【作者】 郑晟；

【导师】 邱全胜；

【作者基本信息】 兰州大学 ， 植物学， 2014， 博士

【摘要】 Na+,K+/H+反向转运体是H+偶联的协同转运蛋白,其生物功能是跨膜转运Na+或K+,同时进行H+的反向转运。Na+,K+/H+反向转运体在维持细胞离子和pH平衡方面起着重要作用,在许多细胞生理生化过程,包括Na+和K+的转运,耐盐性,细胞周期和细胞增殖的调节,膜微囊运输和生物膜融合,以及生物发生等起着重要的调节作用。拟南芥AtKEA基因家族包括6个基因,它们与细菌K+/W反向转运体KefB和KefC的基因序列高度相似,表明AtKEA可能编码K+/H+反向转运体,在植物钾离子调控方面起着重要的作用。然而目前对AtKEA基因家族的离子转运特性和生物功能尚缺乏实验研究。我们运用生物信息学,酵母生长实验,实时定量PCR, GFP标签,GUS染色等分子生物学和细胞生物学技术对拟南芥AtKEA基因家族的离子转运特性,基因表达模式,蛋白质细胞定位以及生物学功能进行全面研究,以期阐明拟南芥AtKEA基因家族在钾吸收,逆境胁迫,pH调节以及渗透调节等方面的生物学功能,为全面揭示植物体内钾离子平衡机理,提高植物钾离子利用效率等奠定重要的理论基础。本研究取得以下研究成果：(1)生物信息学分析发现,AtKEA基因家族的6个基因形成两个亚组：AtKEAl-3和AtKEA4-6。AtKEA1和AtKEA2具有较长的N端结构域。系统发育分析发现,AtKEAs与AtNHXs和AtCHXs分布在不同的进化枝上,暗示AtKEAs的功能可能不同于AtNHXs和AtCHXs。(2)蛋白质结构分析发现,AtKEA1和AtKEA2均由一个N-端结构域,一个Na H交换结构域和一个C-端KTN结构域组成。在早期基因预测时,AtKEA1和AtKEA2基因只含有一个Na H交换结构域和一个C-端KTN结构域,N-端结构域没有包含在内(称为短片段AtKEA1和短片段AtKEA2基因,分别表示为AtsKEA1和AtsKEA2),并且实验也只克隆得到AtsKEA1和AtsKEA2基因。我们试图从拟南芥cDNA上直接克隆含有N-端结构域的全长AtKEA1和AtKEA2基因,但是没有获得与数据库序列一致的全长AtKEA1和AtKEA2基因。我们然后采取分段克隆的方法,在基因的中间选取一个酶切位点,将基因分成两段,分别克隆,最后将两个片段连接起来,得到全长序列基因。通过这个方法我们克隆得到与数据库序列一致的全长AtKEA1基因；但是我们仍然没有获得与数据库序列一致的全长AtKEA2基因。酵母突变体分析发现全长AtKEA1没有离子转运活性。所以本研究中,我们采用缺失N-端区域的AtsKEA1和AtsKEA2进行离子转运实验分析。(3)我们顺利克隆到与数据库序列一致的AtKEA3-6基因,并且用于离子转运实验分析。(4)采用缺失离子转运蛋白的酵母突变体分析AtKEA基因家族的离子转运特性。发现AtKEA基因家族能够抵抗高浓度K+和潮霉素的胁迫,但不能抵抗盐胁迫和Li+胁迫,表明AtKEA基因家族具有K+离子运输功能,并且参与细胞膜微囊运输。实验还发现AtKEA基因家族介导的K+转运与AtNHXs和AtCHXs所介导的K+转运可能具有不同的催化模式：AtKEAs基因在高钾,pH5.8时起作用；AtNHXs在高钾,酸性环境下起作用；AtCHXs只在低钾,碱性条件下有作用。(5) RT-qPC分析发现,AtKEA基因家族在拟南芥根和叶上均有表达。在低浓度K+胁迫下,AtKEA1, AtKEA3和AtKEA4的表达增强；AtKEA2和AtKEA5在山梨醇与ABA处理下表达增强。然而,AtKEA2和AtKEA5在ABA合成缺失突变体aba2-3中未表现出渗透胁迫响应,说明AtKEA2和AtKEA5对渗透胁迫的响应受ABA信号的调节。我们还发现,在SOS突变体中AtKEAs基因的表达不受影响,表明AtKEA基因的表达可能不受SOS途径的调节。(6)启动子-GUS分析发现,AtKEA1和AtKEA3在拟南芥叶片,花及根成熟部位均有表达,暗示AtKEA1和AtKEA3可能在拟南芥营养器官及生殖器官的生长发育过程中发挥作用。(7)GFP融合蛋白分析发现,AtKEA基因家族在酵母细胞中的定位具有多样性,暗示AtKEA基因家族可能具有不同的生理功能。(8)拟南芥原生质体瞬时基因表达和转基因植株分析发现,AtKEA3定位于高尔基体上。总之,我们的研究结果显示,AtKEA基因家族成员具有不同的基因表达模式和亚细胞分布；AtKEA基因在拟南芥中可能具有维持K+平衡和渗透调节的作用。更多还原

