Construction Of A New Transgenic Vector And Transformation Via Sperm-Mediated Pathway In Antheraea Pernyi

Antheraea pernyi, common name called Tussah, is an economically important insect and model organisms for basic research. Its transgenic research has very important significance. Compared to the silkworm, the transgenic research of tussah has just initiated, but shown specialty and good research convenience. We focused on tussah research, hoping to make use of molecular biology techniques in transgenic silkworm breeding, to improve the quality of silk, to enhance its market competitiveness, and to improve the utilization of tussah resources.In this dissertation, tussah transgenic vector was modified by optimizing the promoter, transposons, reporter genes and other important transgenic elements; the mechanism of sperm-mediated pathway was studied by fluorescent labeling; artificial splicing gene fragment of spider draggling silk was cloned, and was transferred into tussah by sperm-mediated pathway. The integration and expression of the reporter gene was verified. The major contents and conclusions are as follows:(1) Tussah actin promoter Al was amplified by chromosome walking approach, and its total length was1927bp. Compare with silkworm actin promoter, their core region homologous rate was92%. Promoter Al had typical eukaryotic features. Promoter expression regulatory region was idetified through the functional areas deletion. F4fragment of Al promoter showed a strong positive regulatory function, which could be used as strong promoter for transgenic research. Strong suppression components might exist at upsteam of TATA box for the negative regulation of gene expression.(2) Fibroin gene5’and3’end fragments lengthened by Tail-PCR were fused with GFP marker gene to construct a new fibroin gene homologous expression cassettes Fib-GFP. The expression cassette was inserted into the piggyBac trnnsposon between two repeated sequences to obtain a silkworm gene transfer vector pBac[Al-DsRed+Fib-GFP]; using tussah actin promoter Al transform auxiliary transfer vector to obtain an assistance carrier pBacAlhelper. Connecting the Al-piggyBac expression cassette with the transformation vector containing inverted repeat sequences to obtain a binary silkworm gene transfer vector pBac [Al-DsRed+Fib-GFP]-Al-piggyBac. The optimized silkworm expression vector was used to transfect Sf9cells. The two plasmids vector and binary gene transfer vector were capable of expressing the red fluorescent protein gene. The binary gene transfer vector had higher integration efficiency than two plasmids vector, suggesting a potent silkworm transgenic application.(3) Tussah Sperm and FITC-labeled exogenous DNA were mixed and laid for a period of time, Fluorescence phenomenon was detected at sperm head by fluorescence microscopy. It showed that tussah sperm has the ability to uptake exogenous DNA. The FITC-labeled exogenous DNA were imported into silkworm moth body through sperm-mediated pathway, and the motion process of exogenous DNA into the female reproductive system was monitored under a fluorescence microscope. The results showed that exogenous DNA could be transfered into the eggs along with sperm movement through sperm-mediated pathway. Then, a vector carried GFP gene was transferred into silkworm eggs by using sperm-mediated pathway. Transient expression of green fluorescence was observed in eggs, RT-PCR and Western blot confirmed its transformation and expression.(4) The towing silk protein gene (ASP) was cloned by nested PCR technique from the big round belly spider Araneus ventricosus, which was837bp in length. The ASP was constructed into the prokaryotic expression vector pGEX-6p-1and eukaryotic expression vector pGFP-N2, and named as pASG and pASN, respectivly. pASG was induced and expressed in E. coli at16℃,24h, and observed successful expression of the fusion protein GST-ASP; Green fluorescent protein (GFP) expression was observed after pASN was transfected into Sf9insect cells in48h.(5) The Draggling silk protein gene (ASP) was then constructed to binary silkworm gene transfer vector and was transfencted tuaash using sperm-mediated pathway. DsRed fluorescence expression was observed in tussah eggs and blood cells of third instar. Further molecular identifications showed silk protein and GFP gene were expressed in tussah individuals.