本文選題：條葉藍芥 + HKT基因　； 參考：《蘭州大學》2017年碩士論文

【摘要】：植物HKT(high-affinity K~+transporter)轉(zhuǎn)運蛋白是Na~+或K~+轉(zhuǎn)運蛋白或Na~+/K~+共轉(zhuǎn)運體,在植物抵抗鹽漬脅迫方面起著重要的作用。擬南芥HKT1定位于韌皮部薄壁細胞的質(zhì)膜上,其作用是通過外排降低葉片Na~+含量,避免高濃度鹽對植物的傷害作用。條葉藍芥(Thellungiella parvula)是十字花科鹽芥屬(Thellungiella)植物,具有很強的耐鹽脅迫能力。由于條葉藍芥是擬南芥近緣植物,所以可以作為研究耐鹽脅迫機理的模式植物。條葉藍芥含有兩個HKT基因:TpHKT1;1和TpHKT1;2。然而,目前對TpHKT1;1和TpHK1;2的離子轉(zhuǎn)運活力和生物功能還很不清楚。我們的目的是研究條葉藍芥HKT基因的離子轉(zhuǎn)運特性和在耐鹽脅迫中的作用。我們采用分子遺傳學方法,分析了TpHKT1;1和TpHKT1;2在組織中的表達模式和亞細胞定位。我們將TpHKT1;1和TpHK1;2基因表達在酵母和擬南芥athkt1-1突變體之中,分析其離子轉(zhuǎn)運特性和生理功能。我們得到的結(jié)果如下:1.將TpHKT1;1和TpHK1;2基因表達在擬南芥野生型Col(gl1)和athkt1-1突變體中,制備TpHKT1;1和TpHK1;2轉(zhuǎn)基因植株。2.采用GUS染色方法分析TpHKT1;1和TpHKT1;2在組織中的表達模式。TpHTK1;1主要在葉尖和根的中柱表達,在果莢頂端和果柄以及雄蕊和柱頭表達較弱;TpHKT1;2在葉片和下胚軸上表達很強,在側(cè)根原基和果莢中表達很弱,但在雄蕊和柱頭上的表達比TpHTK1;1強,表明TpHKT1;1和TpHKT1;2的表達模式相似,但不完全相同。3.將GFP-TpHKT1;1/Col(gl1)轉(zhuǎn)基因植株與表達了細胞器marker的擬南芥植株雜交,制備雙共表達植株,進行TpHKT1;1的亞細胞定位分析。激光共聚焦顯微鏡觀察發(fā)現(xiàn),TpHTK1;1在質(zhì)膜(PM)、高爾基(Golgi)、反式高爾基網(wǎng)狀結(jié)構(gòu)(TGN)、液胞前體(PVC)/多胞體(MVB)上均有分布,表明TpHTK1;1的定位具有多樣性。4.酵母生長實驗發(fā)現(xiàn),表達TpHKT1;1和TpHKT1;2基因能夠提高酵母細胞對潮霉素、NaCl和LiCl的抗性,說明TpHKT1;1和TpHKT1;2可能具有轉(zhuǎn)運Na~+和Li~+的活力,并參與細胞膜微囊的運輸。5.將TpHKT1;1和TpHKT1;2基因過表達在Col(gl1)和athkt1-1中時,在含NaCl的培養(yǎng)基上轉(zhuǎn)基因植株的種子萌發(fā)和幼苗生長都受到明顯抑制。土壤栽培試驗發(fā)現(xiàn),在200 m M NaCl脅迫下,轉(zhuǎn)基因植株的生長明顯受到抑制,表明TpHKT1;1和TpHKT1;2具有將Na~+運輸進入細胞的活力。還發(fā)現(xiàn),轉(zhuǎn)基因植株的幼苗生長對LiCl脅迫敏感;然而過表達TpHKT1;2基因?qū)iCl脅迫的敏感度大于表達TpHKT1;1基因�？傊�,TpHK1;1和TpHK1;2具有向細胞內(nèi)轉(zhuǎn)運Na~+的活力;TpHKT1;1和TpHKT1;2對Na~+和Li~+的轉(zhuǎn)運模式存在差異。
[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.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q943.2

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