Functional Analysis Of Genes Regulating Wing Development In Bombyx Mori

Insects are the only winged invertebrates among arthropods and their flight ability is a key factor underlying their expansion,invasion and reproduction.The silkworm Bombyx mori is an important insect as lepidopteran model insect.However,molecular mechanisms of wing disc remain to be explored in Bombyx mori.Here we mainly studied and identified the genes that regulate the development of silkworm wing in two directions.For the forward genetics,the STS molecular localization technology was used to locate the mutant gene?mw?in the range of 275kb on chromosome 22.A total of 15candidate genes were identified in the 275kb area.By comparison the 15 gene expression at mRNA level of analysis,gene Bmapp are highly expressed in the wing primordium,at the same time in the expression of mutant and wild type obvious difference,thus we speculate Bmapp was the important gene for minute wing.Then,using CRISPR/Cas9 system,the early embryo of silkworm was directly injected with Cas9mRNA synthesized in vitro and a specific sgRNA mixture for Bmapp to obtain the mutant bombyx mori.In the Bmapp mutant,the silkworm's wing development is abnormal,which is similar to that of the mw mutant.Therefore,Bmapp is determined to be the key gene for the mw mutation.We found Bmapp promoter in the mutant gene with 10 bp insertion,and mutant Bmapp promoter activity significantly lower than the wild type,the Hippo signaling pathways activated associated with wing development also decreased obviously.This suggests that the mw mutant promoter regions in 10 bp insertion Bmapp has been affected by the promoter activity,make Bmapp expression decreased in the mutant,ultimately affect the Hippo signaling pathway gene expression,lead to the mw phenotype.Secondly,from the direction of reverse genetics,using of CRISPR/Cas9 systems,we design a sgRNA targets respectively in third and fourth exons of BmHh genes according to the principle of GGNN₁₈NGG,and build Cas9 expression vector and sgRNA expression vector,respectively.Microinjection in the early embryos of mulberry silkworm,we get two independent transgenic silkworm strains.After hybridization of two transgenic silkworms,the mutant silkworms knocked out by BmHh were obtained.The knockout of BmHh resulted in abnormal development of silkworm wings and some adult discs.The specific phenotype is as follows:in the mutant silkworm,the wings of silkworm become smaller or even disappear,the feeler of adult silkworm becomes vesicular,and the external genitalia is deformed.qRT-PCR detection was used to analyze the gene expression differences between the mutant silkworm and the wild type,indicating that the gene expression of multiple signaling pathways related to wing development in the BmHh mutant was significantly down-regulated.This indicates that Hedgehog signaling pathway regulates the wings of silkworm.