本文選題：擬南芥　切入點：ZNE1　出處：《內(nèi)蒙古大學》2017年碩士論文

【摘要】：擬南芥At3g08650被預測編碼鋅轉運蛋白,但是沒有直接的實驗證據(jù)。我們將 At3g08650 命名為 ZINC NUTRITION ESSENTIAL 1(ZNE1)。為了確定 ZNE1蛋白的亞細胞定位,在煙草表皮細胞內(nèi)瞬時表達ZNE1-GFP(綠色熒光蛋白)融合基因。在激光共聚焦顯微鏡下,通過對比發(fā)射紅色熒光的細胞器標記蛋白位置,證明ZNE1定位在高爾基體。進一步將ZNE1在哺乳動物細胞HEK293T中表達,利用免疫熒光證明ZNE1存在于細胞質中。為了確定ZNE1在植物鋅依賴生長中的生理功能,純合鑒定到兩個擬南芥T-DNA插入缺失突變體zne1。進一步在水培條件下,對比擬南芥野生型Co1和zne1鋅依賴生長表型,發(fā)現(xiàn)zne1在高鋅水培液中表現(xiàn)葉片發(fā)黃的敏感表型。將完整的ZNE1編碼序列整合到zne1突變體基因組可以使zne1對高鋅環(huán)境的反應恢復到野生型,證明ZNE1是擬南芥適應高鋅環(huán)境的必需基因。為了探索質外體Zn2+在活體葉片中的分布,我們利用離體葉片葉柄浸泡法和膜不通透性Zn2+特異性熒光染料FluoZin-3,發(fā)現(xiàn)在高鋅處理條件下,擬南zne1一、二級葉脈中Zn2+濃度顯著高于野生型,證明ZNE1是調(diào)節(jié)擬南芥葉子內(nèi)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.
【學位授予單位】：內(nèi)蒙古大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2

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