拟南芥动蛋白AtOvKLP基因的功能研究

NWIPB OpenIR

	拟南芥动蛋白AtOvKLP基因的功能研究
其他题名	Function analysis of kinesin AtOvKLP in Arabidopsis thaliana
	刘瑞娟
学位类型	硕士
导师	窦全文
	2007-06-04
学位授予单位	中国科学院西北高原生物研究所
学位授予地点	西北高原生物研究所
关键词	动蛋白拟南芥胚囊发育基因表达 T-dna插入突变
摘要	动蛋白是一类依赖于微管的马达蛋白，参与生物体诸多的生物学过程。植物中动蛋白的研究主要集中在细胞极性生长与胞质分裂方面，拟南芥基因组测序工作的完成为研究植物动蛋白的结构和功能提供了有利条件。据有关报道在拟南芥基因组中编码动蛋白的基因有61个，在完成基因组测序的真核生物中拟南芥基因组中动蛋白基因所占比例最高。如何解释以拟南芥为代表的高等植物中庞大的动蛋白亚家族中各个成员的功能，成为今后植物动蛋白研究的焦点。本研究选择拟南芥动蛋白KHC亚家族下AtOvKLP基因作为研究对象，对其表达部位以及生物学功能进行了如下研究：首先利用半定量RT-PCR的方法对拟南芥不同器官中AtOvKLP的表达进行了分析，结果显示AtOvKLP在花中特异表达。为了确定AtOvKLP在拟南芥体内具体表达部位，将AtOvKLP启动子区域与GUS基因融合，转入拟南芥后对转基因植株进行了GUS表达分析，发现AtOvKLP主要在花中的胚珠部位表达。在微分干涉显微镜下观察胚珠中GUS的表达情况，发现胚囊部位着色较深。为进一步揭示AtOvKLP的功能，对AtovKLP基因位点T-DNA插入突变体纯合后代的胚囊发育过程进行了观察。结果显示突变体胚囊发育从大孢子有丝分裂开始出现异常，最终导致不能形成正常的胚囊结构。最终我们得到结论：AtOvKLP作用于胚囊的形成过程，其功能缺失致使胚囊败育。此外，为今后生化方面动蛋白特性的研究，我们做了AtOvKLP基因原核表达试验，并成功得到了体外表达蛋白。
其他摘要	Kinesin is a kind of microtubule-based motor proteins that perform diverse biological processes. The study on kinesins in plants is focus on cell polar growth and cytokinesis. The completed sequencing of Arabidopsis thaliana genome has given us convenience to elucidate the structure and function of kinesins in plant cellular processes. 61 kinesin-like genes have been identified in Arabidopsis, and surprisingly the Arabidopsis genome contains the largest number of kinesins among all eukaryote genomes that have been sequenced. It is becoming one of the focuses to elucidate the functions of kinesins that are belonged to a number of subfamilies. AtOvKLP is one of 61 Arabidopsis kinesin-like proteins that belonged to KHC subfamily, and our study is focus on its expression and biological function. Expression of AtOvKLP in different organs of Arabidopsis was analyzed by semi-quantitative RT-PCR, and the result showed that AtOvKLP was expressed especially in inflorescences. To known the expressing position of AtOvKLP in vivo, the promoter region of AtOvKLP was cloned and fused to GUS (Pk-GUS). Then transformed into Arabidopsis and GUS expression in transgenic Arabidopsis was analyzed. The results showed GUS was expressed in ovule and mainly in embryo sac parts observed by DIC. For further reveal the function of AtOvKLP, mutant of AtOvKLP T-DNA insertion was confirmed by PCR and the formation of embryo sac had been observed by CLSM. The result suggested that the embryo sac of mutant can not develop a normal structure. The functional megaspore cell was abnormity that can not continue mitosis. The results demonstrated that AtOvKLP acts on the development events leading to embryo sac formation in Arabidopsis, and loss function of AtOvKLP results in abortion of embryo sac. For further study in validating the biochemistry activity of AtOvKLP, we expressed the AtOvKLP cDNA sequence in E.coil and obtained desired products in vitro.
页数	57
语种	中文
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/3120
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	刘瑞娟. 拟南芥动蛋白AtOvKLP基因的功能研究[D]. 西北高原生物研究所. 中国科学院西北高原生物研究所,2007.

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