Functional Research Of Homologous Genes Of Sphingomyelin Synthase And Sphingomyelinase In Nilaparvata Lugens (St?l)

Bioactive sphingolipids such as ceramides and sphingomyelins are not only one of the essential components of the plasma membrane in eukaryotic cells,but also appreciated as signaling modulators involved in multiple cell biological processes.As two vital types of enzymes in sphingolipid metabolism,sphingomyelin synthase(SMS)family and sphingomyelinases(SMases)play important roles in maintaining homeostasis between ceramides and sphingomyelins in organisms.The subject of the present research is the brown planthopper(BPH)Nilaparvata lugens(St?l)(Hemiptera: Delphacidae),one of the major pests for rice.The present study cloned and identified homologous genes of SMS family and SMases in BPH,and conducted a functional research of the two set of genes in modulating BPH biological processes based on RNA interference(RNAi)tech.The main results were as follows:1.Two SMS family homologous genes,NlSMSL1 and NlSMSL2,were cloned in BPH,and they showed different spatiotemporal expression levels and enzymic activities in vivo.NlSMSL1 was 1434 bp,encoding 477 amino acids,which were highly expressed in BPH adults and reproductive organs,while NlSMSL2 was 1188 bp,encoding 395 amino acids,and were highly expressed in BPH adults and malpighian tubules.There were no significant changes in the concentrations of ceramides and sphingomyelins when NlSMSL1 was inhibited by RNAi.However,after NlSMSL2 was interfered,a significant increase was found in the total concentrations of ceramides in BPH,especially d18:0/18:1 ceramide and d18:1/18:0 ceramide.2.After interfering NlSMSL1 and NlSMSL2 in BPH by microinjection of dsRNA,we found that these two genes played crucial roles in modulating survival rate,weight,fecundity,hatchability,and development of reproductive organs.The decrease of NlSMSL1 was followed by a significant increase in the weight of female BPH,but there were no significant changes in the total concentrations of free fatty acids,which are mostly C16 and C18.Besides,the fecundity and hatchability of female BPH also significantly decreased.The more retarded development of ovaries for the experimental group(microinjection of ds NlSMSL1)as compared to the control group(microinjection of ds GFP)was observed under an optical microscope,along with the significant decrease in vitellogein related genes and protein levels.After the interference of NlSMSL2,the survival rate of female BPH significantly decreased.There was no significant change in the fecundity and hatchability of NlSMSL2-interfered female BPH,however,the fecundity of normal female BPH significantly increased when mated with NlSMSL2-interfered male.3.Four SMases homologous genes(NlSMase1,NlSMase2,NlSMase3 and NlSMase4)in BPH were cloned,and their key functions in ovarian developments were identified.NlSMase1,2,3,4,was 2196,1687,1275 and 1446 bp,encoding 731,550,424 and 481 amino acids,respectively,which were highly expressed in females and ovaries,in nymphs and midguts,in females and wings,and in females and ovaries,respectively.Although NlSMase1~4 shared less than 35% similarity in amino acid alignments,the interferences of all the four genes could delay ovarian developments in different degrees,and the decrease of NlSMase4 showed the most significant effect.Accordingly,we analyzed the transcriptome of NlSMase4-interfered ovaries and found 211 different expressing genes,among which 112 genes were upregulated while 99 genes were downregulated.GO and KEGG analysis showed most of changed genes enriched in Protein kinase C signaling,Diacylglycerol kinase activity,Glycerophospholipid metabolism,Phosphatidylinositol signaling system and Glycerolipid metabolism.Therefore,NlSMase4 might be involved in development of ovaries by regulating of multiple lipids metabolism.