryza Sativa L.)

Inositolphosphorylceramide synthase(IPCS)is an essential enzyme in sphingolipid biosynthesis that catalyse ceramides and phosphatidylinositol(PI)into inositolphosphorylceramide(IPC)in plants.Genes encoding this enzyme have been known to be widely distributed in plants.However,reports on identification,expression,and function analysis of plant IPCS family genes are very limited.The rice genome includes 3 OsIPCS genes: OsIPCS1,OsIPCS2 and OsIPCS3,little is known about the molecular characteristics,expression profiles and gene functions of them.In this study,a total of three OsIPCS genes were identified in the rice database.Similarity analysis of three Os IPCS genes on both DNA and protein level,domain structure and motif analysis were conducted with the help of bioinformatics tools.The expression profiles of OsIPCS were studied by quantitative real-time RT-PCR(qRT-PCR).The transgenetic rice plants overexpressing and down-regulating OsIPCS genes were obtained by agrobacterium-mediated genetic transformation.The subcellular localizations of OsIPCS proteins were analyzed using the transient transformation of Nicotiana benthamiana leaves.Main results are as follows:1.Bioinformatic analysis of OsIPCS family genes in RiceThe OsIPCSs are distributed on the 1st and 5th rice chromosomes.These genes had 12-13 exons.It shows that most plant IPCS genes,including rice,Arabidopsis,maize and soybean,contain 11 introns,this may be a conservative characteristics of plant IPCS family genes.The results of OsIPCS protein sequences analysis also indicated that 3 prediction OsIPCS proteins are typical plant IPCS family proteins,as two typical and conserved motifs(D3 and D4)in plants were found in all the 3 putative Os IPCS proteins.phylogenetic analysis confirmed that most plant IPCS proteins could be classed into two sub-groups: the dicot group and monocot group,OsIPCS1 and Os IPCS2 clustered in a branch and Os IPCS3 in another.Cis-elements like ABRE,MBS,Skn-1_motif and circadian were found in the three Os IPCS promoters,suggesting that OsIPCS genes may be involved in plant growth and development,hormone response and abiotic stress adaptation.The cis-acting element involved in fungal elicitor responsive Box_W1 and the cis-acting element involved in defense and stress responsiveness TC-rich repeats were found in promoters of OsIPCS3 and OsIPCS1,respectively.This indicates that Os IPCS genes may also be involved in biotic stress adaptation.2.Expression profiling of OsIPCS family genes in different tissues of rice under normal and adverse conditionsExpression profiling revealed that OsIPCS genes are differentially expressed in various tissue types isolated from rice.This may suggest that OsIPCS genes play very important roles during the process of rice growth and development.The transcripts of OsIPCS genes appeared in different rice organs including seedling roots,stems and leaves,and young panicles,but each gene showed a unique organ-specific expression pattern.OsIPCS1 and Os IPCS2 showed similar expression patterns,and their highest expressions were found in the young panicles,followed by the young leaves,roots and stems.Whereas,OsIPCS3 showed the highest expression in the young leaves,followed by young panicles,roots and stems.Expression patterns of OsIPCS genes were different under all the measured abiotic stresses like drought,cold and salt.For example,under cold stress,OsIPCS1 and OsIPCS2 were significantly up-regulated in roots and stems but down-regulated in leaves.However,OsIPCS3 were significantly up-regulated in all the measured organs including roots,stems and leaves.3.Vectors construction and genetic transformation of over-expressing and down-regulation of OsIPCS2 and OsIPCS3All OsIPCS genes were cloned by RT-PCR,OsIPCS2 and OsIPCS3 constructed into the expression vector pCXUN,gene silence of OsIPCS2 and OsIPCS3 vectors were constructed based on RNA interfering technology.All the reconstructed vectors were transformed into rice by agrobacterium-mediated method.Following,screened 160 T0 transgenetic seeds,all T1 transgenetic seeds have been harvested,3 T2 lines of overexpressing OsIPCS2 and 2 of OsIPCS3 were obtained.4.Subcellular localization of OsIPCS proteinsThe online software WoLF PSORT was used to predict subcellular localizations of Os IPCS proteins.It showed that OsIPCS1 was expressed in cytoplasm,endoplasmic reticulum,mitochondrion,plasma membrane and vacuole;OsIPCS2 was expressed in cytoplasm,nucleus,plasma membrane,endoplasmic reticulum and vacuole;Os IPCS3 was expressed in mitochondrion,mitochondrion_chloroplast,chloroplast,and cytoplasm_mitochondrion.The fusion expression vectors of OsIPCS proteins and green fluorescent protein GFP were constructed respectively.Subcellular localizations of Os IPCS proteins were observed using the fluorescence microscope by transient transformation of Nicotiana benthamiana leaves.Our results showed that OsIPCS2 was expressed on the nucleus,plasma membrane and other membranes;OsIPCS1 was mainly expressed on the nucleus,while the expression on plasma membrane and other membranes was weak.Further research of the subcellular localization of OsIPCSs protein is needed.