BcICE1基因表达模式及调控转基因水稻抗冷通路研究

Study on Expression of BcICE1 Gene and Its Regulation Function of Cold Responsive Signalling Pathway in Transgenic Rice

以大白菜幼苗和水稻T1代转BcICE1基因株系为材料,通过半定量RT-PCR及实时定量PCR等方法,研究BcICE1基因表达模式、水稻转基因株系中BcICE1基因的遗传及冷反应基因的表达情况。半定量RT-PCR分析表明,BcICE1基因在大白菜的根、茎、叶中为组成型表达,其中茎和叶的表达量较强;BcICE1基因的表达水平能被冷、ABA和NaCl处理所上调,但不被脱水处理上调。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,3个抗冷转基因株系中BcICE1基因均是以单拷贝、单位点整合整合到水稻基因组中,并正常表达。实时定量PCR分析表明,冷胁迫下,BcICE1基因的超表达对水稻的OsDREB1F基因影响较大,表明BcICE1基因对转基因水稻抗寒性的影响依赖OsDERB1冷反应通路。

The expression profiles ofBclCE1 gene, hereditary conditions ofBclCE1 gene and expression con- ditions of cold regulatory genes in transgenic rice lines were studied by semi-quantitative RT-PCR and real time quantitative PCR with Brassica pekinensis seedlings and T1 rice lines overexpressing BclCE1 gene. The semi- quantitative RT-PCR results suggested that BclCE1 was expressed constitutively in the examined tissues, such as the roots, stems and leaves, and BclCE1 gene mRNA levels were dominant in stems and leaves of Brassica pekinensis seedling. The expression levels of BclCE1 were upregulated by cold, ABA, NaC1 treatments, but not by the dehydration treatment. ~2 test showed that the transgenic T1 rice plants existed hygromycin resistant segregation model for 3：1 ratio. The Southern and Northern blot analysises showed that BclCE1 gene had been integrated into the genomes of three cold-resitant To transgenie lines, and expressed normally. Real time quantitative PCR results showed that after cold stress, overexpression of BclCE1 gene had significant effect on the expression of OsDR- EBIF, suggesting that the function of BclCE1 in cold stress was dependent on the OsDREB1 cold responsive signalling pathway in transgenic rice.