Transcriptomic And Expression Analysis Of The Salivary Glands In Nilaparvata Lugens And Sogatella Furcifera

The brown planthopper(BPH),Nilaparvata lugens(Stal)(Homoptera:Delphacidae)and the white-backed planthopper(WBPH),Sogatella furcifera(Horvath)are the major sap-sucking pests of rice,causing direct damage by probing and indirect damage by transmitting viruses.Saliva of hemiptera insect is thought to play a key role in insect-host iteractions.In this study,the transcriptomes of salivary glands from both planthopper were obtained by Illumina sequencing and genes related to herbivore feeding and plant defense response were studied,and their expression levels in different tissues were also analysed.In addition,the role of multicopper oxidase 4(MC04),a specific expressed gene in the salivary gland of WBPH,were validated by RNAi,which laid the foundation for the study of its role in the development of whitebacked planthopper-rice.The main findings are as follows:(1)The transcriptome of the salivary gland in BPH was obtainedA total of 55,913 transcripts(mean length 751 bp)were obtained from Illumina sequencing of the BPH salivary gland,which were assembled into 45,421(mean length 668 bp),and 16,210 unigenes(35.68%of the total)were matched the known sequences in NCBI,among which 12,758 were annotated as GO terms,and 6,917 unigenes were located in the KEGG pathways.Nineteen genes involved in the sugar metabolism,detoxification,plant defense and other metabolic process were obtained from the salivary gland transcriptome of BPH.Tissue expression profiles of 19 salivary genes revealed that the expression level of alpha-glucosidase 31 had no difference in the five tissues,suggesting that it may have functions in the whole-body.Glucose dehydrogenase[FAD,quinone]-like was highest expressed in the salivary gland,seven of 19 genes were primarily expressed in gut,which might play a major role in insect digestion and detoxification process.In addition,seven candidate reference genes including RPL9(Ribosomal protein L9),RPS11(ribosomal protein S11),RPS15(ribosomal protein S11),ACT(actin 1),MACT(muscle actin),TUB(?-tubulin)and 18S(18S ribosomal RNA)were evaluated by qRT-PCR,and their expression stability in different tissues of BPH were identified by using four alternative methods(BestKeeper,geNorm,NormFinder,and the delta Ct method).RPL9 and RPS15 showed the highest stability for acquisition of different tissues.(2)The transcriptome of the salivary gland in WBPHIn this study,the cDNA library of salivary glands of WBPH was constructed,sequenced and assembled.A total of 51,842 Unigene were obtained with an average length of 746 bp,and 17,767 sequences(34.27%of the total number of Unigene)were noted in the nr database,14,753 sequences were given GO notes,and 7,876 Unigene notes into the KEGG pathways.In the present study,we identified 32 salivary genes involved in in sugar metabolism,digestion,detoxification,and suppression of plant defense responses.Tissue expression profiles analysis by using qPCR revealed 32 genes had a big difference in different tissues.Multicopper oxidase 4,multicopper oxidase 6,carboxylesterase and uridine phosphorylase were primarily expressed in the salivary gland,suggesting that they played putative role in insect-rice interactions.Thirteen genes were primarily expressed in gut,which might play putative role in digestive and detoxify mechanism.Development expression profiles analysis revealed that the expression level of 26 salivary protein genes had no significant difference,suggesting that they may play roles in different developmental stages of WBPH.The other six genes have a higher expression level in the salivary gland of adult compared to nymphs.Except putative acetylcholinesterase 1,all the other genes have no significant difference in male and female insects,suggesting that their expression level have no difference between sexes.(3)RNA interference analysis of MCO4 gene in WBPHFirstly the 3rd instar nymphs of WBPH were fed with artificial diet containing 0.5?g/?L dsRNA.Poor gene silencing effect was checked.Then we use microinjection method.The expression level of MCO4 mRNA was the lowest at the 4th day,and only 5.53%of the control group.The survival rate was not significantly different between the experimental group and the control group,suggesting that MCO4 gene might not be a lethal gene of the WBPH.