Constructions Of Sf9 Cell Line And Explorations On Constructing Transgenic Strain Of The Armyworm Mythimna Separate(Walker)to Express Tet On 3G Stably

Lepidopteran is the second order among Insecta and most of lepidopteran insects are phytophagous and harmful to agriculture.Chemical control which caused serious environmental problems is still the main prevent and cure method of these insect pests.One reason is that other environmentally friendly methods must be based on intensive research of molecular mechanism of lepidopteran pests which has not been studied deeply enough.The replication controllable AcMNPV vector based on Tet-On system constructed before to study molecular mechanism of lepidopteran pests can work effectively.However,basal expression is too high in this system.Tet-On 3G system which has lower basal expression,more maximal expression after induction and higher sensitivity to doxycycline is used to reduce the basal expression in Tet-On system.In this study,AcMNPV genome was recombined by Bac-to-Bac Baculovirus Expression Systemby.We inserted the dnapol or lef3 gene to make them under the control of PTRE3G(an inducible promoter of the Tet-On 3G system)and enhanced green fluorescent protein(EGFP)was also inserted as report gene.The recombined AcMNPV was tranfected into the transgenic Sf9 cell line to detect the replication controllablely on a cellular level.To avoid the superposition of basal expression,the expression of Tet on 3G which is indispensable in the Tet-On 3G system was transferred into hosts(Sf9 cell or armyworm)by transgenic technology.Multiple different PiggyBac shuttle vectors and two transgenic Sf9 cell line,Sf9(PIE1-Tet 3G)and Sf9(opie2-Tet3g),were constructed in this study.The expression of Tet on 3G in transgenic cell line was verified on the RNA,protein and cellular level.That the feasibility of the replication controllable AcMNPV vector was also verified on cellular level.Construction of a transgenic strain of the armyworm mythimna separata(walker)to express Tet on 3g stably was tried through microinjecting Armyworm embryos with PiggyBac system.The constructions of transgenic Sf9 cell line to express Tet on 3g stably provide a terrace to verify and use the replication controllable AcMNPV vector and accelerate the research process of molecular mechanism of lepidopteran insects.The constructions of these cell lines provide the basis for the exploitation of better control method,and have important meaning in agricultural and forestry production.