【作者】 陶亚忠；

【导师】 王国英；

【作者基本信息】 中国农业大学 ， 生物化学和分子生物学， 2004， 博士

【摘要】 油菜素类固醇（BR）是植物生长发育所必需的一类植物固醇类激素，而油菜素类酯（Brassinolide, BL）是现在发现的这类类固醇中最具活性的一种形式。BR生物合成或信号传递相关基因的缺失，将使植物矮化。在双子叶植物中BR相关突变体的分子遗传学及其生理功能的研究已经证明了BR对于双子叶植物正常的生长和发育起着关键的作用。然而，除了最近报道的水稻的3个突变体外，BR对玉米以及别的单子叶植物的作用还知之甚少。拟南芥的DWF1／DIM是一个双功能蛋白，在BR的生物合成途径中催化Δ²⁴异构反应以及随后的Δ^24（25）还原反应。 为了更好的理解DWF1在单子叶植物的BR生物合成途径中的作用。我们首先利用RACE和RT-PCR的方法克隆了玉米中DWF1的同源基因ZmDWF1，这是在玉米中克隆的第一个与BR相关的基因。推测ZmDWF1编码562个氨基酸，与水稻OsDWF1和拟南芥DWF1蛋白氨基酸序列相似性分别为95％和86％。Southern blot分析证明ZmDWF1在玉米的基因组中以单拷贝形式存在。而Northern blot分析表明，ZmDWF1基因在不同时期不同组织中具有表达特异性，并且主要在幼嫩的根尖表达。根尖免疫荧光实验进一步直接的表明在玉米根冠细胞中存在大量明亮的红色颗粒状荧光，而在别的部位没有发现这种颗粒。因此我们推测，BR的生物合成可能主要在玉米的根中，尤其是根尖（根冠细胞）进行。 其次，尽管蛋白氨基酸序列的疏水性分析表明，ZmDWF1是一种整合膜蛋白，我们依然将ZmDWF1全长序列克隆到原核表达载体pET30（+）中，并成功的在大肠杆菌BL21（DE3）中表达了这种预测的膜蛋白全长。随后，利用Ni-NTA his tag亲和层析柱纯化了这种蛋白。在四次免疫兔子后，得到了抗ZmDWF1的多克隆抗体。同时，采用免疫胶体金技术将ZmDWF1定位在根尖细胞的内质网上。这是第一次将BR相关的蛋白用直接的方法进行定位研究。 第三，构建了ZmDWF1基因的双链发夹环干涉载体p3301RNAi，用农杆菌介导的方法转化玉米。PCR和Southern blot的检测结果表明，RNAi构建体已经被整合进玉米的基因组中。而转基因矮化玉米不同时期的Western bot检测结果表明，ZmDWF1蛋白的表达受到了不同程度的抑制，并且这种抑制的程度与矮化的株高对应。转基因后代表现出一系列不同程度的矮化，在器官的发育和生长中表现一系列的异常。最显著的是茎和叶的伸长异常。以及维管束的分化和成熟受到影响。光镜的观察表明这些异常都是由于茎和叶细胞的组织以及极性伸长受到影响而造成的。电镜观察则显示气孔和叶绿体的生长也受到了影响。基于以上的实验研究，我们讨论了BR在玉米中的作用。 最后，转同样构建体的拟南芥植株具有与dwfl突变体类似的表型，PCR-Southem blot的检测结果表明，RNAi构建体已经整合进拟南芥的基因组中。而转基因矮化植株Western bot检测结果表明，DWF1蛋白的表达受到了不同程度的抑制。这表明高度相似的异源基因序列之间完全可以产生沉默。因此，我们的构建体有可能在其他作物的矮化基因工程中应用。更多还原

【Abstract】 Brassinosteroids (BR) are natural plant growth regulators required for plant growth and development. Brassinolide (BL) is the most bioactive form found to date. BR-deficient or BR-insensitive mutants display dwarfism. Molecular genetic and physiological studies on BR-related mutants of dicot plants play a critical role in normal plant growth and development. However, little is known about the function of BR in monocots (grasses), except for the phenotypic analysis of three rice mutants. The Arabidopsis DWF1/DIM is a bifunctional protein which catalyses both the A24 isomeration, and subsequently, the △24 (25) reduction in BR biosynthesis pathway.To better understand the role of DWF1 of monocot plants in BR biosynthesis, we firstly cloned a maize gene, ZmDWFl, which has extensive sequence similarity to Arabidopsis DWF1. ZmDWFl is predicted to encode a 562-amino-acid protein that has 95% and 86% similarity with rice OsDWFl and Arabidopsis DWF1, respectively. Southern blot result showed that one copy of ZmDWFl is present in maize genome. Northern blot analyses revealed that expression of ZmDWFl is temporally and spatially regulated during development, and especially in young root tip. Immunofluorescence localization experiment in maize root tip showed that there are large amount of bright red granule-like fluorescence in the cells of root tip (calyptrogen), and weak or no such fluorescence in the other cells. So these results maybe imply the presence of complete BR biosynthetic pathway in maize root, especially in root tip (calyptrogen).Secondly, although protein sequence hydropathy prediction result showed ZmDWFl is an integrated membrane protein, we still successfully expressed the full-length protein fused into the plasmid pET30a (+), then purified the 70 KD fusion proteins by Ni-NTA agarose affinity chromatography column. After immunning rabbit four times, the rabbit polyclonal antibodies were obtained. We also localized the putative membrane protein in the endoplasmic reticulum membrane using immunogold-labeling method. This is the first reported directly localized experiment of BR-related protein.Thirdly, transgenic maize plants with double-stranded ZmDWFl RNA were generated mediated by Agrobacterium. PCR and Southern blot analysis demonstrated the integration of this RNAi construct into maize genome. Western blot result in the early and late stage maize leaves also demonstrated the expected cosuppression of ZmDWFl gene in transgenic dwarfed plants, and the suppressed degree is consistently with dwarfed degree. Transgenic plants had dwarfed phenotype varying in severity, showed a range of abnormalities in organ growth and development, the most striking of which were defects in the elongation of the stem and leaves, and in the differentiation of the vascular bundles. Light microscopic observations revealed that these abnormalities were primarily owing to a failure in the organization and polar elongation of the leaf and stem cells. Electron microscopic observationsfurther showed the abnormalities in stoma and chloroplast. Based on this, we discuss the biological function of BR in maize plants.Finally, transgenic Arabidopsis plants with the same construct showed dwarfed phenotype like dwfl mutant. PCR-Southern blot analysis demonstrated that the integration of this RNAi construct into Arabidopsis genome. Western blot results also demonstrated the expected cosuppression of ZmDWFl gene in transgenic dwarfed plants. This fact demonstrated the heterologous gene silencing is possible based on the high sequence similarity between the maize and Arabidopsis DWF1 homologues. So maybe this construct can be used in dwarfed gene engineering of other crops.更多还原