Manikandan R, Balakrishnan N, Sudhakar D, Udayasuriyan V
Department of Plant Biotechnology, Centre for Plant Molecular Biology and Biotechnology, Tamil Nadu Agricultural University, Coimbatore, 641 003, India.
3 Biotech. 2016 Jun;6(1):10. doi: 10.1007/s13205-015-0315-4. Epub 2016 Jan 5.
Bacillus thuringiensis is a major source of insecticidal genes imparting insect resistance in transgenic plants. Level of expression of transgenes in transgenic plants is important to achieve desirable level of resistance against target insects. In order to achieve desirable level of expression, rice chloroplast transit peptide sequence was fused with synthetic cry2AX1 gene to target its protein in chloroplasts. Sixteen PCR positive lines of rice were generated by Agrobacterium mediated transformation using immature embryos. Southern blot hybridization analysis of T transgenic plants confirmed the integration of cry2AX1 gene in two to five locations of rice genome and ELISA demonstrated its expression. Concentration of Cry2AX1 in transgenic rice events ranged 5.0-120 ng/g of fresh leaf tissue. Insect bioassay of T transgenic rice plants against neonate larvae of rice leaffolder showed larval mortality ranging between 20 and 80 % in comparison to control plant. Stable inheritance and expression of cry2AX1 gene was demonstrated in T progenies through Southern and ELISA. In T progenies, the highest concentration of Cry2AX1 and mortality of rice leaffolder larvae were recorded as 150 ng/g of fresh leaf tissue and 80 %, respectively. The Cry2AX1 expression even at a very low concentration (120-150 ng/g) in transgenic rice plants was found effective against rice leaffolder larvae.
苏云金芽孢杆菌是转基因植物中赋予抗虫性的杀虫基因的主要来源。转基因植物中转基因的表达水平对于实现对目标昆虫的理想抗性水平很重要。为了达到理想的表达水平,将水稻叶绿体转运肽序列与合成的cry2AX1基因融合,使其蛋白靶向叶绿体。利用未成熟胚通过农杆菌介导的转化产生了16个水稻PCR阳性株系。对T代转基因植物进行Southern杂交分析,证实cry2AX1基因整合到水稻基因组的2至5个位点,ELISA检测表明其表达。转基因水稻事件中Cry2AX1的浓度在鲜叶组织中为5.0 - 120 ng/g。T代转基因水稻植株对稻纵卷叶螟初孵幼虫的昆虫生物测定显示,与对照植株相比,幼虫死亡率在20%至80%之间。通过Southern杂交和ELISA在T代子代中证实了cry2AX1基因的稳定遗传和表达。在T代子代中,Cry2AX1的最高浓度和稻纵卷叶螟幼虫的死亡率分别记录为150 ng/g鲜叶组织和80%。发现在转基因水稻植株中即使Cry2AX1浓度非常低(120 - 150 ng/g)也对稻纵卷叶螟幼虫有效。