Function Study Of Inter-Alpha-Trypsin Inhibitor Heavy Chain 4 And Transcriptomic Analysis Of Ovary Epithelial Plug From Nilaparavata Lugens(St?l)

Nilaparvata lugens St?l,also known as brown planthopper(BPH),is an important agricultural pest of the Hemiptera.It is the most serious rice pest not only in China but also in Asia.When BPH sucks rice sap through its piercing-sucking mouthparts,rice virus such as rice ragged stunt virus can be spread.More than that,the excrement of the BPH contains a variety of amino acids and honeydew,which often leads to the breeding of mold.Those behave lead to a significant reduction in rice production.So far,China's conventional method for controlling brown planthoppers is chemic control,but it has brought problems such as drug resistance of BPH and environmental pollution.As a Homoptera,BPH is monophagos with auxotrophy,which only relies on the phloem sap of rice.However,Yeast-like Symbionts(YLSs)which inside its body provide essential amino acids and cholesterol.YLSs can also accelerate the nitrogen cycle and participate in the sedimentation of yolk.YLSs inside female BPH are coated with fat bodies.They will be released into haemolymph at the second day after BPH eclosions.The very places where they invade into ovaries are epithelial plugs.After invading,YLSs duplicate themselves in the oocyte.This behavior is called vertical transmission,which indicates that epithelial plug plays a key role in the vertical transmission of YLSs.Previous studies uncover that the inter-alpha-trypsin inhibitor heavy chain(ITIH4)gene significantly increases on the second day after eclosion of the BPH,indicating that it may be related to the reproduction of the BPH,or may affect the behavior of YLSs.In this study,the function of ITIH4 was tested and the specific gene expression of ovarian epithelial plug was analyzed.The results are as follows:(1)The whole ITIH4 was cloned.The opening reading frame last 2724 bp,which can be translated into a 907-amino-acid protein with an isoelectric point of 6.17.Signal peptide site is located ai the 24 th amino acid.By sequence alignment and establishment of phylogenetic trees,it was found that ITIH4 is conserved in Hemiptera.The protein sequence transcribed from ITIH4 of the BPH was blasted on NCBI,and two conserved domains was found: the Vault protein inter-alpha-trypsin domain(VIT)and the von Willebrand factor A type(v WFA).Quantitative fluorescence experiments revealed that ITIH4 is the most abundant at 2nd instar larvae,while least at 4th instar larvae.And it was significantly upregulated in female adultson the second day after eclosion.When ITIH4 gene expression was interfered by RNA interference,the mortality rate of BPH was very high,exceeding 87% on the 4th day after eclosion.The average number of eggs laid in the experimental group was 29.4 in ten days after the injection,which was 57.3% lower than the control group.The egg hatching rate of the experimental group was 63.5%,which was also lower than that of control group(87.7%).The RNA interference of BPH retards ovarian development.QPCR confirmed that the expression of the vitellogenin's precursor,vitellogenin(Vg)and vitellogenin receptor(Vg R)were down-regulated in the BPH when ITIH4 silenced.It shows that ITIH4 may affect the reproduction of BPH based on the above genes.Based on the statistics of fecundity and hatching rate,it is speculated that the interference of ITIH4 will prevent the formation and accumulation of Vg,which affect the formation of the BPH eggs,and even make egg death.The precipitation of insect yolk is regulated by juvenile hormone and the juvenile hormone is Methoprene-tolerant(Met)mediates juvenile hormones in the body of the brown planthopper.The expression of juvenile hormone receptors was detected by fluorescent q PCR.No significant differences were found except for the third day afer intereference,test group expressed significant lower than control.The KEGG pathway analysis showed that genes regulating Vg and ovarian development such as insulin growth factor,IGF,transforming growth factor,TGFB,and ITIH4 may co-express in macromolecular phagocytosis.In summary,ITIH4 plays an important role in the development of the ovary,synthesis and accumulation of Vg in the reproductive process of the BPH.(2)The follicular cells of three tissues,petiole,epithelial plug,and follicle,were obtained from the ovary of the BPH by microdissection.The Illumina single-cell transcriptome high-throughput sequencing was performed to screen and analyze the specific expression genes of the epithelial plug.This study used high-throughput sequencing to obtain 78427277 sequences with an average length of 517 bp.After being assembled and assembled by the Trinity method,these genes were compared with public databases(KEGG,KOG,Pfam,NR,and SWISS-Prot)and 151422 genes were annotated.By analysing and annotating,it was found that the gene expression of endosome membrane,endocytosis,and cytoplasmic membrane-bounded vesicle was significantly higher than that of follicular and ovarian tubule plexus at epithelial thrombus.These genes may be related to YLS invasion of the ovary site.There may beinteractions between DNAJB1,TGF-?1 proteins and ITIH4.