過量表現 HvICE1 或 AtICE1 ( Inducer of CBF Expression ) 水稻轉殖株之分子鑑定及非生物逆境耐受性分析

Abstract

Through cold acclimation, plant can increase its tolerance capability upon exposure to low temperature. Previous study showed that CBFs/DREBs (C-repeat binding factor/ dehydration response element binding factor) were the major transcription factors that involved in cold acclimation process. The function of CBFs/DREBs evolved highly conserve between dicot and monocot. Recent study from Arabidopsis has defined ICE1 (inducer of CBF expression), a bHLH (basic helix-loop-helix) protein, as an important transcriptional factor that acts on the promoter of CBF gene and regulates its expression. In this study, based on rice functional genomic approach with transgenic rice analysis, we aimed to understand physiological function of Arabidopsis AtICE1 and Barly HvICE1 under different abiotic stresses. Meanwhile, the action mold of AtICE1 and HvICE1 will be compared under various stresses. To reach this goal, first, by bioinformatics search at least four of OsICE genes were found in rice genome. From currently available rice microarray data revealed that OsICE1 expression was highly induced by salt and drought but not affected in low temperature. OsICE2 and OsICE3 transcripts were repressed upon exposure to salt and drought environment. On the other hand, OsICE4 expression was salt and drought induced. Besides, OsICE3 and OsICE4 gene expression were both increased under low temperature stress. Then, we used TNG67 (Oryza sativa L., japonica; cold and salt tolerant but drought sensitive) and TCN1 (Oryza sativa L., indica; cold and salt sensitive but drought resistant) rice cultivars to investigate OsICEs; OsDREBs and OsDREBs regulon-related downstream genes expression profiles under abiotic stress treatments. The results indicated that OsICE2 expression level were down-regulated quickly in TCN1 at low temperature. And the amount of OsDREB1F、OsDREB1G、OsDREB1H、OsDREB1I and OsDREB1J expressions in TCN1 were also less than those of TNG67. To further elucidate the physiological effects of AtICE1 and HvICE1 under various abiotic stresses, we generated ICEs-overexpressed transgenic rice lines, 35S::AtICE1 and 35S::HvICE1. By Southern blotting analysis, TAIL-PCR, and PCR-based genotyping, we determined the copy numbers of transgene, T-DNA inserted flanking sequence and obtained either one or two copies of homozygous transgenic lines. RT-PCR result showed under normal growth condition indeed we can detect the overexpression of ICE genes in 35S::AtICE1 and 35S:: HvICE1 transgenic rices. However; compared to low-temperature stress-treated wild type plant, the whole gene expression profile of ICE-corresponding downstream genes (OsDREBs) did not obviously changed. The physiological analysis of abiotic stress tolerance assay, including chlorophyll, malondialdehyde (MDA) and proline content measurements showed that 35S::AtICE1 transgene rice with OsDREB1A; 1B; 1C and 2B transcripts enhanced could increase cold tolerance but not for drought and salt tolerance. 35S::HvICE1 transgene rice that with slightly OsDREB 1B; 1C and 1E gene expression increased could raise up its drought and cold tolerance but not salt tolerance. Taken together, the above results suggested that AtICE1 and HvICE1 may function not exactly the same in cold acclimation pathway. This may due to other ICEs co-operate in the regulation of CBFs/DREBs and CBFs/DREBs regulon-related gene expression or ICEs activity can be adjusted through post-translation modifications that lead to different responses when exposure to different abiotic stresses.