The Study Of Transformer On Sex Determination In Bactrocera Dorsalis Based On CRISPR/Cas9 System

Sexual reproduction is an essential and universal biological process to maintain population number and species diversity.In insects,the sex determination that generates female and male dimorphism has been one of the most well-known genetic mechanisms and is an essential process of sexual reproduction.In many insect species including dipteran,the transformer(tra)that served as a sex determine switch is a key gene to regular sexual differentiation and sustain sexual identity.Bactrocera dorsalis(Hendel),the oriental fruit fly,is a worldwide quarantine pest that threatens fruit and vegetable industries.In tephritidae species,the structure and function of the sex determination transformer gene in Bactrocera dorsalis(Bdtra)has been isolated and identified.In this study,we targeted the nonmodel organism B.dorsalis as the research object and applied the CRISPR/Cas9 system to the genome editing in B.dorsalis by using embryo microinjection for knock-out of white gene and Bdtra gene.The main results are as follows:Microinjection of wl-sgRNA,w2-sgRNA mixed with Cas9 mRNA into embryos could induce the mutation of the white gene in the somatic cells and produce the mosaic phenotype,and the chimeras accounted for 5.5%and 26.3%respectively.Cross experiments gave rise to male and female offspring with complete white eyes and allowed us to establish the homozygous mutant lines,indicating that the white mutations could be transmitted to subsequent generation through germline.By using CRISPR/Cas9 system to knock out the tra gene,the ratio of male is up to 63%,69.6%,55.3%and 74%of the injected tral,tra2,tra3 and co-inject tral+2 treatment.Moreover,the proportion of mosaic individuals is 6.8%,11.6%,2.1%and 12%,respectively.During fertility test,approximately 13.6%,20.1%and 20%of the treated males produced female-only offspring,indicating that these males were XX pseudomales.The intersex individuals exhibited the male black-colored short bristles with malformed genitalia.Upon dissection,we found that XX pseudomales developed with the poorly developed testis and the intersex individuals with various degrees of intersexuality with female and male reproduce tissues.In addition,the female ovaries were not fully developed and could not produce mature eggs.2.1%flies exhibited intersexualityIn this paper,the CRISPR/Cas9 genome editing system was successfully applied to B.dorsalis genome for modification and specific gene point mutation.The obtained mutants will further perfect the B.dorsalis database and improve the application scope of the sterile insect technique(SIT)as well as provide theoretical and technical support for the control of B.dorsalis.