BtCry1Ac、BADH双价基因对烟草的遗传转化及表达检测

Genetic transformation and expression detection of tobacco transformed by BtCry1Ac and BADHbivalent gene

利用农杆菌介导法,将植物转化载体p209-BtCry1Ac-BADH转化烟草,获得了Kan筛选的完整再生植株。经PCR检测证明,4个株系中检测到BtCry1Ac和BADH基因。荧光定量PCR检测表明,检测到目的基因的株系中有3个株系的2个目的基因均在转录水平得到表达,且存在表达差异;有1个株系未检测到BADH基因的表达。ELISA毒蛋白检测表明,经荧光定量PCR筛选的3个株系均检测到毒蛋白的表达,含量最高为414.63ng/g。室内饲虫试验表明,3个转基因株系中只有2个株系对斜纹夜蛾幼虫表现出抗虫性,校正死亡率最高为38.2%,其余均未表现出明显抗虫性。选取2个株系进行组培苗耐盐试验,表明在不同NaCl浓度下,转基因株系与对照之间没有明显差异。研究中,有些系号经PCR检测说明BtCry1Ac和BADH基因已整合到植物基因组中,但荧光定量PCR和ELISA检测表明虽然基因整合到植物基因组中却未表达,有可能发生了基因沉默。

Tobacco was transformed by plant transformation vector p209-BtCry1Ac-BADH through Agrobacterium-mediated,the complete regenerated plants were obtained by screening of Kanamycin.4transgenic lines that detected BtCry1Ac and BADH were obtained after PCR detection.Fluorescence quantitative PCR indicated that two target genes were expressed at the transcriptional level in 3lines that detected all the target genes,and there were some difference among these lines.However,the expression of BADHin a line was not detected.ELISA detection of toxic protein showed that the expression of toxic protein in3lines screened by fluorescence quantitative PCR was detected,with the maximum content of toxic protein of 414.63ng/g.And the expression of toxic protein in the line with detected BtCry1Ac was detected.The indoor insects-resistance test showed that only 2lines in the 3transgenic lines showed the resistance against Prodenia litura（Fabricius）,with the maximum corrected mortality of 38.2%.The others showed no obvious resistance to the larve.Additionaly,the results of the plantlets salt-tolerance test in 2lines selected revealed that there were no significant differences between the transgenic lines and the control.In the study,BtCry1Ac gene and BADHgene of some lines had been integrated into the plant genome by PCR,but the quantitative PCR and ELISA showed that although the gene into the plant genome no expression was detwecxted,which was possiblely due to the gene silencing.