Regulation Mechanisms Of Knickkopf On The Formation Of Drosophila Wing And Haltere

knk is a key gene for chitin arrangement in the insect group.Mutant study of knk showed that the formation of procuticle chitin was affected in the cuticle,as well the arrangement of chitin in the tracheal system during the embryonic period in Drosophila,with being lethal in the early larval stage.However,it has not been reported that the function of knk in the important organs like wing and haltere.Gal4/UAS system,a transgenic expression tool,was used widely in the study of gene function in Drosophila,and allowed the target gene to be specifically expressed based on the appointed promoter properties.In this study,the regulation rules of the expression level of the target gene with temperature in the Gal4/UAS system were studied firstly,and then the rules were fully utilized to the further study of the mechanism of knk regulation in the formation of wing and haltere.The main contents are as follows:Firstly,taking dpp-Gal4 and target gene wg as an example,the larvae of cross progeny were cultured at a series of temperatures,and fluorescence immunohistochemistry was used to detect wg transgene expression levels in the 3rd instar larvae,which was further quantified and analyzed.The results showed that,the overexpression levels of transgene wg was not affected by temperature,while the expression level of wg RNAi decreased significantly with the rise of temperature.In addition,the system of Gal4 and Gal80ts combination could flexibly manipulate UAS transgene expression levels between 25-30?.Secondly,the detection results of knk expression pattern in Drosophila by RT-qPCR showed that knk highly expressed at the pupal stage,and the period of knk function on wing was from about 30 hours before pupation and 48 hours after pupation by the Gal80 ts and temperature shift.According to the rules of target gene expression level with temperature in Gal4/UAS system,the overexpression and RNAi were performed in the whole wing respectively.Both of them had no any visible phenotype in the wing discs at larval stage,as well knk overexpression at the pupal and adult stage.While knk was RNA interfered,the pupal wings within shell were deformed when been pressed and become lighter in the color of vein.Further,there were three defect phenotypes in the adult wing: 1)failure to molt and lethal due to the drag from wing;2)molting without wings that left within the pupal shell;3)molting with defect wing that were crimped or expanded but lighter color.When driven by ap-Gal4,working in dorsal layer of wing,the wings of knk over-expression were upturned,on the contrary,the wings of knk RNAi were downturned.Thirdly,the further tests showed the physical structure and some properties of wing were effected by knk over-expression and RNAi.The pupal wing became thickener and its space of two layers got wider when knk was over-expressed;wing veins color of adult wing slightly got darker;the FB28 dye was impermeable from vein to membrane;the nanostructure-bumps on wing membrane has deformed,but chitin lamella and lipid regionalization had no visible difference from control.While knk was RNA interfered,pupal wing became thinner and its space of two layer of pupal wing pupal got narrowed,,the adult wing vein structure was damaged,the hairs on the wing membrane seemed to be outstanding,it was easier for FB28 dye to penetrate,bumps were smaller and shorter,in addition,lipid regionalization on the wing changed,and the procuticle got thinner and its chitin lamella disappeared.Finally,haltere,as the degraded wing in Diptera,the size of flatten haltere were measured and the results showed that,the normal size of the male is shorter than the female significantly,there were no difference when knk was over-expressed.While knk was RNA interfered,the size in both of male and the female became shorter significantly,and there was no difference between the male and the female anymore,and the chitin lamella disappeared like the wing.In conclusion,the expression level of target gene in Gal4/UAS system,could be manipulated flexibly by the temperature control.When knk expression level was regulated,over-expression(up-regulation)or RNAi(down-regulation),the normal molting of body,the chitin lamella,nanostructure bumps on wing membrane,the permeability to dye and lipid distribution were effected,as well the halteres size.These results will complement the contents in the areas of insect wing and haltere,gene function study of knk and chitin formation.