利用拮抗多肽技术解析CLE多肽激素在种子和维管束发育中的功能

The establishment of seed and vascular bundle systems in higher plants during the evolutions is probably the main reason to allow higher plants to be so successful in land environment. Studies in recent years revealed that the formations of seeds and vascular bundles are regulated by multiple intracellular and extracellular signals. In the past two years, with the support of the National Natural Science Foundation of China we established an effective antagonistic peptide technology for efficient analyses of CLE peptides. The wildtype Arabidopsis transformed with a construct carrying the full-length CLV3 with the 6th glycine residues in the peptide-coding region replaced by threonine exhibited a dominant clv3-like phenotype. Using the antagonistic peptide technology, we found 3 CLE genes, CLE19, CLE25 and CLE27, regulating the development of embryo, endosperm and vascular bundle. CLE19G6T transgenic plants exhibited embryo lethal phenotype. Defected vascular bundle formations were observed in CLE25G6T and CLE27C6T transgenic plants. In this project, we will elucidate functions of CLE19, CLE25 and CLE27 peptide in seed and vascular bundle formations. We will perform detailed cell biological analyses in embryo, endosperm and vascular bundle, identify receptors that sense these peptide signals, and characterize potential peptidases that are responsible for processing of these peptide hormones. We hope to establish the molecular relationship between CLE peptide hormones and other signal molecules such as auxin. We believe this project will advance our understanding how CLE peptides coordinate the development of embryo, endosperm and vascular bundle.

在进化过程中，种子和维管束的出现是高等植物成功的重要原因。近年来的研究表明这两个发育事件受到胞内和胞间多重信号的严格控制。在青年基金项目资助下，我们建立了一种高效拮抗多肽技术，即把CLV3编码多肽激素的CLE结构域的甘氨酸替换为苏氨酸（CLV3G6T），在其自身启动子驱动下可以使转基因的野生型植物表现出显性的clv3突变体表型。利用该技术我们发现3个CLV3/ESR(CLE)基因参与种子和维管束发育。其中CLE19G6T转基因植物胚胎致死；CLE25G6T和CLE27C6T转基因植物维管束不连续。本项目将在此基础上深入解析这三个多肽激素在胚胎胚乳协调发育和维管束形成中的功能；鉴定CLE多肽激素可能的受体和参与其加工的多肽酶；鉴定其信号通路下游因子；分析小分子多肽激素与生长素等其他信号分子间的关系。相信本项目将帮助我们从机理上了解CLE多肽激素在调控胚胎胚乳协调发育和维管束形成中的作用。

种子和维管束是高等植物区别于低等植物的重要特征，对种子和维管束发育的调控过程进行研究具有重要科学意义。多肽激素作为信号分子，通常在各个生物学过程的上游发挥调控作用。本课题利用之前建立的拮抗多肽技术，构建了拟南芥CLE家族32个成员的拮抗肽转基因植物，对其中的CLE19、CLE25和CLE27三个多肽激素进行了详细的功能研究。发现CLE19多肽激素在成熟花粉和萌发花粉管的营养核、球形期至子叶期胚胎的表皮细胞层表达，参与调控种子发育早期胚胎胚乳协调发育过程及种子发育晚期的子叶发育；解析了CLE25多肽激素在韧皮部起始及后期发育中的调控作用，发现其通过受体复合体CLE25R/CLV2及OPS、CVP2等组分最终调控韧皮部起始发育；CLE27通过调控PIN1-GFP的极性定位调控生长素的极性分布，进而调控花瓣基部维管束发育及最终与植物主体维管束的连接。通过本课题研究，我们对多肽激素参与高等植物的胚胎和维管束发育这两个重要生物学过程有了一些了解，发现多个CLE多肽激素作为信号分子参与这些过程的早期调控，后期对CLE19、CLE25及CLE27信号途径下游因子的鉴定及深入解析将使我们更全面了解胚胎和维管束发育过程的分子机理。

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