甜瓜钾离子通道MIRK提高植物耐盐性的作用机理研究

K+ channels play an important role in plants salt tolerance. MIRK(Melon Inward Rectifying K+ channel), a K+ channel from melon (Cucumis melo L.), displays a unique property: sensitivity to external Na+. And transfer of MIRK channel gene into the model plant Arabidopsis thaliana (T-DNA mediated transformation) has been shown to improve the plant salt tolerance. But there are something unclear that molecular base of MIRK sensitivity to Na+ and mechanisms of MIRK improve plant salt tolerance, etc. Concerning K+ channels, such regulation is mostly documented at the transcriptional level so far, in the Shaker family. The project will study on identification of the molecular bases of MIRK sensitivity to Na+ through methods of molecular biology and electrophysiology. Physiological evaluation, such as the activity of MIRK and the balance regulation of K+/Na+ in guard cells of transgenic plants overexpressing MIRK channel or MIRK mutants with altered sensitivity to salt, then better understanding, both at the molecular and physiological levels, the mechanisms underlying MIRK K+ channel gene involvement in plant salt tolerance, and more generally the role of K+ transport in plant adaptation to salt stress. In order to better understand the physiological role of MIRK channel in melon salt tolerance and more generally to evaluate the potential of an increase in K+ channel activity in the improvement of plant salt tolerance, contrast anlasis physiological evaluation of MIRK in different melon cultivated varieties will be operated. The results expected to underlie its role in plant salt tolerance, with the aim to evidence a molecular trait that could be later introduced by crossing in melon or possibly also by genetic engineering in other horticultural crop species to improve their tolerance to salt stress.

K+通道对植物耐盐性有重要作用。MIRK(Melon Inward Rectifying K+ channel)是甜瓜(Cucumis melo L.)中的一个K+通道，该通道在所有已知的植物钾离子通道中具有独特特性：对外界Na+敏感；表达MIRK的转基因植株耐盐性明显增强。MIRK对Na+敏感的分子基础、提高植物耐盐性的作用机理等尚未明了，Na+对K+通道的影响文献报道多集中在其转录水平上。本课题研究K+通道MIRK受Na+影响的功能域，通过盐胁迫下植株保卫细胞MIRK的活性、K+/Na+平衡调节等生理响应的分析，探索K+通道作用机理，在生理和分子水平上解释MIRK对Na+敏感和提高植物耐盐性之间的关系，进一步了解K+的转运在植物耐盐中所起的作用。并通过对比分析MIRK在不同甜瓜品种中的耐盐生理响应，了解MIRK在甜瓜耐盐中的的生理作用，为种质创新及培育耐盐、高效的园艺作物品种提供线索。

本项目从葫芦科作物黄瓜（Cucumis sativus L.）和西瓜(Citrullus lanatus)中分别分离一个K+通道基因CsKAT1和ClKAT1。利用双向电压钳进行电生理实验，结果表明，CsKAT1及ClKAT1具有典型的Shaker内向整流钾离子通道缓慢激活动力学特征。二者门控特征具电压依赖性，通道开放概率不依赖于外界钾离子浓度。通过对K+的亲和性及离子选择性分析，证明CsKAT1和ClKAT1是主要吸收K+的低亲和性通道。此外，CsKAT1和ClKAT1内向电流幅度随外界pH酸化而增强，其通道活性受外源Na+抑制，与甜瓜中的MIRK一致，该抑制效应随着外界K+浓度的降低而增强，且不受膜电压的影响。受Na+抑制的程度依次为ClKAT1 ≈ MIRK > CsKAT1，该次序不依赖于外界K+浓度。.利用拟南芥KAT2 不受胞外Na+抑制的特性，将其编码通道外口区(第5跨膜片段-P功能区-第6跨膜片段)替换到MIRK相应片段，得到△KAT2/MIRK突变体。通过电生理实验证实该突变体基因所编码的钾离子通道对内转运钾时不再受外源Na+抑制，并且具有MIRK基因转运钾离子的特征，明确了MIRK受外源钠离子抑制的作用位点位于S5-P-S6区域片段。.对盐胁迫下的kat2缺失型拟南芥和经MIRK或△KAT2/MIRK互补后的株系进行研究。结果表明，在150 mM 和300 mM NaCl处理下，kat2功能缺失型拟南芥和经MIRK或△KAT2/MIRK互补后的株系之间耐盐性有较大差异。离子含量检测发现，转MIRK或△KAT2/MIRK的kat2功能缺失型拟南芥含有较高的钾钠比。.通过两个耐盐差异性的甜瓜品种冰雪脆和玉露，研究MIRK转录水平，钾钠含量分布和气孔开度，对MIRK与甜瓜耐盐性进行相关性分析。结果表明，经盐处理后，冰雪脆生物量的减少小于玉露。在整体植株水平上，两品种中Na+含量相同，但在冰雪脆叶片上含有较少的Na+，有较高的K+/Na+比。盐处理后，两品种叶片上的MIRK表达量均下降，气孔开度均减少；比起玉露，冰雪脆中气孔开度和MIRK的表达量减少较小。此外，MIRK转录水平与气孔开度呈显著相关性。盐胁迫下，MIRK的表达量及活性的抑制减少了保卫细胞中钾离子的吸收，从而控制气孔开度的变化，进一步平衡蒸腾及CO2的进出，有利于植株适应外界盐胁迫。