RsICE1基因表达和转基因水稻抗冷通路研究

Expression of RsICE1 Gene and Its Regulation Function of Cold Responsive Signalling Pathway in Transgenic Rice

以萝卜幼苗和水稻T1代转RsICE1基因(从抗寒萝卜植株中分离的一个编码碱性-螺旋-环-螺旋低温胁迫转录因子,HQ891287)株系为材料,通过半定量RT-PCR及实时定量PCR等方法,分析RsICE1基因的表达、水稻转基因株系中RsICE1基因的遗传及冷诱导基因的表达情况。结果显示:(1)半定量RT-PCR分析表明,RsICE1基因在萝卜的根、茎、叶中为组成型表达,在幼苗根和茎中表达量较强;RsICE1基因的表达水平能够被冷处理和NaCl处理诱导上调表达,但ABA和脱水处理无上调作用。(2)卡方测验表明,水稻转基因T1代潮霉素抗性发生了3∶1分离模式;Southern和Northern杂交结果表明,4个抗冷转基因株系中RsICE1基因均以单拷贝、单位点整合到水稻基因组并正常表达。(3)实时定量PCR分析表明,冷胁迫下,RsICE1基因超表达但对水稻OsDREB1A(AF300970)、OsDREB1B(AF300972)、OsDREB1F(AY785897)基因表达没有影响,表明RsICE1基因对转基因水稻抗寒性的影响不依赖OsDERB1冷反应通路。

With radish seedlings and T1 rice lines overexpressing RsICE1 gene （a low temperature stress transcription factor encoding basic-helix-loop helix from cold-tolerant radish） as materials,we studied ex pression profiles of RsICE1 gene,hereditary conditions of RsICE1 gene and expression conditions of cold regulatory genes in transgenic rice lines by semi-quantitative RT-PCR,molecule hybridization, and real time quantitative PCR. The results are as follows： （1）The semi-quantitative RT-PCR results suggested that RsICE1 was expressed constitutively in the examined tissues, such as roots, stems and leaves, and RsICE1 gene mRNA levels were dominant in roots and stems of radish seedling. The expression levels of RsICE1were upregulated by cold and NaC1 treatments,but not by the ABA and dehydration treatments. （2）X^2 testshowed that the transgenic T1 rice lines existed resistant segregation model for 3 ： 1 ratio, Tne Soutnern and Northern blot analysis showed that RsICE1 gene had been integrated into the genomes of four cold- resitant To transgenic lines,and expressed normally. （3）Real time quantitative PCR results showed that af- ter cold stress,overexpression of RslCE1 gene had no significant effect on the expression of OsDREB1A, OsDREB1B,OsDREBIF suggesting that the function of RslCE1 in cold stress was independent on the Os- DERB1 cold responsive signalling pathway in transgenic rice.