The Establishment Of Anopheles Sinensis Transgene And CRISPR/Cas9 Technology System

Anopheles sinensis(Diptera: Culicidae) is one of the main malaria transmission vectors in China and Southeast Asia.Effective prevention and control of An.sinensis and cutting off the transmission route are the key to malaria control in the endemic region.The main method of combating malaria is to use insecticides to control vector mosquitoes.However,extensive use of insecticides has subjected mosquitoes to intensive selection pressure,resulting in the development of resistance,so a new mosquito control strategy is urgently needed.With the development of molecular biology,the improvement of genetic engineering technology and the release of An.sinensis genome and transcriptome data,it has become possible to use transgene and CRISPR/Cas9 gene editing technology for genetic control of mosquitoes.Transgene and CRISPR/Cas9 gene technology has been successfully applied to a variety of mosquitoes,but there is no relevant report in An.sinensis.This study aims to establish the microinjection method of An.sinensis eggs,establish the transgene and CRISPR/Cas9 technology system,and lay the foundation for the genetic function research of An.sinensis and genetic control based on gene drive.The main technical systems studied are as follows:1 Microinjection of An.sinensis eggsA set of microinjection system suitable for collection,arrangement and injection of eggs was assembled by matching all kinds of equipment needed.On this basis,the injection parameters and injection methods were explored and optimized,and the hardware conditions and injection methods suitable for microinjection of An.sinensis eggs were established.The establishment of microinjection technology of An.sinensis eggs laid the foundation for the application of transgene,CRISPR/Cas9 and other technologies.2 Establishment of An.sinensis transgenic technology systemBased on the established microinjection technology system of An.sinensis eggs,the transgenic technology system of An.sinensis was established by injecting p Bac[3x P3-EGFP afm] transgenic vector and phsp-p Bac helper,including microinjection ofeggs and transgenic screening,analysis of insertion sites and establishment of homozygous lines.Two homozygous EGFP transgenic lines were successfully established with a transformation rate of 21.43%.On this basis,a transgenic strain specifically expressing Cas9 gene in the ovary of An.sinensis was established,which laid the foundation for perfecting the CRISPR/Cas9 gene editing technology system of An.sinensis.3 Establishment of An.sinensis CRISPR/Cas9 technology systemThe sg RNA target sites of As Yellow and As OR7 genes of An.sinensis were analyzed by online software,and the CRISPR/Cas9-mediated genome editing of An.sinensis was achieved for the first time by injection of sg RNA and Cas9 protein,resulting the establishement of CRISPR/Cas9 technology system for An.sinensis.Analysis of the target sites of As Yellow and As OR7 genes,there were frameshift mutations caused by base insertion or deletion,with mutation rates of 8% and 18.75%,respectively.Analysis of potential off-target sites revealed no non-specific mutations.