【作者】 肖雪；

【导师】 杨清； 黄丛林；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2006， 硕士

【摘要】 本实验室从玉米中克隆得到四个与逆境相关的蛋白激酶基因(ZmSPK1，ZmPti1，ZmASK1，ZmSIMK1)，受到盐、甘露醇、脱落酸及低温等胁迫因子诱导时表达上调，表明它们可能参与逆境信号传导过程。为了进一步研究它们在体内的功能，我们利用生物信息学方法初步分析了它们在胞内可能的定位情况，然后利用绿色荧光蛋白基因gfp作为报告基因，构建融合基因的植物表达载体，采用花粉浸醮的方法，通过农杆菌介导转入模式植物拟南芥中，观察其在拟南芥细胞内的定位，结果如下： 利用生物信息学软件分析ZmPti1、ZmASK1、ZmSIMK1和ZmSPK1的初级结构，发现ZmPti1和ZmASK1的N末端都具有线粒体前导序列，均为可溶性蛋白，推测它们可能定位于线粒体基质内。ZmSIMK1和ZmSPK1定位情况较为复杂，初步推测它们可能是定位于细胞质和细胞核内。 构建了以35S启动子驱动的四个融合蛋白ZmX-GFP和游离GFP的植物表达载体pGZmX-GFP和pG35s-GFP。转入拟南芥中均筛选得到稳定转化株系，用体式荧光显微镜观察它们在组织器官内的荧光信号，发现它们在根尖、茎尖等幼嫩组织内表达量高。 激光共聚焦荧光显微镜观测结果表明，融合蛋白ZmPti1-GFP的荧光信号主要分布在细胞质中，且分布不均匀，推测该蛋白激酶为可溶性蛋白，定位在特定的细胞器内。结合生物信息学分析及该细胞器大小，我们推测定位于线粒体内。融合蛋白ZmSPK1-GFP的荧光信号的分布与游离GFP对照相似，在细胞基质及细胞核内均可见。但是与单独表达GFP的对照相比，融合蛋白ZmSPK1-GFP在核内信号更强。据此，我们推测ZmSPK1是可溶性蛋白，定位在核内。 由于未得到荧光表达较强的转ZmSIMK1融合基因和转ZmASK1融合基因的株系，无法进行这两个基因产物的精细定位。 蛋白质的细胞区域分布是其功能体现的一个方面，通过本实验的研究为进一步研究它们在逆境信号传递中的作用，及其在植物体内可能的生物学功能奠定基础。更多还原

【Abstract】 Four maize protein kinase genes (ZmSPK1, ZmPti1, ZmASK1, ZmSIMK1) responsing to salt, mannitol, ABA or cold stresses have been cloned in our laboratory. The obtained results show that the genes may play roles in the stress signaling pathways. In order to further study their functions in vivo, the primary research on cellular compartment of the four kinases has been carried out by bioinformation and GFP (Green Fluorescence Protein) tag.With bioinformation softwares (PSORT, TargetP or MitoProt) analysis, the results show that ZmPti1 and ZmASK1 with eventual N-terminal mitochondrial targeting sequences might be localized in mitochondrial matrix, and ZmSIMK1 and ZmSPK1 might be localized in nucleus and cytoplasm.To determine the subcellular localization of the four protein kinases, using GFP as a reporter and 35S as a promoter we constructed five plant expression plasmids: pGZmX-GFP (ZmX represents the four kinases) and pG35s-GFP, and transformed them respectively into Arabidopsis by floral dip and screened stable transgenic lines by Kan resistance and PCR. Subcellular localization was determined by Confocal laser scanning microscopy. The fused protein ZmPti1-GFP is mainly localized within cytoplasm with unequal distribution, which suggests ZmPtil might localize in one special organelle. According to bioinformation softwares analysis and the size of the organelle we presumed it localized in mitochondrion. The fused ZmSPK1-GFP showed a nuclear and cytoplasmic distribution similar to that of free GFP. Compared with free GFP, ZmSPK1-GFP was more strongly accumulated in the nucleus, which suggests that ZmSPK1 might be a soluble protein and localize in the nucleus. It’s a pity for failing to get comparably strong expression of ZmSIMK1-GFP and ZmSPK1-GFP and hard to achive their cellular target.The native subcellular compartment of a protein is an important characteristic with functional implications. This work will provide basis for further study on their roles in the stress signaling pathways and on their potential biological functions.更多还原