【作者】 秦治翔；

【导师】 王学臣；

【作者基本信息】 中国农业大学 ， 植物学， 2005， 博士

【摘要】 1,3,4-三磷酸肌醇5/6-激酶(简称5/6-激酶)是磷酸肌醇代谢过程中的一种重要的调控酶。动物细胞中,它具有肌醇激酶和蛋白激酶双重活性,还能进行自身磷酸化,是一个从植物到人甚至线虫都保守的酶;而且5/6-激酶还是第一个鉴定出的与COP9信号体(简称CSN)互作的蛋白激酶,但它们互作的生物学意义还不清楚。植物细胞中,仅知道5/6-激酶是一种广泛存在的肌醇激酶,有关它的功能没有更多的报道。因此,研究5/6-激酶是否具有蛋白激酶的活性、是否与CSN互作,并参与植物的光形态建成,对揭示5/6-激酶的生物学功能,阐明植物光形态建成机理等方面具有重要的理论意义。 本文以拟南芥为材料,克隆了5/6-激酶的基因(命名为AtItpk-1),并进行了原核表达(His标签),纯化融合蛋白并制备了多克隆抗体。通过对AtItpk-1在不同光质下的表达分析,发现它受红光强诱导,说明AtItpk-1可能参与了植物对红光的应答。通过对AtItpk-1在拟南芥中的定位研究,发现AtItpk-1定位在细胞核中。 为了研究5/6-激酶在拟南芥中的生物学意义,对AtItpk-1的T-DNA插入突变体atitpk-1-1和atitpk-1-2进行了筛选和鉴定,并通过转基因技术获得了超表达AtItpk-1基因的转基因植株。表型分析表明,与野生型相比,atitpk-1突变体表现为红光抑制下胚轴伸长,超表达植株则表现为红光促进下胚轴伸长。另外,当将AtItpk-1基因转到突变体atitpk-1-1中,下胚轴伸长便恢复到与野生型一样。这些结果表明拟南芥5/6-激酶AtItpk-1可能参与了红光下的光形态建成。 利用我们制备的有活性的GST-AtItpk-1融合蛋白,进行了自身磷酸化实验,发现AtItpk-1具有蛋白激酶的活性。这为进一步解释5/6-激酶在光形态建成中的可能作用提供了线索。 以fus6/CSN1-3-4为材料,利用AtItpk-1的抗体和CSN4、CSN5的抗体,进行了5/6-激酶和CSN的免疫共沉淀实验,结果发现拟南芥的5/6-激酶可以和CSN共沉淀。基于免疫共沉淀和AtItpk-1、CSN都是核蛋白的结果,我们认为在拟南芥中存在5/6-激酶和CSN互作的可能性。 本研究发现了拟南芥的5/6-激酶参与了红光下的光形态建成,这一作用可能是通过与CSN互作完成的,并且5/6-激酶在光形态建成中的功能可能是通过它的蛋白激酶活性来实现的。但是,植物的光形态建成是一个非常复杂的过程,5/6-激酶参与光形态建成的具体机制仍需要进一步探讨。更多还原

【Abstract】 Inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) is a key regulatory enzyme in the process of the inositol phosphate metabolism. In mammalian cells, the 5/6-kinase exhibits the activity of both inositol kinase and protein kinase, and is also reported to have the activity of autophosphorylation. It is conserved from plants to humans and is found even in Entamoeba histolytica. Moreover, the 5/6-kinase is also the first identified COP9 signalosome (CSN) -associated kinase in mammalian cells. But the biological function of their association still remains unclear. In plants, it is only reported that the 5/6-kinase is the ubiquitous inositol kinase. But there are not more reports on the function of this enzyme. So, the works that study if the 5/6-kinase exhibits the activity of protein kinase, and if it associates with CSN and then involves in plant photomorphogenesis will be significative for opening out the biological function of 5/6-kinase and elucidating the mechanism of plant photomorphogenesis.In the research for the dissertation, we cloned the 5/6-kinase gene AtItpk-1 fiom Arabidopsis. We also expressed the His-AtItpk-1 protein in E.coli, obtained the purified fusion protein and prepared the anti-AtItpk-1 antibody. In order to analyze the expression of AtItpk-1 under different light quality, RT-PCR and Western blot were performed. The results indicated that the expression of AtItpk-1 was strongly induced by red light, which suggested that AtItpk-1 may be involved in the response of plant to red light. In addition, the study of localization showed that AtItpk-1 is located in the nucleus in Arabidopsis.In order to study the biological function of 5/6-kinase in Arabidopsis, two T-DNA insertion mutants of AtItpk-1, designated (by us) as atitpk-1-1 and atitpk-1-2, were screened and identified. Also, the transgenic plants overexpressing AtItpk-1 were generated by transgenic technology. The phenotypic results indicated when compared with wild-type plants, the atitpk-1 mutants exhibit the phenotype that red light inhibits the elongation of hypocotyls, and the over-expression lines of AtItpk-1 show the phenotype that red light promotes the elongation of hypocotyls. In addition, when the Atltpk-l gene was transformed into atitpk-1-1 mutant, the length of hypocotyls was restored to that of the wild-type. These results suggested that the 5/6-kinase Atltpk-1 may be involved in photomorphogenesis under red light in Arabidopsis.Using the active GST-AtItpk-1 fusion protein expressed by ourselves, the autophosphorylation assay was performed. The results showed that Atltpk-l has protein kinase activity, which can provide the clues for interpreting the possible function of 5/6-kinase in the photomorphogenesis.The fus6/CSN1-3-4 was used to do the co-immunoprecipitation (co-IP) of 5/6-kinase and CSN using the anti-AtItpk-1, anti-CSN4, and anti-CSN5 antibodies. The result showed that 5/6-kinase can co-immunoprecipitated with CSN in Arabidopsis. Based on co-IP and the result that both Atltpk-1 and CSN were located in the nucleus, we think that it is possible for the association of 5/6-kinase and CSN in Arabidopsis.In our research, it was found the 5/6-kinase may be involved in photomorphogenesis under red light in Arabidopsis, possibly via interaction with CSN. The role of 5/6-kinase in photomorphogenesis is possibly based on its activity of protein kinase. But the process of plant photomorphogenesis is very complex and the detailed mechanism for 5/6-kinase involving in photomorphogenesis still needs to study further.更多还原