Molecular Cloning And Function Of Ultraspiracle Gene Of Monochamus Alternatus

Monochamus alternatus Hope?Coleoptera:Cerambycidae?,an efficient vector of pine wilt disease,is an important forest pest.Ultraspiracle?USP?in the process of cascade regulation starting position of insect development,metamorphosis and reproduction,has the specificity of organization and time.USP was found in a variety of insects and some arthropods.There is a need for more comprehensive investigation of the structure and function features of USP of different insects in order to gain deeper understanding of its action mode.It also has very important practical value and theory value in researches of both the growth and development of beneficial insects as well as insect pests prevention.In this paper,our study of the protein cDNA sequence characteristics and functions of USP gene of Monochamus alternatus has an important guiding role in the development of environmentally friendly new drug and gene regulation switch.A full length sequence of USP was cloned from Monochamus alternatus by RACE?rapid-amplification of cDNA ends?method;in addition,we also studied both the the physical and chemical properties of USP protein by bioinformatics analysis and functions of USP gene by RNA interference?RNAi?.The main results are as follows:?1?Molecular cloning of MaUSP:A total length of 1583 bp of USP gene sequences,which contains 186 bp 5?end non-coding regions,241 bp 3?end non-coding regions,as well as 12 bp Poly real A tail and a characteristic sequence,plus tail signal named AATAAA from the 1547 to 1552 nucleic acid sites was cloned from Monochamus alternatus by amplifying 3?end and 5?end of MaUSP.The open reading frame?ORF?of the sequence is1155 bp,encoding 384 amino acids,with a calculated molecular mass of 42.87 kDa and an isoelectric point of 8.49.Its molecular formula is C₁₈₇₅H₃₀₂₈N₅₃₆O₅₆₆S₂₃.The similarity is extremely high by online tool NCBI BLAST comparing USP amino acid sequences of seven kinds of insects,with the potato beetle?Leptinotarsa decemlineata?USP having the highest similarity with MaUSP,90%.Phylogenetic tree shows that Monochamus alternatus and Leptinotarsa decemlineata gather together and shows the closest genetic distance among all the 8 kinds of insects in the phylogenetic tree,and Monochamus alternatus shows a closer genetic distance with coleopterous insects while a farther genetic distance with other non-coleopterous insects.?2?Physical and chemical analysis of MaUSP:The MaUSP sequence was translated into protein,and its amino acid composition,the hydrophobic trans-membrane region,glycosylation modofoed phosphorylation prediction,signal peptide,second structure and tertiary structure,subcellular localization,conserved domain were analyzed.The results of amino acid composition showed that the protein contains 20 amino acids of the protein composition of organisms,and the leucine content is the highest,followed by serine.MaUSP was amphoteric amino acid which average hydrophilic coefficient was-0.364.There is no transmembrane region of the protein,MaUSP is a outer membrane protein with a probability of 100%.Phosphorylation predictive analysis showed 38 phosphorylation sites including 20 serine?Ser?phosphorylation sites,14 threonine?Thr?phosphorylation sites and 4 Tyrosine?Tyr?phosphorylation sites.There were 2 N-glycosylation in the 256and 379 amino acid sites.MaUSP is mainly composed of alpha helix and random coil.The protein is predicted to be a nuclear protein according to protein subcellular localization prediction.There are two conserved domains of MaUSP amino acids,one is the DNA binding domain?DBD?which consists of 77 amino acids from 66^th to 142^nd bits,another is the ligand binding domain?LBD?which consists of 202 amino acids from 162^nd to 363^rd bits.?3?RNA interference experiment of DNA binding domain?DBD?,Ligand binding domain?LBD?of Monochamus alternatus USP:The relative expression levels of MaUSP-DBD,MaUSP-LBD and MaEcR were detected respectively after Monochamus alternatus 4^th instar larvae and 5^th instar larvae injecting with MaUSP-DBD dsRNA and MaUSP-LBD dsRNA.The results showed that the relative expression was down-regulated,the RNA interference efficiency of the 4^th instar larvae was higher than that of the larvae of the age of 5,and the mortality rate was also higher than that of the 5^th instar larvae.The larvae treated with dsRNA suffered abnormal growth and development as well as phenotypic defects.Treated larvae could not molt.These results suggest that USP gene plays a key role in the larval larval molting process and larval pupal metamorphosis of Monochamus alternatus.It is speculated that abnormal phenotypes appears when USP and EcR are in different titer levels;when the two are both at the same titer level,they can keep the normal shape of the insect;USP precedes EcR transcription and functions,the USP-DBD domain precedes the USP-LBD domain and activates the USP-LBD domain to regulate the level of EcR titer,the change of EcR titer level may regulate the level of USP titer by stimulating USP-DBD to maintain both USP and EcR at the same titer level.