双价抗虫基因共转化烟草叶绿体的研究

The Research of Bt and OC Gene Cotransformation in Tobacco Chloroplast

分别构建苏云金芽孢杆菌晶体毒蛋白基因 [BtCryIA(C) ]和水稻巯基蛋白酶基因 (Oryzacystatin ,OC)叶绿体转化载体 ,其中Bt基因叶绿体转化载体以烟草叶绿体基因trnH psbA trnK为同源片段 ,以水稻 (OryzasativaL .)叶绿体 psbA基因启动子和终止子为调控基因 ;OC基因叶绿体表达载体以烟草叶绿体基因片段 psbA ORF5 12为同源片段 ,以烟草叶绿体 16S启动子和终止子为调控基因 ;两载体均以壮观霉素抗性基因 (aadA)为筛选基因。利用基因枪方法 ,共转化烟草叶片 ,获得壮观霉素抗性植株。经Southern、Western检测、表达蛋白活性测定证明 ,双价抗虫基因己整合到烟草叶绿体中并得到表达。转基因植株抗棉铃虫试验表明 。

The Bt Cry IA(C) chloroplast expression cassette and OC chloroplast expression cassette were constructed. The Bt expression cassette contained the 3.5 kb wild type Bt CryIA(C) gene under the control of the strong light induced psbA promoter and terminator from rice (Oryza sativa L.) chloroplast, the gene: trnH psbA trnk from tobacco(Nicotiana tabacum L.) as the homologous fragment; The OC chloroplast expression cassette contained the OC gene under the control of 16S promoter and terminator from tobacco, the tobacco gene: psb AORF512 as homologous fragment. Both of the two cassettes had the aadA gene expression cassette as the selectable marker. Leaves of tobacco were cotransformed with particle bombardment method. After selection by spectinomycin, the transformants were obtained. The integration of Bt and OC gene were confirmed by Southern blotting analysis, Western blotting analysis. Proteinase inhibitor assays showed that the Bt and OC gene had expressed. Bioassays showed that the transgenic tobacco had a significant resistance to the larvae of cotton bollworm (helicoverpa zea).