Functional Analysis Of Cht6 And Cht10 In Wing Cuticle Development In Drosophila

The cuticle plays an essential biological role in insects.As an external barrier,it maintains water balance in body and resists the invasion of external pathogenic microorganisms.As the exoskeleton of insects,it provides support and movement.These functions depend on its composition and structure of the cuticle.One of the main components of insect cuticles is chitin.Chitin interacts with chitin binding proteins to deposit on the apical surface of epidermis and trachea cells in a fixed and orderly manner to form a cuticle.As a kind of specialized cuticles,adult wings of Drosophila melanogaster(D.melanogaster)has a relatively simple structure,which is only composed of cystic double layer epithelium without any attached muscle.Wing,as an important flying organ,is necessary for life and behavior in D.melanogaster.Many proteins and enzymes have been identified to participate in the terminal differentiation of wing cuticle.In this paper,the biological functions of Chitinase 6(Cht6)and Chitinase 10(Cht10)were studied by using the model D.melanogaster as the research object.The main results are as follows:1.Expression pattern and structure analysis of Dm Cht6 and Dm Cht10 genes.The coding sequences(CDS)of Dm Cht6 and Dm Cht10 genes were obtained by searching Drosophila transcriptome and genome database(http://flybase.org).The length of Dm Cht6 CDS was 11112 bp,encoding3703 amino acids,and Dm Cht10 CDS was 6861 bp,encoding 2286 amino acids.Based on the analysis of the structure of Cht6 and Cht10,it was found that Cht6 had typical conservative structure in different species,including signal peptide,chitin binding domain(CBD)and Glyco₁8 catalytic domain,while Cht10 also had typical conservative structure in different species,including signal peptide,chitin binding domain(CBD)and Glyco₁8catalytic domain.The expression patterns of Cht6 and Cht10 in wild-type D.melanogaster were analyzed,and it was found that both chitinases were highly expressed in the pupal stage,in which the expression of Cht6 was the highest on the third day of the pupal stage,followed by the first,second and fourth day of the pupal stage,and the highest expression of Cht10 was on the second day of the pupal stage,followed by the first day,the third day and the fourth day of the pupal stage.2.Study on the function of Dm Cht6 in wing cuticle.Ci-gal4(Gal4 specifically expressed in anterior compartment)and en-gal4(Gal4 specifically expressed in posterior compartment)were hybridized with UAS-Cht6-RNAi,respectively.After specific knockout of target gene,phenotypic observation showed that wing size changed significantly after specific knockout of Cht6 in anterior compartment.After the specific knockout of Cht6 in the posterior compartment,there was not only a significant change in the size of the wing,but also an obvious wrinkle in the posterior compartment.The silencing efficiency test showed that the silencing effect of Cht6 in pupa stage was very obvious.Using hematoxylin-eosin staining(HE staining),it was found that the permeability of wing cuticle interfering with Cht6 was significantly enhanced.The results of transmission electron microscopy showed that Cht6 could affect the internal microstructure of the wing.Therefore,Cht6 plays a very important role in the development of D.melanogaster wing cuticle.3.Study on the function of Dm Cht10 in wing cuticleBased on the literature research and previous studies,we found the expression of Cht10 coincided with the expression of chitin synthase gene kkv.This suggests that the two proteins may cooperate with each other in the process of wing differentiation.en-gal4(Gal4 specifically expressed in posterior compartment)and nub-gal4(Gal4 specifically expressed in wing pouch)were hybridized with UAS-Cht10-RNAi,respectively,and obvious phenotypes were observed in adult wings.The results of eosin staining showed that interfering with Cht10 can affect the permeability of wing cuticle.Chitin staining and chitin content detection showed that the inhibition of Cht10 can lead to an excessive accumulation of chitin in wing cuticle.The results of electron microscopy showed that interference with Cht10 could also affect the microstructure of the interior and surface of the wing.Moreover,D.melanogaster with impaired Cht10 function cannot fly.To sum up,Cht10 plays a very important role in the development of D.melanogaster wing cuticle.