液泡膜水通道蛋白CsTIP1;2和CsTIP1;3在茶树抗旱中的功能研究

Drought is one of the main environmental factors limiting tea plant growth and seriously affecting the yield and quality of tea. Tonoplast intrinsic proteins (TIPs) control water transport across vacuole membrane and thus contribute to plant resistance in response to dehydration. However, the function of TIPs in tea plant response to drought stress is yet to be revealed. In the previous study, we cloned 20 CsAQP genes from tea plant. Among of these genes, CsTIP1;2 and CsTIP1;3 showed close correlation with tea plant drought responses based on their expression patterns; they were differently expressed under stress treatment, these indicated that they might play different roles in tea plant response to drought; overexpression of CsTIP1;2 lines had higher dehydration tolerance than wild type in Arabidopsis. On this basis, we plan to comprehensively detect the expression profiles of CsTIP1;2 and CsTIP1;3 in different organs and cultivars respectively by qRT-PCR and in situ hybridization, clone their promoter sequences and analyze their spatial expression patterns, explore their transport activity using Xenopus oocyte and yeast expression systems, and investigate their function by Arabidopsis transformation. The results will be benefit for understanding the role and regulation mechanism of water transport in vacuole mediated by CsTIPs in response to drought stress in tea plant, and providing theoretical basis for the tea plant cultivation measures improvement and higher drought tolerance varieties development.

干旱是限制茶树正常生长，影响茶叶品质和产量的一个主要环境因子。TIPs是控制水分进出液泡的关键蛋白，在提高植物对干旱等逆境胁迫的抵御能力中发挥重要作用，但它们在茶树响应干旱中的功能还有待揭示。前期，我们克隆了20个茶树CsAQPs基因，从中发现CsTIP1;2和CsTIP1;3的表达与茶树抗旱响应密切相关，而且它们在响应干旱过程中有着不同的表达模式，在茶树抗旱中可能具有不同的作用；CsTIP1;2转基因拟南芥株系具有较强的抗旱能力。在此基础上，本项目拟采用qRT-PCR、原位杂交等方法分析这2个基因在组织、品种间的表达模式，克隆它们的启动子序列，研究其时空表达特异性，借助爪蟾卵母细胞和酵母系统解析它们的转运功能，发展转基因拟南芥株系揭示这2个基因在抗旱响应中的功能。阐明CsTIPs基因介导的水分跨液泡膜转运在茶树响应干旱胁迫中的功能及其调控机理，为改进茶树栽培措施及抗旱品种选育提供指导。

干旱胁迫严重限制茶树的正常生长，同时影响茶叶的品质和产量。水通道蛋白介导的水分转运在植物抗旱响应中发挥了重要的功能。本项目通过研究液泡膜水通道蛋白CsTIP1;2和CsTIP1;3在茶树干旱胁迫响应中的功能，为揭示茶树抗旱机制提供理论参考。克隆分析了CsTIP1;2和CsTIP1;3的基因组全长序列和启动子序列，发现CsTIP1;2不含ABA调控的响应元件而CsTIP1;3含有3个ABA响应元件，表明它们可能具有不同的抗旱响应模式。亚细胞定位分析显示，CsTIP1;2和CsTIP1;3均可能定位在液泡膜上。表达分析表明，CsTIP1;2和CsTIP1;3的表达均受干旱、低温和盐胁迫的诱导，CsTIP1;2不受ABA调节，而CsTIP1;3受ABA诱导；它们在不同抗旱性品种间的表达水平有一定差异，可以指导后续抗旱品种的早期筛选；其在夏秋季的表达水平较高，表明它们与茶树年生长周期密切相关。开展了转基因拟南芥株系筛选，将对它们在抗旱响应中的功能进行验证。本项目还挖掘了茶树中6个未知的水通道蛋白AQP基因，鉴定了60个与茶树抗旱响应密切相关的基因或基因家族，为后续研究茶树抗旱提供了充分的目标基因。同时我们还鉴定出与茶树抗旱密切相关的miRNAs，提出了干旱胁迫下茶树生理变化及其特异性miRNA的作用机制，并研究了茶树茸毛的发育调控机制，有利于揭示茶树茸毛等组织发育在茶树抗逆中的作用。本项目的完成有助于揭示茶树抗旱机理，为改进茶树栽培措施及抗旱品种选育提供指导。