拟南芥锌转运蛋白ZNE1的亚细胞定位及其功能研究

发布时间：2018-04-03 11:11

本文选题：拟南芥　切入点：ZNE1　出处：《内蒙古大学》2017年硕士论文

【摘要】：拟南芥At3g08650被预测编码锌转运蛋白,但是没有直接的实验证据。我们将 At3g08650 命名为 ZINC NUTRITION ESSENTIAL 1(ZNE1)。为了确定 ZNE1蛋白的亚细胞定位,在烟草表皮细胞内瞬时表达ZNE1-GFP(绿色荧光蛋白)融合基因。在激光共聚焦显微镜下,通过对比发射红色荧光的细胞器标记蛋白位置,证明ZNE1定位在高尔基体。进一步将ZNE1在哺乳动物细胞HEK293T中表达,利用免疫荧光证明ZNE1存在于细胞质中。为了确定ZNE1在植物锌依赖生长中的生理功能,纯合鉴定到两个拟南芥T-DNA插入缺失突变体zne1。进一步在水培条件下,对比拟南芥野生型Co1和zne1锌依赖生长表型,发现zne1在高锌水培液中表现叶片发黄的敏感表型。将完整的ZNE1编码序列整合到zne1突变体基因组可以使zne1对高锌环境的反应恢复到野生型,证明ZNE1是拟南芥适应高锌环境的必需基因。为了探索质外体Zn2+在活体叶片中的分布,我们利用离体叶片叶柄浸泡法和膜不通透性Zn2+特异性荧光染料FluoZin-3,发现在高锌处理条件下,拟南zne1一、二级叶脉中Zn2+浓度显著高于野生型,证明ZNE1是调节拟南芥叶子内Zn2+分布的重要基因。
[Abstract]:Arabidopsis At3g08650 is predicted to encode zinc transporter, but there is no direct experimental evidence.We named At3g08650 as ZINC NUTRITION ESSENTIAL 1.In order to determine the subcellular localization of ZNE1 protein, ZNE1-GFP fusion gene was transient expressed in tobacco epidermal cells.The localization of ZNE1 in Golgi body was proved by comparing the position of labeled proteins in organelle of red fluorescence under laser confocal microscope.ZNE1 was further expressed in mammalian HEK293T and ZNE1 was demonstrated to exist in the cytoplasm by immunofluorescence.In order to determine the physiological function of ZNE1 in plant zinc dependent growth, two Arabidopsis T-DNA insertion deletion mutants Zne1 were identified by homozygote.Furthermore, under hydroponic conditions, compared with wild type Co1 and zne1 zinc dependent growth phenotype of Arabidopsis thaliana, it was found that zne1 showed a sensitive phenotype of leaf yellowing in high zinc hydroponics.Integration of complete ZNE1 coding sequence into the genome of zne1 mutants can restore the response of zne1 to high zinc environment to wild type, which proves that ZNE1 is an essential gene for Arabidopsis to adapt to high zinc environment.In order to investigate the distribution of Zn2 in living leaves, we used the method of leaf petiole immersion in vitro and membrane impermeability Zn2 specific fluorescent dye FluoZin-3.The concentration of Zn2 in secondary leaf vein was significantly higher than that in wild type, which suggested that ZNE1 was an important gene regulating the distribution of Zn2 in leaves of Arabidopsis thaliana.
【学位授予单位】：内蒙古大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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