【作者】 李绪彦；

【导师】 刘国琴； 任东涛；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 动蛋白是一类依赖于微管的马达蛋白,有许多异型体,这个超家族在真核细胞中参与了众多的生物学过程,如细胞器运输、有丝分裂和减数分裂、微管的动态调节以及信号转导等。植物动蛋白的研究主要集中在细胞极性生长和胞质分裂等方面。动蛋白的功能具有多样性,研究拟南芥动蛋白异型体基因的具体生物学功能,对于了解动蛋白超家族及揭示拟南芥发育的分子机理具有重要的意义。本实验室从拟南芥中克隆了一个动蛋白基因,定名为AtKP1。本研究对动蛋白异型体AtKP1的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AtKP1的表达进行了分析,结果显示,拟南芥动蛋白异型体AtKP1是一个在幼嫩器官广泛表达的基因。为了解AtKP1在体内的具体表达活性,我们克隆了AtKP1启动子区域,PLACE在线软件分析表明该区域具有TATA box和CAATbox等基本元件,同时还具有生长素反应元件;将该启动子区域与gus基因融合(prokpl-gus),转入拟南芥后对转基因植株进行了GUS活性分析,结果显示,AtKP1基因主要在幼嫩中柱和幼嫩叶毛中表达,表明AtKP1可能参与了中柱和叶毛的发育过程。对转prokpl-gus拟南芥植株进行逆境和生长素处理(10 μM ABA,1 mM IAA,300 mM NaCl),结果显示,AtKP1基因的表达响应IAA的诱导,但对ABA和高盐处理没有明显反应:通过RT-PCR研究表明,野生型体内AtKP1基因在IAA诱导48 h表达量才增加,说明AtKP1是响应IAA反应的晚期基因。为了进一步了解AtKP1基因的功能,通过PCR、Southern blotting和Northern blotting鉴定,筛选出拟南芥AtKP1 T-DNA插入突变纯合体。表型观察结果显示,突变体的种子萌发对外源IAA表现出一定抗性,说明AtKP1介入了IAA的生理反应过程,但AtKP1基因敲除并未影响IAA阜期反应基因IAA2和IAA16的变化;在正常生长条件下,AtKP1基因的缺失并未影响植株的生长、发育,表明AtKP1基因的功能可以被其它基因所替代。KatD是一个和AtKP1高度同源的拟南芥动蛋白异型体。为了解AtKP1的功能是否被KatD替代,我们克隆了KatD的启动子区域,用gus作为报告基因,对KatD的表达进行了分析。结果显示,KatD基因在花药中大量表达;但同时我们在幼嫩叶毛和幼嫩维管束中同样观察到KatD的大量表达,和AtKP1基因的表达部位基本相似。鉴定、筛选出拟南芥KatD的T-DNA插入突变体,表型观察结果显示,KatD的突变不影响植株的正常生长、发育,表明KatD基因的功能也可被其它基因所替代。将KatD与AtKP1突变体进行杂交,通过PCR、半定量RT-PCR的鉴定获得了双突变纯合体。对T2代双突变体植株表型初步观察显示,植株有明显矮化现象,但维管束和叶毛的发育基本正常。T3代植株的详细表型有待进一步详细分析。更多还原

【Abstract】 Kinesin is a kind of microtubule-dependent motor proteins that constitute a superfamily with many isoforms. Members of the kinesin superfamily perform many diverse cellular functions, such as transport of membrane organelles, mitosis, meiosis, microtubule dynamics and signal transduction etc. The study on kinesins in plants is focus on cell polar growth and cytokinesis. Functions of kinesin isoforms show diversity, so the study on special functions of kinesin isoforms in Arabidopsis would provide valuable information for understanding the function of the kinesin superfamily and the development mechanism of the Arabidopsis. AtKPl is a gene with characteristics of kinesin, which had been cloned from the stem of Arabidopsis in our laboratory. This study is focus on expression of kinesin isoform AtKPl and its biological function, and the results were as follows:Expression of AtKPl in different organs of Arabidopsis was analyzed by semi-quantitative RT-PCR, and the result demonstrated that AtKPl was expressed widely in young organs. In order to know the expression activity of AtKPl in vivo, the promoter region of AtKPl was cloned. Analysis by PLACE with Internet software indicated that the promoter region had not only basic elements like TATA box and CAAT box, but also auxin-response element. The promoter region was fused to gus (prokpl-gus) , and transformed into Arabidopsis. GUS expression pattern in transgenic Arabidopsis was analyzed. The results demonstrated that GUS was expressed mainly in young leaf trichomes and young stele, suggesting that AtKPl may be involved in development of stele and leaf trichome .Prokpl-gus transgenic plant were exposed to environment stress and auxin (10 u.M ABA, 1 mM IAA, 300 mM NaCl). Expression of AtKPl obviously responded to IAA, but not to ABA and high salt. Based on semi-quantitative RT-PCR analysis, AtKPl expression was responded only after induced for 48 h with IAA, indicating that AtKPl is a late response gene of IAA. To further reveal the function of AtKPl, mutant of AtKPl T-DNA insertion was confirmed by PCR, southern blotting, northern blotting, and its phenotype was observed. The germination of mutant seeds showed resistance to IAA, which suggested that AtKPl may be involved in IAA physiological process, but deletion of AtKPl didn’t affect the change of the early response gene IAA2 JAA16 ; In normal growth conditions, the deletion of AtKPl didn’t affect the normal growth and the development of Arabidopsis plants, suggesting that the function of AtKPl may be substituted by other homologous gene.KatD is another kinesin isoform with high identity to AtKPl. To know if KatD could substitute AtKPl, we cloned the promoter region of katD. With gus as reporter gene, expression pattern of KatD in Arabidopsis was analyzed. KatD had high expression in anther. Its strong expression was also occurred in young leaf trichome and young vasculature, similar to that of AtKPl.katD T-DNA insertion mutant was confirmed by the method of PCR and semi-quantitative RT-PCR. Observation of phenotype demonstrated that deletion of katD didn’t affect normal development of Arabidopsis plant, suggesting that the function of KatD may be substituted with that of its homologous gene. KatD mutant was crossed with AtKPl mutant, and the double mutant was obtained. Primary observation to T2-generation showed that double mutant plant had obvious dwarf phenotype, but had no defect in leaf trichome and vasculature. The T3-generation double mutants were waiting to be observed in detail.更多还原