Cloning And Functional Study Of Chitin Synthase 2 Gene In Rusty Grain Beetle(Cryptolestes Ferrugineus)

Cryptodestes ferrugineus?Stephens?is the most common stored grain pest in the world.Its Adults and larvae mainly feed on the embryos of the grain.Owing to their strong reproductive ability,they are easy to reproduce in the grain depots,causing serious harm to the safety of stored grain.Due to the long-term use of phosphine,coupled with the irregular operation of the applicator during use,C.ferrugineus has generally developed high resistance.The widespread resistance to pesticides and the rampant population of C.ferrugineus have become the most serious problems in grain storage at present.Therefore,the development of new,environmentally friendly pesticides has become a top priority.Chitin mainly exists in the cell wall of fungi,the shell of crustaceans and the body wall of insects.Higher mammals do not contain chitin.Therefore,the development of new insecticides based on chitin synthesis synthesis has become a hot topic.In this study,the characteristics and functions of chitin synthase 2?CHS2?gene,a key enzyme in the chitin biosynthesis synthesis pathway,were studied by RNAi technology and real-time quantitative PCR?qPCR?.The results provide theoretical basis and scientific basis for the future use of chitin synthase to prevent and control C.ferrugineus.As a kind of chitin synthesis inhibitor,chlorfluazuron has been widely used.But the effects of chlorfluazuron on the growth and development of C.ferrugineus,and possible targets have not been systematically studied.In this study,C.ferrugineus was treated with sub-lethal dosage of chlorfluazuron to study the effects of chlorfluazuron on the growth and development of C.ferrugineus,as well as its possible targets,so as to provide a theoretical basis for the future use of chlorfluazuron in the prevention and control of stored grain pests.The details are as follows:1.Cloning of Chitin Synthase 2 Gene from C.ferrugineusAccording to the homologous comparison of transcriptome sequencing sequence with other insects'CHS2 gene,conservative primers were found,and three fragments of CHS2gene were amplified.The complete open reading frame of chitin synthase 2 gene was obtained by DNA MAN software splicing.The CHS2 gene sequence was translated into amino acids and compared with the amino acids of some insects,such as Tribolium castaneum.The results showed that the CHS2 gene was closely related to the protein of Tribolium castaneum,Leptiarsa decemlineata and Anthonomus grandis.It is CfCSH2?GenBank landing number MH234580?.The analysis of ORF Finder software in NCBI shows that ORF contains 4428 BP and encodes 1475 amino acids.There are 11 predicted glycosylation sites and 14 transmembrane structures in the amino acid sequence,indicating that the protein encoded by the amino acid sequence is a transmembrane protein.Among them,322?523-845?amino acids are conserved sequences of chitin synthase,containing EDR and QRRRW,which are specific label sequences of chitin synthase.2.Spatial and temporal expression of chitin synthase 2 gene in C.ferrugineusThe expression of chitin synthase 2?CfCHS2?gene was analyzed by Real-time Quantitative polymerase chain reaction?qPCR?at different developmental stages of T.rubrum.The results showed that CfCSH2 was expressed in different developmental stages of egg,larvae,pupae and adults,but the expression level was significantly different?P<0.05?.In larval stage,the relative expression of CfCSH2 was the highest at the 1st instar,then gradually decreased,and the lowest expression was in pupa stage,with a small expression in egg stage and the highest expression in adult stage.The results showed that the gene was expressed more in the feeding period and less in the non-feeding period.The expression of CfCSH2 in different tissues was significantly different?P<0.05?.There was little expression in the epidermis of adults,and a little expression in the head,chest and fat body.The highest expression was in the abdomen,followed by the midgut.The high expression in the abdomen might be related to the larger volume of the sample and more tissue.3.Functional Study of Chitin Synthase 2 Gene from C.ferrugineusThe function of chitin synthase gene and its effect on insect growth and development were studied by RNAi interference technique.CfCHS2 gene silencing was achieved by in vitro injection and feeding in the larvae of C.ferrugineus.The results showed that no matter by injection or feeding,the larvae were curled up after RNA silencing,and their abdomen was empty and could not crawl normally.At the same time,the optimal interference efficiency of injecting dsRNA was 64.83%,which was significantly higher than that of feeding group?49.59%?.After 7 days of continuous feeding,the mortality rate of the experimental group reached 63.33%,which was significantly higher than that of the control group?6.67%?.During the continuous feeding of dsRNA,the expression of CfCHS2 was found to be over-expressed.The expression of CfCHS2 gene decreased first,then increased and then decreased.This indicates that after the silence of CfCHS2 by dsRNA,insects increased the expression of CfCHS2 in order to maintain the stability of the body,thus forming a compensatory feedback mechanism.Toluidine blue staining and transmission electron microscopy?TEM?were used to study the midgut of C.ferrugineus treated with RNAi.The results showed that the midgut peritrophic membrane of larvae after CfCHS2gene silencing was absent,the intestinal epithelial cells were not protected by the peritrophic membrane and could not adhere to the transverse muscle of the midgut.These results suggest that chitin synthase 2 plays an important role in the formation of peritrophic membrane in the midgut of C.ferrugineus.Silencing of CfCHS2 gene can hinder the development and cause death of C.ferrugineus.In the experiment,nano-chitosan was used as carrier,and the expression of target gene was significantly decreased by feeding method.The mortality rate of C.ferrugineus was significantly increased,which provided a scientific basis for the prevention and control of C.ferrugineus based on RNAi technology in the future.4.Effect of Chlorfluazuron on the Growth and Development of C.ferrugineus and Its Action MechanismsThe toxicity equation of chlorfluazuron to C.ferrugineus was established:y=7.514+1.81x,r=0.974.The calculated LD₅₀0 was 0.041 mg/kg,95%confidence interval was 0.025-0.059 mg/kg,and the sublethal doses of LD₁₀,LD₂₀0 and LD₃₀0 were 0.008 mg/kg,0.014 mg/kg and 0.021 mg/kg.In addition,it was found that sublethal dosage of chlorfluazuron treatment could significantly prolong the growth and development period of C.ferrugineus and significantly reduce its body weight?P<0.05?.After long-term treatment with LD₃₀0 of chlorfluazuron,the body weight of C.ferrugineus could be reduced by 25%.Low dosage of chlorfluazuron could significantly reduce the content of chitin?P<0.05?,which indicated that chlorfluazuron had a significant inhibitory effect on the growth and development of C.ferrugineus,and could be used to control the stored grain pest.It was found that the expression of CfCHS1 gene in the third instar larvae of C.ferrugineus treated with LD₃₀0 chlorfluazuron was significantly up-regulated,which was 1.787 times higher than that in the control group.The expression of CfCHS2 gene was also up-regulated,which was1.274 times higher than that of the control group.Therefore,the target of chlorfluazuron is chitin synthase from C.ferrugineus,which inhibits the activity of CHS and makes CHS unable to synthesize chitin normally.