【作者】 张扬；

【导师】 陈建民； 姚泉洪；

【作者基本信息】 扬州大学 ， 细胞生物学， 2008， 硕士

【摘要】 转录调控是植物生长发育、逆境反应、信号转导、抗病性等一系列基因表达的最主要调控形式,转录因子是参与基因转录水平调控过程的重要因子。MYB转录因子是植物转录因子中最大的家族之一,可以特异的识别结合基因启动子中的MYB顺式作用元件,调控与生物或非生物逆境相关的一系列基因表达。本研究对水稻转录因子OsMYB2进行研究,对OsMYB2 cDNA的氨基酸序列与其他植物中已知的MYB家族的蛋白进行对比,结果表明该转录因子具有一个非常保守的R2R3MYB结构域。序列分析显示OsMYB2在N端保守域包含两个不完全的色氨酸重复,色氨酸重复区域形成螺旋-转角-螺旋的DNA结合域,还含有一个潜在的ATP-GTP结合位点,一个对氧化还原反应敏感的半胱氨酸,和一个核定位序列。对水稻进行各种逆境处理和提取不同组织的RNA,将其反转录为cDNA进行RT-PCR分析。结果是OsMYB2受干旱(PEG)、盐(200mM NaCl)和ABA(200μM)处理诱导,但不受低温诱导。组织表达特异性检测表明,OsMYB2在根、茎、叶、开花期穗、灌浆期穗均有表达,但是在根中的转录水平最高。为了深入了解所克隆的水稻OsMYB2基因的生理功能及与非生物逆境应答的关系,构建了OsMYB2基因的植物表达载体,通过农杆菌蘸花法转化拟南芥,经潮霉素筛选、GUS活性检测和PCR检测验证筛选到转基因阳性植株。生理功能分析表明,与野生型相比,转基因株系对ABA表现出超敏,对逆境的反应表现更弱,但是对糖的反应表现更强。同时,对OsMYB2可能调控的下游基因进行了表达谱分析,结果是RD22和RD29A的表达被下调,而ADH的表达被上调。MYB转录因子能特异的与MYB顺式元件相结合。本文选择SV40的增强子中的一段MYB顺式元件TAACTG来构建酵母鱼饵质粒,利用酵母单杂交方法,对OsMYB2识别结合MYB元件的特异性进行了研究。结果表明,OsMYB2与MYB元件能特异结合,且结合后导致下游报告基因表达量减少,推测此基因为转录抑制子,在基因表达中起负调控的作用。更多还原

【Abstract】 Regulation of gene expression at the level of transcription controls many crucial biological processes including growth and development, stress response, signal transduction and disease resistance. A number of different factors are required for the processes of transcription, in which the transcription factors play an important role. The MYB transcription factors comprise one of the largest families in plant transcription factors. It can specifically interact with MYB cis-acting elements and induce expression of multiple drought- or cold-responsive genes. Constitutive overexpression of MYB genes has been reported to trigger the MYB regulons and enhance freezing, drought, and salt tolerance in some of the transgenic plants. In this article, we analyzed the function of OsMYB2, a MYB transcription factor in rice.Sequence analysis reveals that OsMYB2 contains two imperfect tryptophan repeats (R2 and R3) in the N-terminal conserved region, which is characteristic of most plant MYB transcription factors. Furthermore, secondary structure analysis of OsMYB2 predicts that the tryptophan repeats form helix-turn-helix DNA-binding motifs. It also contains a potential ATP-GTP-binding site, and a nuclear localization.Real-time RT-PCR analyses were performed to determine the expression profiles of OsMYB2. In three-week-old seedlings, expression of OsMYB2 was induced by dehydration ( PEG), high salt ( 200mM NaCl ), and ABA(200μM), but not by cold (4oC). In addition, we examined tissue-specific expression of OsMYB2 in rice using real-time RT-PCR. The OsMYB2 transcripts were detected in all organs tested including root, stem, leaf and spike, but the highest level was in root.Since OsMYB2 is a transcription factor, it might be modulating the expression of genes involved in these processes. To test this hypothesis, gene expression analysis of possible regulated genes in transgenic plants was carried out by real-time RT-PCR. Under ABA or high salt treatment, RD29A and RD22 were down-regulated, whereas ADH was up-regulated.Transgenic Arabidopsis thaliana, Columbia plants overexpressing OsMYB2 were also obtained to further investigate the precise function of OsMYB2. In contrast to non-transgenic plants, these transgenic lines showed hypersensitive to ABA. Overexpression of OsMYB2 decreases tolerance to drought, salt and heavy metal, but increases tolerance to suger.Since MYB transcription factors have been determined to interact specifically with MYB cis-element, the DNA-binding specificity of OsMYB2 was examined by using yeast-one-hybrid assay. The TAACTG cis-element from the enhancer of SV40 was used to construct bait plasmid. The experimental results indicated that OsMYB2 specifically bound to TAACTG. On averageβ-galactosidase activity was decreased 2-fold relative to the control for the MYB cis element. It suggested that OsMYB2 was likely to be a transcriptional repressor and played function of negative regulation in gene expression.更多还原