Studies On The Role Of Transformer Of Zeugodacus Cucurbitace In Its Sex Determination And Genetic Engineering Pest Control

Melon fruit fly Zeugodacus（Bactrocera）cucurbitace is one of the most important quarantine pests in China,which mainly infects Cucurbitaceae,Solanaceae and other melons and fruits through its larvae.Transgenic insect sterility technology has been widely used in Diptera,Hymenoptera and Lepidoptera insects,but it has not been reported in the control of Z.cucurbitace.In this study,the transformer（tra）gene was knocked out by using CRISPR/Cas9 technology,and the female-specific lethal vector was constructed.To establish female-specific lethal strain of Z.cucurbitace and evaluate its lethality,the embryos of Z.cucurbitace were microinjected by microinjection technique,and the populations with different fluorescence intensity were screened by fluorescence microscope.The main results were as follows:1 In order to a large number of consistent insect sources,a quantitative indoor artificial rearing method of Z.cucurbitae was established.（1）Selecting for solid medium formula for Z.cucurbitae larvaePumpkin,corn flour,propionic acid and sodium benzoate with different quality are added into the solid culture medium,it was found that the optimal medium was composed of 20g pumpkin,20g yeast extract,12g sugar,5g glucose,13g corn flour,6g soybean flour,0.1g Ca Cl₂·2H₂O,1g yeast extract,1.1g agar powder and 1.2ml propionic acid.The pupation rate,average pupal weight and eclosion rate of Z.cucurbitae larvae fed with this formula were higher than those of the control.The formula has the advantages of simple and convenient operation,high survival rate and quantification and is an effective method for realizing scale and annual subculture breeding of test insects.（2）Solid artificial diet for rearing larval Z.cucurbitae assessed by two-sex life tableThe effects of natural food such as pumpkin,cucumber and solid medium on the popμlation dynamics of Z.cucurbitae were studied by two-sex life table.The results showed that the development time from egg to pupa was 19.11 d when larvae were reared on the artificial diet,which was slightly longer than that of pumpkin（17.73 days）and cucumber（17.13 days）.The pupal weight,length and width of Z.cucurbitae fed on solid medium were higher than those of the two natural foods.In addition,the survival rate of Z.cucurbitae fed on solid medium were higher than those on cucumber,but lower than those on pumpkin.Compared with the two natural diets,the solid medium had no significant effect on the fecundity and population dynamics of Z.cucurbitae.Therefore,the solid artificial diet we developed can be used for laboratory rearing and large-scale rearing of Z.cucurbitae larvae,which is conducive to the research and control of Z.cucurbitae.2 Th function of transformer（tra）gene was verified by CRISPR/Cas9 technologyIn the female transcript of tra gene of Z.cucurbitae,two target sites were selected in exon 5,which were Bcutra1 and Bcutra2,and Bcutra3 in exon 4.The tra gene was knocked out by CRISPR/Cas9.In Bcutra1+2 and Bcutra1+2+3 treatment groups,the proportion of males were 60.87%,46.48%and 55.22%,respectively;the rates of intersex individuals were 5.43%,43.66%and 34.33%,respectively.Intersexual individuals have different degrees of abnormal genitalia,the females with the male black-colored short bristles and males without the male black-colored short bristles.The CRISPR/Cas9 gene editing system was successfμlly applied to the genome of Z.cucurbitae,and the site-directed mutation of tra gene was realized.3 Construction of female-specific lethal genetic strain vectorThree female-specific expression vectors tetO5,tetO14 and tetO21 were successfully constructed by the methods of seamless cloning and fusion PCR,which laid a foundation for the further study of female-specific genetic strains of Z.cucurbitace.4.Selection of female-specific lethal genetic linesUsing the method of embryo microinjection,the transgenic melon fly early fluorescent marker plasmids tetO14 and tetO21 were transformed.In the G1 generation,the fluorescence of melon fly pupae was observed by fluorescence microscope.28 kinds of transgenic melon fly individuals with different fluorescence expression intensity were screened,and 14 lines of G2 generation,9 lines of G3 generation and 6 lines of G4generation were selected.Fluorescent protein gene Ds Red2 and toxic protein gene t TAV were detected by PCR.The homozygous strain of Z.cucurbitace was 100%lethal and died in the egg stage.The genetic transformation system of Z.cucurbitace was successfully established.