Identification Of Chitinase Genes And Function Of DcCht5 And DcCht10 In Eclosion Stage Of Diaphorina Citri

Diaphorina citri infests the rutaceae plants and is the major vector of citrus Huanglongbing(HLB),which is serious threat to the development of citrus industry.At present,prevention of D.citri is one of the main measures to control the spread and outbreak of HLB.D.citri has evolved resistance to a variety of pesticides(such as chlorpyrifos,imidacloprid)due to the unreasonable use of chemicals.Therefore,it is of great significance to excavate new insecticidal targets for the control of D.citri.Chitin is a directly connected polysaccharide composed of N-acetylglucosamine,which is involved in the formation of insect epidermis,midgut peritrophic membrane(PM)and trachea.The growth and development of insects require periodic replacement of chitin,including the degradation of old epidermis and the formation of new epidermis.Chitinases hydrolyze chitin by cleavage of β-1,4-glycosidic bonds between N-acetylglucosamine.Insect chitinase family genes are involved in important physiological processes such as molting,perinophagous membrane degradation,pupation,eclosion and immune defense.The growth of human beings and other higher class animals do not depend on chitin,so it is relatively safe to use chitinase as a target for pest control.Insect chitinase,as a novel insecticide target,has great research potential and practical significance.Based on the NCBI genome of D.citri,10 chitinase genes including 8 chitinase genes(Chts)and 2 imaginal disc growth factors genes(IDGFs)were identified by bioinformatics analysis.The expression patterns of these chitinase genes in different developmental stages,tissues,high and low temperature treatment,dry stress and 20 E treatment were analyzed by qPCR technique.Proteome sequences before and after eclosion of D.citri were determined to screen the differentially expressed chitinase proteins,their encoding genes function in the eclosion stage was explored by using RNAi technology.The main findings are as follows:1.Genome-wide identification of chitinase genes in D.citriBased on the D.citri genome,the nucleic acid and amino acid sequences of the D.citri chitinase gene were obtained by tblastn and tblastp using the chitinase gene of pea aphid and brown planthopper as references.According to domains,exosn,introns and phylogenetic analysis,10 chitinase genes were identified,the length of encoded amino acids was 236 to 2,708 bp,the relative molecular weight of the proteins was 49.1 to 306.5 k Da,and the theoretical isoelectric point was 5.70 to 8.96.The number of exons varies greatly,for example,DcCht8 has only 3 exons,while DcCht6 and DcCht10 have more than 20.All the 10 genes contained chitinase catalytic domains,DcCht1,DcCht7 and DcCht10 contained 4,3 and 5 catalytic domains,respectively,while the other genes contained only one.Multiple sequence alignment analysis showed that the DWEYP sequence in the conserved domain II of DcCht1-a,DcCht7-a,DcCht8,DcCht9,DcCht10-a,DcCht10-b and Dc IDGF1 was replaced by other amino acids,suggesting the loss of chitinase activity.Phylogenetic tree copolymerization of insect chitinase showed 11 Groups,while10 chitinase genes clustered in 7 Groups,namely Group I,II,III,IV,V,VI and VIII.2.Expression pattern analysis of chitinase gene in D.citriThe expression patterns of chitinase genes in different developmental stages and tissues were analyzed by qPCR technique.The expression levels of DcCht1,DcCht5 and DcCht10 in eggs were significantly higher than those of other genes.The transcription levels of 10 chitinase genes were all low from the 1～4 instar of nymph.The expression levels of DcCht5 and DcCht9 were up-regulated and DcCht10 was down-regulated from the 5 instar nymphs to the newly emerged female adults.Compared with other genes,DcCht5,DcCht7 and DcCht9 were highly expressed in day 7 female adults.Dc IDGF1 and Dc IDGF2 are specifically highly expressed in the fat body of adult D.citri.The expression levels of 10 chitinase genes were low in midgut and martensian tubule.DcCht5,DcCht9,DcCht10 and Dc IDGF2 are highly expressed specifically in the epidermis,suggesting that they may be involved in the metabolic process of insect epidermis.The expression patterns of chitinase under extreme temperature and humidity stress and after20 E treatment were analyzed.It was found that DcCht6,DcCht7,DcCht9 and Dc IDGF1 responded to low temperature stress at 4℃ and 16℃ to different degrees.The expression of Dc IDGF1 increased significantly after 10 h of 35℃ stress,suggesting it may be involved in the response to high temperature.The expression levels of DcCht6,DcCht7,DcCht8,DcCht9,Dc IDGF1 and Dc IDGF2 under drying stress at 12 h were significantly lower than those at 6 h.After 20 E treatment,except DcCht2 and Dc IDGF2,other genes showed varying degrees of changes.The expression of DcCht10 was significantly higher than control group at 3 h,6 h and 8h,suggesting that DcCht10 was closely related to 20 E regulation.3.Functional exploration of DcCht5 and DcCht10 during eclosionA total of 4,253 proteins were identified in the proteome of D.citri before and after eclosion,2513,2011 and 2192 annotated proteins were obtained by GO,COG and KEGG databases,respectively.By protein expression of the ratio of the amount(|FC| > 1.2,P< 0.05)screening to1059 differentially expressed proteins,including 571 up-regulated proteins and 488 down regulated proteins.Among them,27 epiderm-related proteins were found,including 23 epiderm related proteins and 4 chitin related proteins.According to the protein domains and sequence alignment revealed that chitin related proteins contained 2 chitinase,namely DcCht5 and DcCht10.The function of DcCht5 and DcCht10 in the eclosion of D.citri were investigated by RNAi technique.The expression levels of DcCht5 and DcCht10 decreased significantly 24 hours after ds RNA injection,and the silencing efficiency was 76.63% and 80.92%,respectively.Interference with DcCht5 and DcCht10 resulted in the reduced emergence and survival rate.In addition,the malformation rate of DcCht5 treatment group was significantly higher than control group.In conclusion,this dissertation identified 10 chitinase genes of D.citri and their expression patterns in different developmental stages,tissues,environmental stresses and 20 E treatments were analyzed.Based on proteomics,bioinformatics analysis and RNAi technology before and after eclosion,the functions of DcCht5 and DcCht10 in eclosion stage were elucidated.It provides a theoretical basis for the development of new insect control agents targeting chitinase.