USP Functional Analysis Using RNAi Technology And Study On The Resistance Of Transgenic Poplar Expressed DsRNA

Gypsy moth(Lymantrla dispar)was a kind of wide distribution pest.At present,the main method of preventing and controling of gypsy moth is application of chemical insecticides.However,use of chemical pesticides is easy to cause insecticide resistance and environmental pollutions.Therefore,new effective and friendly control strategy need to be explored.With the rapid development of molecular biology,the exploration of molecular targets will contribute to find novel control ways.The ideal molecular target is the gene product which involved in vital physiological function.By interferring the expression of this typical gene,the normal physiological mechanism of pests is seriously affected.Transgenic pant expressed dsRNA make it possible that using RNAi technology to control pests in the field.In this paper,the Asian gypsy moth is selected as object,the transcriptomes were constructed of the Asian gypsy moth treated with sublethal dose of rotenone which is one of the botanical insecticide to analyze the differential expression gene by the high through put sequencing technology.The expression quantity of LdUSP gene in the Asian gypsy moth was screened declined specially.The heterodimer composed by ultraspiracle protein(USP)and ecdysone receptor(EcR)plays a crucial role in the process of ecdysone signal transduction.Therefore,ultraspiracle gene plays an important role in the molecular regulation mechanism of incomplete metamorphosis.In this paper,the functions of LdUSP gene was specially analyzed.And the insect-resistant of the transgenic poplar expressed dsRNA was explored.The main results are as the following:1.The tag method was used to determine the toxicity of rotenone to the 3rd-instar L.dispar larvae by bioactivity,the CAT activity and the fresh weight changes of larval.The results showed that rotenone had effective toxic effect on the 3rd-instar L.dispar larvae,12.?24?36 and 48h LC50 of rotenone for the 3rd-instar larvae were 14.10?10.44?9.75 and 9.51 mg/L,respectively.Further the CAT activity was investigated,the results showed that the CAT activity of rotenone treatment group increased first and then decreased,and obviously higher than normal control group.Compared with ddH2O and 5%DMSO treatments,the fresh weight of 3 rd-instar gyspy moth larvae treated by rotenone were lower.2.The transcriptomes data base of the 3rd instar Asian gypsy moth treated by rotenone(4 mg/L)and the control were constructed;10.23 Gb data were obtained.Total 103492 of Transcript and total 40830 of Unigene were obtained,and the length of 12239 among them was over lkb.N50 of Transcript and Unigene were 1976 and 1146,respectively.Total 710 differential expression genes were detected,total 325 were down-regulated expressed genes,total 385 were up-regulated expressed genes.Among them,548 genes were annotated in Nr protein bank,404 genes were annotated in SwissProt protein bank,160 genes were annotated in KEGG protein bank,222 genes were annotated in COG protein bank,257 genes were annotated in GO protein bank.3.Through the analysis of the gypsy moth transcriptome library,the full-length of LdUSP gene was got.The reading frame(ORF)length of LdUSP gene is 1395 bp,and coded with 464 amino acids.The conservative domain predicted that the LdUSP belongs to the nuclear receptor family members.LdUSP protein does not contain signal peptide sequence,and was hydrophilic.The multiple sequence alignment and phylogenetic tree analysis showed LdUSP has close relationship with Agrtis ipsilon USP(AGA17964.1).4.An in-depth profiling study of the transcript levels of LdUSP in different developmental stages,and a temporal analysis of third-instar larvae and different tissues isolated throughout the third-instar stage of L.dispar were then carried out.Relatively high expression levels of LdUSP were observed at 72 h in the third-instar larvae after ecdysis.In the adult male,the expression levels of LdUSP was the highest.And in the egg,prepupa,pupa,the expression levels of LdUSP were low.Analyzed with the RT-qPCR method,LdUSP specially expressed in the thorax.After the 3rd instar larvae were treated with three sublethal concentrations of rotenone(4,10,20 mg/L),the specific expression of LdUSP gene in 3rd instar larvae of Lymantria dispar were analyzed using real-time PCR.The results showed that LdUSP decreased expression in the three doses of rotenone.5.To identify the function of LdUSP in molting and growth of L.dispar,the LdUSP gene in third instar larvae was knocked down by RNA interference.Silencing of LdUSP significantly downregulated the transcript level of E74,E75 and FTZ-F1,three ecdysone-inducible genes and Shd,Sad and Phm,three Halloween genes.The present results indicated that LdUSP gene has direct or indirect control effect to ecdysone response early-related genes,and has feedback regulation to Halloween genes.In addition,the LdUSP knockdown increased larval mortality and increased the duration of the third-instar larval stage.After 8 days of LdUSP silencing,the weights of the larvae were significantly and continuously lower than that of the control larvae fed RNase-free water.The present results indicated that LdUSP might affect the development of L.dispar directly or indirectly.6.LdUSP-3 was chosed as target gene to construct hpRNAs.The Agrobacterium-mediated transformation method was chosed to infection.The transgenic poplars were detected by PCR.The results showed that the transcript levels of the USP gene were reduced after the 3rd gypsy moth fed on transgenic poplar plants.The weight of gypsy moth were not significant affected after fed on transgenic poplar plants for six days.After fed on the transgenic poplar,the phenotype of gypsy moth were not be changed,however,the survival rate were declined.The results showed that LdUSP gene responsed to rotenone stress.The function analysis of LdUSP gene by RNAi indicated that LdUSP gene has direct or indirect control effect to ecdysone response early-related genes,and has feedback regulation to Halloween genes.In addition,the weight of gypsy moth were not been significantly affected after the LdUSP were knockdown,while the survival rate were found to be reduced.The transcript levels of LdUSP gene in third instar larvae were reduced after gypsy moth fed on the transgenic poplar plants.These results will provide a theoretical basis to pest control.