Expression Of The Cry1Ac Gene From Transgenic Cotton In E.coli And Arabidopsis

As the transgenic plant which got approve of commercialization earliest,transgenic insect-resistant cotton generated huge profit but attracted extensive attention to its safety problem.A great amount of Bt protein are needed to assess the safety of transgenic cotton.However,due to the various kinds of proteins and the low amount Bt protein expressed in transgenic cotton,it is difficult to extract large quantity of purified Bt protein to do the related assessment work.Therefore,in order to provide materials for research of safety,Bt genes are transported to microbes to produce high level Bt protein.In researches of transgenic plants' long-term environmental effects,traditional crop plants are usually selected as material,but their growth cycle are too long.In this study,Bt gene was transported into Arabidopsis thaliana to shorten experimental periods and facilitate related researches.In this study,cotton with cy1Ac gene is used as material.The cry1Ac gene was cloned from transgenic cotton and transported to E.coli.Then,Bt protein was measured to assess the expression level and purified according to the His-tag at C region.After that,Cry1Ac protein was quantified with ELISA and its physiological activity was assessed by feeding sensitive Helicoverpa armigera line,cry1Ac gene was transported to Arabidopsis thaliana via infecting inflorescence method to get crylAc-introduced Arabidopsis thaliana.Main results include:?1?Expression and purification of cry1Ac gene from transgenic cotton in E.coliP35 promotor and Nos terminator primers were designed according to the alien gene sequences in transgenic cotton.Cry1Ac gene?full length:3546b,theoretical molecular mass 130.68kDa?was cloned from the total DNA template extracted from transgenic cotton leaves.Then the cry1Ac gene from transgenic cotton was connected with expression carrier pET,29a and pET-42b.We expressed the target protein in the way of auto inductionSDS-PAGE showed the expression of target protein was low.According to the preference of E.coli's codon,we synthesized crylAc gene and performed auto induction in E,coli by using pET expression carrier.SDS-PAGE showed a notable elevated expression of target protein.CrylAc protein's proportion in the total protein enhanced from 0.002254%to 0.35%.We performed auto induction of the engineered bacteria and homogenized them via ultra-sonic instrument.After this treatment,target protein was in soluable phase.The cry1Ac protein was primer purified thought Ni-NTA chromatography,because of the problem about expression level,the purified protein was still contained other hybrid proteins.We studied the effect of LC50 about the purified CrylAc protein using the method of bioassay by adding different concertration purified CrylAc protein into the feed which eat by Helicoverpa armigera.Experiments found that the LC50 is 0.157 ?g/g?95%confidence limits of 0.128-0.191?,LC90 is 0.952 ?g/g?95%confidence limits of 0.694-1.448?,and it consistents with the linear relation.According to the preference of E.coli's codon,Zhou Shuilian optimizated the crylAb/c gene?1830bp?which got from the Bt transgenic rice,then the gene expressed in E.coli.Quantitative Elisa was used to compare the expression of CrylAc and CrylAb/c in the same conditions in E.coli.Cry1Ac?1178a.a?and CrylAb/c?609a.a?protein were 0.3529%and 0.3265%of total protein respectively and difference was found..Therefore,the length of the gene about Bt protein is not the main factor in E.coli's expression.?2?acquire and verify the Arabidopsis plant contain cry1Ac geneThe total DNA from transgenic cotton was used as template to amplify crylAc gene?3546bp?.The gene was then linked to pCAMBIA1302 plasmid to construct and then transformed into Agrobacterium GV3101.Transgenic Arabidopsis was acquired by infecting Arabidopsis inflorescence which genome contained crylAc gene.The acquired seeds were selected in MS medium with kana?30 mg/L-50 mg/L?to get Ti generation.PCR was used to select 8 positive plants in T₁ generation.