【作者】 杨梅；

【导师】 熊立仲；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 非生物逆境一直是造成作物减产的重要原因,并且随着全球环境恶化和气候异常将日益危害着粮食生产安全。因此研究作物的抗逆机理和鉴定抗逆相关基因具有重要的意义。挖掘逆境应答中的重要基因和启动子元件并加以利用,可以提高植物的抗逆性。本研究分为两部分内容：一是MYB类转录因子的分析和功能鉴定,通过对14个MYB候选基因的逆境表达谱分析结合其转基因超表达植株的抗逆性鉴定,筛选出对低温和高盐胁迫调控有关的基因CST1(Cold and salt tolerance 1,OsMYB30),并对其进行了初步的功能鉴定；二是逆境诱导型启动子的分离和鉴定,通过对候选启动子的分离和逆境表达活性鉴定,发现了一个受干旱强烈诱导的启动子。研究的主要结果发下：1.对14个候选MYB基因进行了超表达载体的构建并转化了水稻粳稻品种中花11；2.通过对超表达转基因植株的苗期逆境抗性筛选,发现了两个与抗逆相关的候选基因。CST1超表达植株对低温和高盐胁迫呈现出敏感的表型,是本研究的重点。3.通过生物信息学分析CST1属于R2R3-MYB,从DNA结合结构域的进化关系上来分析,该蛋白与拟南芥中MYB15和水稻中的Osmyb4关系最近。4.CST1基因的表达呈现一定的组织特异性,在颖壳和二次支梗中表达量相对较高,在发芽后的幼芽和花药中也相对较高。同时该基因受到低温胁迫的持续上升诱导表达,在高盐胁迫时下调表达。5.该基因的T-DNA插入突变体cstl显著降低了CST1的表达量,其纯合家系对低温和高盐胁迫的耐受性相对于野生型明显增强。6.通过Real-time PCR分析了一些冷应答相关基因的表达量,发现DREB/CBF转录因子以及其他已发表的基因并没有差异表达,表明这些基因很可能并不受CST1的调控。7.通过芯片数据分析CST1超表达、突变体和野生型植株在正常生长条件下以及低温胁迫条件下基因的表达量差异,大量的基因在表达水平发生了变化,表明CST1在冷胁迫应答中发挥着重要的转录调节的作用。8.对12个候选启动子进行了分离和构建GUS报告基因的表达载体,并转化了水稻粳稻品种中花11。9.对5个候选启动子的转基因植株进行了逆境条件下的GUS活性定量分析,发现了一个受干旱强烈诱导的启动子Oshox24P,其余均受逆境诱导比较微弱。更多还原

【Abstract】 Abiotic stresses are major limiting factors for plant productivity, and with global environmental degradation and abnormal climate changes, they will increasingly endanger food security. So, it is important for us to know how plants sense and adapt to the adverse environmental conditions and which genes act with crucial roles during the stress sensing and adaptation processes. A number of studies suggest that stress resistance can be significantly improved by engineering such important genes.This study is comprised of two parts. The first part is isolation and characterization of stress resistance candidate genes. Fourteen stress-responsive MYB genes were overexpressed in rice to test their effectiveness on improving stress resistance. CST1 (Cold and salt tolerance 1, OsMYB30) was found to be one of the MYB genes involved in cold and salt tolerance. The second part is isolation and characterization of stress-inducible promoters, and a drought-inducible promoter named Oshox24P was identified. The main results are as follows:1. The over-expression vectors of 14 candidate genes were constructed and introduced into japonica rice Zhonghua 11 by Agorobacterium-mediated transformation.2. After stress testing at seeding stage, transgenic rice of two candidate genes showed increased or deceased stress tolerance. One of the candidate genes, CST1, was chosen for further analyses.3. Bioinformatics analysis showed that CST1 belongs to R2R3-MYB. The phylogenetic tree based on the MYB-binding domain revealed that CST1 is close to MYB 15 from Arabidopsis and OsMYB4 from rice.4. The CST1 transcript was detected in most organs and tissues of rice, but the highest level was in lemma and palea, secondary branches, and anthers, and the lowest was in panicle. The expression level of CST1 was increased during cold stress but decreased during salt stress. 5. T-DNA insertion mutant cstl showed significantly increased cold and salt tolerance compared to the wild type plants.6. A number of cold-related genes were detected by Real-time PCR concluding DREB/CBF genes and other genes reported, however, the expression of these genes had no significant different between the overexpression and mutant plants. So, it is suggest that these genes weren’t regulated by CST1 during cold stress.7. Based on DNA chip analysis, large member of genes changed their expression on transcriptional level; this result indicated that CST1 act as an important role in cold stress tolerance.8. Promoter::GUS fusion constructs for 12 stress-inducible promoters were made and introduced into japonica rice Zhonghua 11.9. GUS activities of transgenic plants of 5 candidate promoters were analyzed under different stresses. Promoter Oshox24P was found to be strongly induced under drought condition, while the others were induced slightly.更多还原