Study On Alternative Splicing And Function Of Notch In Brown Planthopper,Nilaparvata Lugen

Notch encodes a highly conserved,single-pass transmembrane receptor protein with an extracellular domain and an intracellular domain.It has fundamental functions in differentiation,proliferation and apoptosis;abnormal expression can lead to death or cancer.It can use canonical and noncanonical Notch signal pathways to submit signals.Notch has four paralogs in mammals but only one in Drosophila melanogaster.Much research considered that Notch codes for only one protein,which as a receptor and contact two ligands（Delta and Serrate）to transmit signals in the past three decades.However,the simple model was difficult to explain how Notch worked in insect to instead of the four in mammals and the complex functions of Notch.This work had deepened the knowledge of Nl Notch in brown planthopper（BPH）,Nilaparvata lugens.Macropterous and brachypterous BPH were maintained for 35 generations,then we got a stable brachypterous BPH population and a macropterous BPH population with a macropterous rate more than 60%.Then we used the populations to do transcriptome analyses and digital gene expression profiling analyses,Nl Notch was chose as the target for the research at last.Through overlapping sequence,we found Nl Notch is a complex alternatively spliced gene that uses different methods,such as alternative initiation,alternative termination,exon skips,exons selectively lengthened or shortened,intron retention and so on to generate variants and code a series of proteins.Nl Notch has 3transcriptional start sites（T1,T2 and T3）,21 transcriptional endpoints and 4 exon skips to generate variants.5 of the 21 transcriptional endpoints can form Non-stop RNAs,and 4 of them can generate very short false 3’UTRs.Nl Notch can use different 3’UTRs to form variants,such as Nl N21 used exclusive exons and trans-splicing to generate 4 different3’UTRs.And full length variants Nl NF had five 3’UTRs from two exons that were cleaved into 12 components and recombined,but no long components could hold only in a single3’UTR and with many short repeat sequences in them.When translated the variants into proteins,we found the two transcriptional start sites T1 and T2 can be used to transcribe the entire gene and use the same start codon,but the third transcriptional start site only transcribe a partly intracellular domain as the full length variants Nl NF.At last,we obtained 10 normal coding sequences and 5 nonstop coding sequences,most of them without intracellular domains.Number of coding sequences was less than the variants indicating that some coding sequences had more than one 3’UTR.Number of conserved domains was utilized to name the variants.Variants with the same conserved domains were distinguished by the length of 3’UTRs.Notch needs an extracellular domain and an intracellular domain.Some ds RNAs were designed and targeted different regions of Nl Notch to detect the function.When 400ng ds RNA was injected into the 3^rd instar nymphs.Insects treated with ds T1 had no phenotype,but ds T2,ds RNAs targeted the coding sequences and the 5 3’UTRs of Nl NF all caused high mortality.Then reduced the injected dose to 100 ng and delayed injection time to the fourth instar,when insects were molting,sensation circles on the antennas near to the root decayed,bristles on forewings shortened,thickened or disappeared,accompanied by thickening veins.When detected the relative expression of Nl Notch in different regions found it has a significant difference among different developmental stages for NICD,but T1 and T2were not.These indicated Nl Notch may have a potential balance system to control the expression of variants.Relative expression of Nl Notch will down regulate after RNA interference means it was successful to disturb Nl Notch.Different antibodies were used to detect the expression of Nl Notch and found Nl Notch coded a series of proteins with the difference among developmental stages.At the 4^th day after RNA interference,the expression of Nl Notch was down regulated.So we suggested the phenotype resulted from RNA interference which will down regulate the expression of Nl Notch.The study results provided evidence that Nl Notch is a complex,alternatively spliced gene and codes a series of proteins.It requires an extracellular domain and an intracellular domain when disturbed it can down regulate the expression of Nl Notch and caused serious phenotype.So Nl Notch uses alternative splicing to instead of the 4 paralogs in mammals.