条叶蓝芥HKT基因的生物功能研究

发布时间：2018-06-21 14:13

本文选题：条叶蓝芥 + HKT基因　；参考：《兰州大学》2017年硕士论文

【摘要】：植物HKT(high-affinity K~+transporter)转运蛋白是Na~+或K~+转运蛋白或Na~+/K~+共转运体,在植物抵抗盐渍胁迫方面起着重要的作用。拟南芥HKT1定位于韧皮部薄壁细胞的质膜上,其作用是通过外排降低叶片Na~+含量,避免高浓度盐对植物的伤害作用。条叶蓝芥(Thellungiella parvula)是十字花科盐芥属(Thellungiella)植物,具有很强的耐盐胁迫能力。由于条叶蓝芥是拟南芥近缘植物,所以可以作为研究耐盐胁迫机理的模式植物。条叶蓝芥含有两个HKT基因:TpHKT1;1和TpHKT1;2。然而,目前对TpHKT1;1和TpHK1;2的离子转运活力和生物功能还很不清楚。我们的目的是研究条叶蓝芥HKT基因的离子转运特性和在耐盐胁迫中的作用。我们采用分子遗传学方法,分析了TpHKT1;1和TpHKT1;2在组织中的表达模式和亚细胞定位。我们将TpHKT1;1和TpHK1;2基因表达在酵母和拟南芥athkt1-1突变体之中,分析其离子转运特性和生理功能。我们得到的结果如下:1.将TpHKT1;1和TpHK1;2基因表达在拟南芥野生型Col(gl1)和athkt1-1突变体中,制备TpHKT1;1和TpHK1;2转基因植株。2.采用GUS染色方法分析TpHKT1;1和TpHKT1;2在组织中的表达模式。TpHTK1;1主要在叶尖和根的中柱表达,在果荚顶端和果柄以及雄蕊和柱头表达较弱;TpHKT1;2在叶片和下胚轴上表达很强,在侧根原基和果荚中表达很弱,但在雄蕊和柱头上的表达比TpHTK1;1强,表明TpHKT1;1和TpHKT1;2的表达模式相似,但不完全相同。3.将GFP-TpHKT1;1/Col(gl1)转基因植株与表达了细胞器marker的拟南芥植株杂交,制备双共表达植株,进行TpHKT1;1的亚细胞定位分析。激光共聚焦显微镜观察发现,TpHTK1;1在质膜(PM)、高尔基(Golgi)、反式高尔基网状结构(TGN)、液胞前体(PVC)/多胞体(MVB)上均有分布,表明TpHTK1;1的定位具有多样性。4.酵母生长实验发现,表达TpHKT1;1和TpHKT1;2基因能够提高酵母细胞对潮霉素、NaCl和LiCl的抗性,说明TpHKT1;1和TpHKT1;2可能具有转运Na~+和Li~+的活力,并参与细胞膜微囊的运输。5.将TpHKT1;1和TpHKT1;2基因过表达在Col(gl1)和athkt1-1中时,在含NaCl的培养基上转基因植株的种子萌发和幼苗生长都受到明显抑制。土壤栽培试验发现,在200 m M NaCl胁迫下,转基因植株的生长明显受到抑制,表明TpHKT1;1和TpHKT1;2具有将Na~+运输进入细胞的活力。还发现,转基因植株的幼苗生长对LiCl胁迫敏感;然而过表达TpHKT1;2基因对LiCl胁迫的敏感度大于表达TpHKT1;1基因。总之,TpHK1;1和TpHK1;2具有向细胞内转运Na~+的活力;TpHKT1;1和TpHKT1;2对Na~+和Li~+的转运模式存在差异。
[Abstract]:Plant HKT high-affinity K ~ transporter is a Na ~ or K ~ -transporter or a Na ~ / K ~ -co-transporter, which plays an important role in plant resistance to salt stress. Arabidopsis thaliana HKT1 is located on the plasmalemma of phloem parenchyma cells. Its function is to reduce the Na ~ content of leaves and avoid the harm of high concentration salt to plants through efflux. Thellungiella parvula (Thellungiella parvula) is a plant of the genus Thellungiella (Cruciferae), which has strong tolerance to salt stress. As Arabidopsis thaliana is a relative plant, it can be used as a model plant to study the mechanism of salt tolerance. There are two HKT genes: TpHKT1 and TpHKT1. However, the ion transport activity and biological function of TpHKT1O1 and TpHK1K2 are still unclear. Our aim is to study the ion transport characteristics of HKT gene and its role in salt tolerance. The expression patterns and subcellular localization of TpHKT1O1 and TpHKT1O2 in tissues were analyzed by molecular genetics. We expressed TpHKT1O1 and TpHK1t2 genes in yeast and Arabidopsis athkt1-1 mutants, and analyzed their ion transport characteristics and physiological functions. Our results are as follows: 1. TpHKT1O1 and TpHK1t2 genes were expressed in Arabidopsis thaliana wild-type Colngl1) and athkt1-1 mutants, and the transgenic plants of TpHKT1O1 and TpHK1t2 were prepared. Gus staining was used to analyze the expression patterns of TpHKT1 and TpHKT1O2 in tissues. The expression of TpHKK1 was mainly in the middle column of leaf tip and root, and the expression of TpHKT1O2 was weak in apical and petiole of fruit pod, stamen and stigma, and the expression of TpHKT1O2 was strong in leaf and Hypocotyl. The expression in lateral root primordia and fruit pods was very weak, but the expression in stamen and stigma was stronger than that in TpHTK1, which indicated that the expression patterns of TpHKT1O1 and TpHKT1T1 were similar, but not identical. 3. GFP-TpHKT1 / 1 / Colgl1) transgenic plants were hybridized with Arabidopsis thaliana plants expressing organelle marker. The results of laser confocal microscopy showed that TpHTK1 was distributed on plasma membrane, Golgil, TGNN and PVC / MVB, indicating that the localization of TpHTK1 / MVB was diverse. Yeast growth assay showed that the expression of TpHKT1O1 and TpHKT1O2 genes could enhance the resistance of yeast cells to hygromycin chloride and LiCl, suggesting that TpHKT1O1 and TpHKT1T1O2 may have the activity of transporting Na ~ and Li ~, and participate in the transport of cell membrane microcapsules. 5. The expression of TpHKT _ 1 and TpHKT _ 1 can increase the resistance of yeast cells to hygromycin chloride and LiCl. The seed germination and seedling growth of transgenic plants on NaCl medium were significantly inhibited by overexpression of TpHKT1O1 and TpHKT1O2 genes in Collomgl1 and athkt1-1. Soil culture experiments showed that the growth of transgenic plants was significantly inhibited under 200 mm NaCl stress, which indicated that TpHKT1O1 and TpHKT1O2 had the activity of transporting Na ~ into cells. It was also found that the seedling growth of transgenic plants was sensitive to LiCl stress, but the overexpression of TpHKT1O2 gene was more sensitive to LiCl stress than that of TpHKT1 gene. In a word, TpHK1T1 and TpHK1T2 have the activity of transporting Na ~ into cells. The transport patterns of Na ~ and Li ~ are different between TpHKT1 and TpHKT1.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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