细胞膜融合不仅是细胞生长所必需的膜组分最重要的来源，也是细胞信号传导的重要过程，同时还是细胞分泌等众多物质运输过程的重要途径之一。Synaptotagmins（SYTs，囊泡分泌蛋白）是一类重要的膜转运蛋白家族，该家族在动物细胞中已被证明是作为调节膜融合的钙感受器。植物细胞中，通过对拟南芥基因组分析表明，Synaptotagmins家族含有5种SYTs，而且植物细胞中的SYTs的序列与动物非常相似。目前已知拟南芥Syt1蛋白在膜损伤修复及调节胞吞作用等方面担负着重要功能，并且和植物病毒运动蛋白对细胞侵染有关。实验室前期的研究表明，拟南芥Syt2参与conventional secretion途径，且在花粉萌发和花粉管生长中起着非常重要的调节作用。前期研究的一个重要的发现是Syt2的表达仅在受精前，且仅在花粉和胚囊中表达，而受精后其表达立即停止。然而，拟南芥Syt2在受精前后的这种精准时空表达的调控机制还不清楚。因此，本论文主要从分析其启动子序列及蛋白结构入手，采取了GUS报告系统的检测、原生质体的转化、点突载体的构建及转基因植物的构建等分子生物学及生物化学方法，进行相关分析，获得以下结果： 1. 为了探究Syt2表达的特异性，在PlantCare网站对Syt2启动子进行了元件预测。分析结果表明，在Syt2全长启动子中存在4种与激素调控相关元件（脱落酸响应元件、赤霉素响应元件、水杨酸响应元件以及茉莉酸甲酯响应元件）以及13种与光响应相关的元件，却没有发现直接与基因时空特异性表达相关的元件； 2. 在避免破坏已预测元件的原则下，将Syt2启动子分为4个片段：pSyt2（-2014~-1）、pSyt2（-1495~-1）、pSyt2（-1100~-1）、pSyt2（-494~-1）。将四个片段分别与GUS报告基因相连接，构建植物表达载体，并转化拟南芥获得转基因植株。经三代筛选获得纯合单插入的转基因拟南芥，利用GUS染色的结果表明，启动子pSyt2（-1495 ~ -1）在花粉和胚囊组织特异启动GUS基因的表达，而启动子pSyt2（-1100 ~ -1）和pSyt2（-494 ~ -1）可以在拟南芥的幼苗、莲座叶、茎生叶、茎、花组织及角果等器官中广泛表达。由此推测，在Syt2启动子的pSyt2（-1495~-1100）位置可能存在某种抑制元件，起着抑制Syt2在幼苗、叶片、茎及角果等组织中表达的调节作用； 3. 对pSyt2（-1495~-1100）进行进一步分析发现，片段pSyt2（-1495~-1306）具有较强的抑制效应，推测抑制元件可能在启动子片段pSyt2（-1495~-1306）中。 4. 对SYT2蛋白序列进行同源序列比对分析，发现C2A结构域钙结合位点高度保守，C2B结构域钙结合位点则相对不保守。 综上所述，当拟南芥Syt2启动子长度低于1100 bp时，Syt2表达的特异性消失，而Syt2启动子长度长于1495 bp时，仍具有表达特异性，对pSyt2（-1495~-1100）进一步分析,发现调控Syt2特异表达的启动子序列可能在（-1495~-1306）位置。这将为Syt2在精确时空表达上的调控研究提供一定的理论依据。

Membrane fusion is not only the most important source of membrane components necessary for cell growth, but also an important process of cellular signal transduction. In addition, it is also one of the key ways for cells to secrete extracellular substances. Synaptotagmins (SYTs, vesicle secretory proteins) is an important family of membrane transporters and it has been shown to act as calcium sensors for regulatory membrane fusion in animal cells. In plant cells, Arabidopsis genomic analysis shows that it contains five kinds of SYTs and the sequence of the SYTs in plant cells is similar to that of animals. It is known that SYT1 protein plays an important role in membrane damage repair and regulation of endocytosis and intercellular transport of viral movement proteins. However, a few research has been reported on other SYTs proteins. Our previous studies show that Arabidopsis thaliana Syt2 participates in the metabolic channel transfers to the cell membrane and plays a very important role in pollen germination and tube growth. In addition, it is found that the expression of Syt2 is only expressed in pollen and embryo sac before fertilization, but the expression is stopped after fertilization. The precise regulation of the spatial and temporal expression of Syt2 in Arabidopsis before and after fertilization is still unclear. We have taken the construction of transgenic plants, detection of GUS reporting systems, the transformation of protoplasts, et.al. By molecular biology research method, developmental biology combined with bioinformatics analysis, we find the following results: 1. To explore the specificity of its expression, the Syt2 promoter is predicted by using the Plant Care website. It is found that there are 4 kinds of elements related to hormone regulation (abscisic acid response element, gibberellin response element, salicylic acid response element, and methyl jasmonate response element) and 13 kinds of light response related elementsthe predicted components.And it do not have elements directly related to gene-specific temporal and spatial expression. 2. The Syt2 promoter is divided into 4 fragments: pSyt2(-2014~-1), pSyt2(-1495~-1), pSyt2(-1100~-1), pSyt2(- 494~-1). The four fragments are respectively ligated with the GUS reporter gene to construct a plant expression vector. And it is transformed Arabidopsis thaliana to obtain transgenic plants. We obtain the homozygous single insertion Arabidopsis thaliana through three generations of screening. The results of GUS staining shows that the pSyt2(-1495 ~ -1) specifically activated GUS gene expression in pollen and embryo sac tissues, while the pSyt2(-1100 ~ -1) and pSyt2(-494 ~ -1) can be widely expressed in the Arabidopsis seedlings, rosette leaves, stem leaves, stems, flower tissue and pods. It is speculated that there may be some inhibitory elements in the position of pSyt2(-1495~-1100) of the Syt2 promoter, which plays an important role in inhibiting the expression of Syt2 in seedlings, leaves, stems and pods. 3. Further analysis of pSyt2(-1495~-1100) reveals that the fragment pSyt2(-1495~-1306) has a strong inhibitory effect. It suggests that the suppressor element may be in the promoter fragment pSyt2(-1495~-1306). 4. Analysis of homologous sequences of SYT2 protein sequence reveals that the calcium binding site of C2A domain is highly conserved and the calcium binding site of C2B domain is relatively unconserved. In summary, when the Arabidopsis Syt2 promoter is less than 1100 bp in length, the specificity of Syt2 expression disappears. However, the Syt2 promoter is longer than 1495 bp and still has specificity of expression. For the Syt2 promoter (-1495 ~-1100), the analysis showes that the fragment that affects the specific expression of Syt2 may be in the (-1495~-1306) position. These results will provide a theoretical basis for the regulation of Syt2 in precise spatial and temporal expression.