| Tetranychus cinnabarinus(Boisduval),belonging to Acari,Tetranychidae,Tetranychus,is a world-wide economically important pest which widely lives on economic crops and ornamental plants such as flowers,beans,cotton,vegetables,mulberry and fruit trees,etc.and could cause severe economical loss.For a long time,insecticides had played an important part in contolling T.cinnabarinus and caused their resistances at different levels to various acaricides.Taking T.cinnabarinus as research object,we firstly explored on the physiological and biochemical reaction of T.cinnabarinus to diflubenzuron stress,based on an analysis of the toxic effects of diflubenzuron on T.cinnabarinus.By cloning the chitin synthase gene from T.cinnabarinus and analyzing its sequence with bioinformatics methods,we secondly researched on the mRNA expression patterns and prokaryotic expression of chitin synthase gene from T.cinnabarinus.Combining with the above research conclusions,this study finally made a comprehensive analysis on the response mechanism of T.cinnabarinus to diflubenzuron,so as to provide reference for better understanding of the response mechanism of insects to insecticids.The main results were summarized as follows.1.Study on the toxicity and sublethal effects of diflubenzuron on T.cinnabarinus The median lethal concentration(LC50)of diflubenzuron to T.cinnabarinus at different developmental stages—eggs,larvae,nymphs,and female adults were 15.83?16.37?18.54 and 24.77 mg/L.It showed that diflubenzuron had toxicity on T.cinnabarinus at different developmental stages,the different toxicities of diflubenzuron to T.cinnabarinus were eggs>larvae>nymphs>female adults.The toxicity of diflubenzuron to eggs was 1.57 times than that of female adults.When eggs,larvae,nymphs and female adults were treated with different sublethal doses of diflubenzuron,the main results were summarized as follows.Egg hatchability rate declined with the increasing of the dosage of diflubenzuron,and the survival rate of larvae hatched with survival eggs treated with sublethal doses of diflubenzuron decreased with the increasing of diflubenzuron.The survival rate of larvae treated with LC50 was only 20.43%of control group.The survival rates of larvae and nymphs also declined with the increasing of the dosage of diflubenzuron when they were treated with the above doses.When female adults were treated with diflubenzuron,their longevity,fecundity and egg hatchability were all affected----with lower doses(LC10?LC20 and LC30),their daily fecundity and total fecundity per female adult were stimulated obviously,in which the group treated with LC30 were the highest values.However,when female adults were treated with LC40 and LC50,their daily fecundity and total fecundity per female adult were inhibited.Meanwhile,mean longevity and oviposition duration of female adults were shortened gradually with the increasing of concentration of diflubenzuron.Egg hatchability of female adults in treated groups decreased gradually with the increasing of concentration of diflubenzuron.These results demonstrated that diflubenzuron was toxic to all the different developmental stages of T.cinnabarinus,and could affect growth,development and reproduction of laboratory populations of T.cinnabarinus.2.Study on the physiological and biochemical reaction of T.cinnabarinus to diflubenzuronCompared with control groups,malondialdehyde(MDA)content in eggs,larvae,nymphs and female adults of T.cinnabarinus increased with the increasing of diflubenzuron concentration when they were respectively treated with LC10?LC20?LC30?LC40and LC50?MDA contents in eggs,larvae,nymphs and female adults were highest in the groups treated with LC50.Wether in the treated groups or the control groups,MDA contents in larvae,nymphs and female adults were higher than in eggs,which suggested that diflubenzuron caused oxidative stress and led to lipid peroxidation of biomembrane in all the different developmental stages of.T.cinnabarinus.Superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)activities in all the different developmental stages of T.cinnabarinus manifested as the changing trend of being stimulated firstly and then surpressed in the end.Compared with control groups,SOD and CAT activities in all the different developmental stages of T.cinnabarinus gradually increased with concentration increase of diflubenzuron from LC10,LC20 to LC30 and reached the highest in the groups with LC30.However,with the concentration increase of diflubenzuron from LC40 to LC50,SOD activities showed a decreasing trend.Wether in treated groups orcontrol groups,SOD activities in nymphs and female adults were higher than in eggs and larvae.Also,compared with control groups,POD' activities in eggs,larvae,nymphs and female adults of T.cinnabarinus increased with the concentration increase of diflubenzuron,when they were respectively treated in order with LC10?LC20?LC30 and LC40,and reached the highest in groups with LC40.POD activities in different