橡胶树干旱胁迫相关水通道蛋白基因的筛选与鉴定

Natural rubber (cis-1,4-polyisoprene) is an important industrial raw material with unique physical properties superior to petroleum-derived synthetic rubber. Among over 2,000 polyisoprene-producing plant species, brazilian rubber tree (Hevea brasiliensis Muell. Arg.) is the only commercial source of natural rubber for its high production and quality of rubber. As a tropical rainforest plant native to Amazon basin, the rubber tree favors the high temperature and high humidity environment. However, trees transplanted in China are often suffered from dry periods, which severely affect the crop growth and latex production. Aquaporins (AQPs), a class of integral membrane proteins that facilitate the passive transport of water, are closely related to plant tolerance to drought stress. In our previous studies, 51 full-length AQP genes were identified from the rubber tree genome. To learn more about their roles in drought stress, in this project, a combination of physiological, biochemical and molecular biology research means are carrying out to identify the AQP genes involved in drought stress. The relationship between gene expression pattern and the drought resistance ability of different varieties is to be investigated using the qRT-PCR technique, and the water transport activity of AQP genes is to be tested with their heterologous expression in Xenopus laevis oocytes. Moreover, the application potential in plant breeding for stress tolerance is to be tested with their overexpression in Arabidopsis thaliana. This study promises to not only improve our knowledge on the molecular response of tropical tree species under drought stress, but also lay a basis for molecular breeding and the development of new drought resistant cultivation technology in Hevea.

天然橡胶是一种重要的工业原料和战略物资，其主要来源是巴西橡胶树。生产上，橡胶（顺-聚异戊二烯）通过用胶刀周期性地切割树皮收集胶乳而获得。与原产地（亚马逊河流域）高温高湿的优良环境相比，我国植胶区地处热带北缘，季节性干旱成为影响橡胶树生长与胶乳产量的关键因素。水通道蛋白（AQP）是一类高效转运水分子的膜内在蛋白。作为细胞间水分跨膜运输的主要通道，AQP与植物的抗旱能力密切相关。本项目基于前期从橡胶树基因组中鉴定的51个AQP基因，通过生理生化与分子生物学相结合的研究手段筛选干旱胁迫相关的AQP基因，研究其表达特性与品系抗旱能力的关系，运用蟾蜍卵母细胞鉴定其水分转运活性，并通过转基因技术探讨其在植物抗旱育种中的可能应用。项目的研究不仅可增进我们对热带木本植物响应干旱胁迫的分子认知，同时还可为抗旱分子育种和橡胶树新型抗旱栽培技术的研发提供理论基础和技术支撑。

我国植胶区地处热带北缘，季节性干旱是影响橡胶树生长与胶乳产量的关键因素。鉴于水通道蛋白（AQP）在植物抗旱中的关键作用，本项目系统分析了橡胶树AQP家族基因的进化特征和表达特性，并对干旱胁迫相关HbAQP基因进行了筛选，深入研究了其水分转运活性以及在抗旱分子育种中的潜力。比较基因组学分析显示，HbAQP基因随ρ全基因组重复而发生了明显的扩张，但其进化模式与共享ρ重复的木薯存在较大的差异；表达模式的分化可能是橡胶树旁系同源基因功能分化的主要原因。基于基因在不同组织、不同发育时期叶片以及响应低温和干旱胁迫的表达模式，研究筛选获得11个干旱胁迫相关的HbAQP基因，其中，HbPIP2;9同时受干旱和低温诱导。序列分析表明，HbPIP2;9编码区长843 bp，编码280个氨基酸，理论分子量和pI分别为29.84 kDa和6.51，含有两个保守的NPA基序以及利于水分转运的F-H-T-R芳香/精氨酸选择性滤孔（ar/R filter）和M-S-A-F-W Froger位点；与其表达模式一致的是，HbPIP2;9 1601 bp的启动子序列含有多个参与干旱（MBS）和低温（LTR）响应的顺式作用元件。体外分析显示，HbPIP2;9在蟾蜍卵母细胞中具有高效的水分转运活性，这与序列分析结果一致。进一步的转基因研究表明，与野生型拟南芥相比，过表达HbPIP2;9的T3代转基因纯合子的抗旱能力显著增强，显示其在植物抗旱育种中的应用潜力。本研究不仅为橡胶树和其他植物的抗旱分子育种提供了新的功能基因，并为进一步揭示橡胶树响应干旱的分子机制奠定了基础。