BmSd Gene Regulates The Silkworm Wing Size By Affecting The Hippo Pathway

Insects have the most extensive distribution range on the earth.They are the only invertebrate animals with wings.Wings are one of the important organs of insects.Wings not only endow insects with unique ability of flying,but also play an essential role in their courtship,self-defense,foraging and communication.The wings of holometamorphosis insects are developed from the wing primordium during morphological changes.The wing disc develops from the adult disc at the embryonic stage.Bombyx mori,a model lepidoptera insect,losts its flying ability after long-term domestication.The wings of different silkworm strains are also various.Recent reports have found different types of wing mutations,like no wing,small wing,chick wing and so on.The key genes causing these mutations have been studied.In this study,we selected p50 as the control group and u11 as the experimental group.P50,also known as dazao,is a normal wing strain from guangdong province.U11 is a naturally occurring recessive mutant with minute wing phenotype.RNA-Seq analysis previously revealed differential Hippo-pathway-related gene expression between the u11 and p50 strains.Hippo pathway is a highly conserved kinase cascade that controls organ size during animal development.In this study,we conducted a functional analysis of Hippo pathway during the development of silkworm wings.We found that mats,warts,and hippo expression levels were higher in u11 compared to p50 wing discs.sd(scalloped)expression,which encodes a prominent transcriptional partner to Yorkie(Yki),gradually decreased during the wandering stage in u11,but exhibited the opposite expression pattern in p50.Therefore,we used RNAi technology to knockdown sd in the wing disc of p50 at wandering stage.When sd was knocked down by small interfering RNA(si RNA)during the wandering stage in the p50 strain,57.1% of the individuals showed minute wings.Additionally,ex,kibra,and wingless expression levels decreased in the sd knockdown mutant.Secondly,the CRISPR/Cas9 system was used to design two Sg RNA in exon 12 and exon 13 of BmSd gene according to GGNN18 NGG principle,and the Cas9 expression vector and Sg RNA expression vector were constructed.The two independent transgenic silkworm strains were obtained by microinjection in early embryo of silkworm.The two transgenic silkworms were hybridized to screen the double positive individuals to obtain the mutant silkworm with BmSd knockout.sd deletion mediated by CRISPR/Cas9 induced 50% of individuals with minute wings,a phenotype similar to the mw mutant.This result demonstrates that BmSd plays pivotal roles in silkworm wing development.The results showed that the expression of the gene decreased significantly after knockout.Functional analysis showed that BmSd was the key gene for the development of small wings.