【Abstract】 Na+,K+/H+antiporters are H+-coupled cotransporters whose biochemical activity is to transfer the Na+or K+across a membrane in exchange for protons (H+). Na+,K+/H+antiporters are critical for ion homeostasis and pH regulation in cells, and function in diverse cellular processes, including Na+and K+movement, salt tolerance, regulation of cell cycle and cell proliferation, vesicle trafficking and fusion, and biogenesis.In the Arabidopsis genome, the AtKEA gene family contains6members. AtKEAs are homologs of bacterial K+/H+antiporters KefB/KefC, indicating that AtKEAs may encode K+/H+antiporters in Arabidopsis, and play an important role in K+homeostasis. However, the physiological functions of the AtKEA gene family remain largely uncharacterizedIn this study, we characterized the ion transport activity, gene expression, cellular localizations and biological functions of the AtKEA gene family using a variety of methods and techniques including bioinformatics, yeast growth, RT-qPCR, GFP labeling and GUS staining. Our goal is to explore the role of the AtKEA gene family in potassium uptake, environmental stresses, cellular pH regulation and osmotic adjustment. Our study will pave the way for the understanding of the cellular mechanisms governing K+homeostasis in plants.The major results are listed below:(1) Bioinformatics analysis shows that the AtKEA family contains six genes forming two subgroups in the cladogram:AtKEAl-3and AtKEA4-6. AtKEAl and AtKEA2have a long N-terminal domain. Phylogenetic analysis finds that AtKEAs separate clearly from the clusters of AtNHXs and AtCHXs with their yeast orthologs, suggesting that AtKEAs may function distinctly from either AtNHXs or AtCHXs.(2) Protein organization analysis showed that AtKEA1andAtKEA2are comprised of a soluble N-terminal domain, a Na_H exchange domain and a C-terminal KTN domain. The long N-terminal domains of AtKEA1and AtKEA2were missed in early gene annotation. The cDNA sequences of the short version AtKEAl and AtKEA2, AtsKEAl and AtsKEA2, lacking the N-terminal domains but containing the Na_H exchange domains, have been cloned in yeast by Dr. John Ward lab. We attempted to clone the full-length cDNAs of AtKEAl and AtKEA2genes. Since the direct amplification from the Arabidopsis cDNA preparation was not successful, we used a two-step strategy. We separated the gene into two pieces by choosing a restriction enzyme site in the middle of the gene; the two pieces were cloned separately. We then combined them to get the full-length cDNA. For AtKEAl, an EcoRI site was chosen to separate the gene into two pieces of A1-C1870and T1871-A3582, and we successfully cloned the full-length cDNA. However, using the same strategy, we did not clone the full length AtKEA2gene. Yeast growth analysis shows that the full length AtKEAl is inactive in ion transport. Thus, the short versions AtKEA1and AtKEA2, AtsKEAl and AtsKEA2, were used in the ion transport assay in this study.(3) We successfully cloned the AtKEA3-6genes and used these gene sequences in ion transport assays.(4) The transport activity was analyzed by expressing the AtKEA genes in yeast mutants lacking multiple ion carriers. AtKEAs conferred resistance to high K+and hygromycin B but not to salt and Li+stress. These results suggest that AtKEA gene family are able to transfer K+, and play a role in vesicle trafficking. Moreover, AtKEAs, AtNHXs and AtCHXs may have different modes of action in facilitating K+homeostasis. AtKEAs function at high K+at pH5.8while AtNHXs function at high K+in acidic environments and AtCHXs at low K+under alkaline conditions.(5) RT-qPCR analysis showed that AtKEAs were expressed in both the shoot and root of Arabidopsis. The expression of AtKEAl, AtKEA3and AtKEA4was enhanced under low K+stress, whereas AtKEA2and AtKEA5were induced by sorbitol and ABA treatments. However, osmotic induction of AtKEA2and AtKEA5was not observed in aba2-3mutants, suggesting an ABA regulated mechanism for their osmotic response. AtKEAs’ expression may not be regulated by the SOS pathway since their expression was not affected in sos mutants.(6) Promoter-GUS analysis showed that AtKEAl and AtKEA3were expressed in leaves, flowers and the mature area of roots in Arabidopsis, indicating that AtKEAs may function in regulating the growth and development of the vegetative and reproductive organs in Arabidopsis.(7) The GFP tagging analysis showed that AtKEAs were distributed diversely in yeast, suggesting that AtKEAs may have diversified roles in growth and development in plants.(8) The Golgi localization of AtKEA3was demonstrated by both the stably transformed seedlings and the transient expression in protoplasts.In summary, AtKEAs expressed and localized diversely, and may play roles in K+homeostasis and osmotic adjustment in Arabidopsis.更多还原