developmental stages of T.cinnabarinus were:eggs<larvae<nymphs<female adults.Compared with control groups,GSH-S-transferase(GSTs)activites in all the different developmental stages of T.cinnabarinus increased gradually with concentration increase of diflubenzuron.When they were treated with LC50,GSTs activites in eggs,larvae,nymphs and female adults reached the highest level.In addition,wether in treated groups or control groups,the changing trend for GSTs activities in different developmental stages of T.cinnabarinus showed:eggs<larvae<nymphs<female adults.Compared with the control group,total antioxidant capacity(T-AOC)in different developmental stages of T.cinnabarinus increased gradually with concentration increase of diflubenzuron.When they were treated with LC50,total antioxidant capacity of eggs,larvae,nymphs and female adults reached the highest level.Additionally,total antioxidant capacity of nymphs and female adults showed always higher than that of eggs and larvae in T.cinnabarinus.On biochemical level,diflubenzuron could reduce the chitin contents in eggs,larvae,nymphs and female adults of T.cinnabarinus under the diflubenzuron stress of different lethal dosages.Compared with control groups,the chitin contents in eggs,larvae,nymphs and female adults were lowest at the concentration of LC50.3.Full length cDNA cloning and sequence analysis of chitin synthase gene in T cinnabarinusIn this study,full length cDNA of chitin synthase gene in T.cinnabarinus was first cloned by the combined techniques of reverse transcriptase-PCR(RT-PCR)with rapid amplification of cDNA ends(RACE),and its GenBank accession in NCBI is KM242062.The TcCHSl cDNA was 4881 bp in length,including 198 bp 5' terminal UTR,4425 bp encoding region including 1474 amino acids and 258 bp 3' terminal UTR.And the theoretical molecular weight of TcCHSl based on the deduced amino acid sequence was calculated to be 168.35 kDa,with an isoelectric point of 6.26.Based on the deduced amino acid sequence encoded by TcCHS1 gene,it was predicted to be a typical transmembrane protein with seventeen transmembrane helixes.There were three domains(A,B and C)in the deduced amino acid sequence encoded by TcCHS1 gene.Domain A was located in the N-terminal and contained 606 amino acids with ten transmembrane helixes.There were 299 amino acids with signature sequences "EDR" and "QRRRW" in the key domain B.Domain C was located in the C-terminal and contained 568 amino acids.O-glycosylation sites of the deduced amino acid sequence of chitin synthase in T.cinnabarinus were NRTG,NLSD,NLST,NITF and NSSG.Signal peptide prediction of the deduced amino acid sequence of chitin synthase in T.cinnabarinus suggested that there was no signal peptide site,indicating that this protein of encoding TcCHS1 gene was not excretive.Alignment of the deduced amino acid sequence of TcCHS1 and those of other insects demonstrated that it.shared 98%and 55%similarity with T.urticae and Metaseiulus occidentalis respectively,and it showed approximately 50%similarity with other insects.Phylogenetic tree analysis reveaed that the putative CHS amino acid sequence of T.cinnabarinus showed high similarity with the sequences of CHS1 in other insects,and was most closely related to that of T.urticae.4.The mRNA expression patterns of TcCHS1 gene from T.cinnabarinusReal-time quantitative PCR demonstrated that TcCHS1 gene was expressed in different developmental stages,including egg,larvae,protonymph,deutonymph,female adult and male adult..The result suggested that the TcCHS1 mRNA were expressed highest in eggs and female adults,but lowest in deutonymph.All the results indicated that TcCHSl could play an important role in the growth and development of T.cinnabarinus.In order.to explore the characterization of chitin synthase gene from T.cinnabarinus responsing to diflubenzuron exposure,eggs,larvae,nymphs and female adults were treated with different dosages of diflubenzuron,mRNA expression level of TcCHS1 gene were detected by RT-qPCR,?-actin gene of T.cinnabarinus was the reference gene.These results suggested that the relative expression level of TcCHS1 gene were increased in the different developmental stages of T.cinnabarinus,comparing with their own control group.All these results demonstrated that chitin synthase gene of T.cinnabarinus could play the vital role in the toxic mechanism of diflubenzuron at molecular level.5.Prokaryotic expression of chitin synthase gene in T.cinnabarinusIn the present.study,the prokaryotic expression vector for TcCHS1 based on pET-32a(?)vector in Escherichia coli was successfully constructed by applying the double digestion EcoR? and Hind? of restriction enzymes with DNA recombination technology,and protein expression induced by IPTG was detected using SDS-PAGE method.The result provided good basis for TcCHS1 expression in vitro,and made it possible for exploring the characteristics and physiological function of TcCHS1 in the future. |
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