【作者】 魏果；

【导师】 向云；

【作者基本信息】 兰州大学 ， 植物学， 2011， 硕士

【摘要】 Annexin家族是一类进化上保守的钙依赖性磷脂、膜结合蛋白,其成员广泛分布在各种真核生物的不同细胞中。大量研究表明,膜联蛋白参与许多重要的细胞活动,例如膜重组、膜运输、分泌活动、囊泡运输等,但是大多数膜联蛋白的确切生理功能仍然是未知之谜。在高等植物中鲜有对于Annexin5基因的研究或报道,因此,本研究项目拟采用分子生物学和细胞生物学方法,首次尝试研究拟南芥花粉特异表达的膜联蛋白家族成员——Annexin5,得到以下结果：1.克隆得到Annexin5的全长cDNA序列以及Annexin5的钙离子结合位点突变、PIP2结合位点突变、缺失突变等突变序列,并将其插入植物表达载体pCAMBIA1300中与GFP基因融合,本实验中我们一共构建了两套植物表达载体,分别用花粉特异启动子LAT52和ZM13启动表达目的基因。2.利用农杆菌介导花序浸泡法转化野生型拟南芥,用潮霉素进行筛选,因其带有GFP荧光定位,也可以用荧光显微镜进一步检测目的基因是否转入拟南芥中,目前已经获得全部的转基因植株。3.筛选稳定表达的转基因植株并观察表型,目前己获得1300-lat52-GFP和1300-lat52-GFP-Ann5的纯合转基因植株,观察这两种植株中的Annexin5过表达对拟南芥花粉萌发以及花粉管生长的影响,旨在为下一步探讨膜联蛋白参与调控花粉管极性生长的分子机制打下基础。本实验为以后进一步揭示膜联蛋白家族成员在花粉管极性生长过程中作为“枢纽”蛋白衔接膜、微丝骨架和钙离子之间相互作用的分子机制研究打下了基础,具有重要的理论价值和应用前景。更多还原

【Abstract】 Annexin are Ca2+ and phospholipid binding proteins forming an evolutionary conserved multigene family, with members of the family being expressed throughout animal and plant kingdoms. It is generally assumed that Annexins are implicated in several processes related to membranes, including regulation of membrane organisation, membrane trafficking, secretion and vesicular trafficking.But we are still far from a comprehensive description of Annexin function in cell processes. There is hardly any research or report about the Annexin5 of higher plant. Therefore, this research project is to use molecular biology and cell biology methods, the first attempt to study Arabidopsis pollen-specific expression of the membrane protein family members-Annexin5. Results are as follows:1. In order to investigate the gene function of Annexin5, the full length of cDNA was cloned and inserted into the plant expression vector of pCAMBIA1300,then fused to GFP gene. We also constructed a set of plant expression vector include mutational or deleted position.for example, mutational in Ca2+ binging position、mutational in PIP2 binging position. In this study,We use pollen special promoter LAT52 and ZM13.2. The recombination vector was introduced into Arabidopsis thaliana by Agrobacterium tumefaciens through the floral dip method. Filtered by hygromyein, meanwhile, fluoroscopic examination proved that the plant expression vectors had been integrated into arabidopsis genome. Now we have got all the transgenic plants.3. Screening transgenic plants stably expressed and observed its phenotype, now we have obtained homozygotes of 1300-lat52-GFP and 1300-lat52-GFP-Ann5. We confirmed that whether the overexpression of Annexin5 has influences on the pollen germination、pollen tube growth and actin cytoskeleton. By observe the effect of Annexin5 on Arabidopsis thaliana pollen tube growth, we wanted to reveal the molecular mechanism of membrane protein involved in pollen germination and pollen tube growth.This study laid a foundation for revealed the molecular mechanism of members of the annexin family, Which act as Hub protein play an important role in the interactions among membrane, actin cytoskeleton and calcium ions during polar growth of pollen tube. has important theoretical value and application prospect.更多还原