【作者】 刘静雅；

【导师】 洪亚辉；

【作者基本信息】 湖南农业大学 ， 细胞生物学， 2010， 硕士

【摘要】 野生稻中蕴藏着丰富的抗病虫、抗逆、抗低温、耐干旱、耐贫瘠等抗逆优良性状基因,在现代水稻抗性育种中具有独特的作用。当今一些模式植物如拟南芥的DREB转录因子的分子生物学研究取得了良好的进展,DREB转录因子的表达受非生物逆境胁迫,如干旱、高盐、低温、冻害等逆境胁迫的诱导,在植物对逆境胁迫与激素应答反应中发挥调控作用。本研究以茶陵野生稻为材料,针对DREB转录因子开展了研究,在低温胁迫下通过分离野生稻的总RNA并反转录成cDNA,克隆了茶陵野生稻中DREB转录因子,全长958bp,编码218个氨基酸,经BLAST及蛋白质同源性分析,该序列与水稻(Oryza sativa) DREB基因的同源性98%,与小麦(Triticum monococcum) CRT/DREB4蛋白的同源性为93%；与大麦(Hordeum vulgare)CBF2A蛋白与CBFIVc-14.1蛋白分别为93%、92%。这表明已经成功克隆了野生稻中DREB转录因子序列。为了进一步研究DREB在不同时间低温胁迫下茶陵野生稻组织中的表达情况,以水稻基因组DNA中的内参Actin序列作为内参基因,应用RT-PCR技术对DREB转录因子的表达进行半定量分析,结果显示在不同时间的低温胁迫条件下,DREB基因的表达有组织特异性,且在根中的表达更为稳定,表达量最高。通过PCR将DREB转录因子从T载体上释放,构建至植物表达载体pWM101载体中。通过对低温胁迫下茶陵野生稻中的各种生化酶的检测,以栽培稻作为对照,分别测定在低温不同积累时间下的过氧化氢酶、过氧化物酶、超氧化物歧化酶活性,结果发现,野生稻体内酶的活性比栽培稻要高,当胁迫累积到24小时之后,酶活性依然能保持在较高的水平,与转录因子不同时间的低温胁迫下的表达量的高低在一定程度上有相似性。更多还原

【Abstract】 Wild rice that play a unique role in resistant breeding is rich in excellent gene such as anti-worm, anti-low temperature and drought resistance, poor resistance, and so on. Today some model plants as Arabidopsis of which DREB transcription factor has made good progress in molecular biology, DREB transcription factor expressed by induction of abiotic stress, such as drought, high salinity, low temperature, freezing and other Stress, which play a regulatory role in response to stress and hormone.Using Chaling wild rice as material in the study to research DREB transcription factor, separated total RNA of wild rice under low temperature stress and reverse transcript it into cDNA, Sequencing analysis showed the cDNA sequence be cloned is 958bp, which can be translated into a putative 218 amino acids protein. The cones-quence of Blast showed:the sequence of rice (Oryza sativa) DREB gene homology 98%, and wheat (Triticum monococcum) CRT/DREB4 protein homology of 93%; and barley (Hordeum vulgare) CBF2A and CBFIVc-14.1 protein were 93%,92%. This indicated that DREB transcription factor sequence has been successfully cloned.To further study the DREB expression model in tissue of Chaling wild rice under low temperature stress at different time, so as to Actin sequence of rice genome DNA sequence as an internal control, make DREB transcription factor expression semi-quantitative analysis with RT-PCR technique. The result showed DREB genes have tissue-specific expression at different time of cold stress conditions and expression in roots is more stable, the highest expression level is detected in root. DREB transcription factor is release from the T vector By PCR, and constructed to the plant expression vector pWM101 vector.Compared with cultivated rice, the CAT (Catalase), SOD (superoxide), POD (Peroxidase dismutase) of Chaling wild rice is detected under low temperature at different time. The results found that the enzyme activity of wild rice is higher than the cultivated rice, when the stress accumulated to 24 hours the enzymes activity of wild rice is still maintained at a high level, which is similar with DREB transcription factors expression model under cold stress at different times.更多还原