特殊资源植物松叶猪毛菜NADP-苹果酸酶基因特性及其应对干旱胁迫的作用机理研究

Salsola laricifolia, a typical C3-C4 intermediate desert plant, is different from C3 and C4 plants in structures and functions. Previous studies showed that it can respond to drought stress by enhancing the C4 Photosynthetic pathway, but only the activity of NADP-malic enzyme (NADP-ME) was significantly increased under different degrees of drought stress, suggesting that the enzyme gene has an important role in response to drought stress. NADP-ME is a key enzyme in the malic metabolic pathway, which is encoded by a small gene family and has complex gene evolution. And the function of each member of the family to cope with drought stress and its role in the malic metabolic pathway is not clear. This study aims to study the characteristics of the NADP-ME genes and reveal its relationship between C3 and C4 plants by gene cloning and subcellualr localization; evaluate the drought-tolerance of NADP-ME genes through analyzing gene expression patten and its response under drought, and clarify its ability by testing the morphological and physiological traits of overexpression Arabdopsis; and study the role of NADP-ME genes in the malic metabolic pathway. The research will shed light on the characteristics of NADP-ME genes and its mechanism of drought stress in S. laricifolia, which provide strong evidence for the further study of the evolutionary rule of desert plants on adapting to drought environment.

松叶猪毛菜是典型的荒漠C3-C4中间型植物，具有区别于C3/C4植物特有的结构功能。前期研究表明它通过增强C4途径来应对干旱胁迫，但光合酶中仅NADP-苹果酸酶（NADP-ME）活性在不同程度干旱胁迫下均显著升高，暗示该酶基因在响应干旱胁迫中具有重要作用。NADP-ME是苹果酸代谢通路中的关键酶，由一个小的基因家族编码，进化关系复杂。目前家族各成员应对干旱胁迫的功能及其在苹果酸代谢通路中的作用仍不清楚。本研究旨在通过松叶猪毛菜NADP-ME基因的克隆和亚细胞定位观察，解析基因特性以及揭示它们同C3、C4植物之间的进化关系；通过分析NADP-ME基因表达模式以及响应干旱胁迫的规律，评价基因的耐旱性并利用转基因拟南芥体系验证抗旱功能；明晰NADP-ME基因在苹果酸代谢通路中的调控作用。最终阐述松叶猪毛菜NADP-ME应对干旱胁迫的作用机理，为深入研究荒漠植物适应干旱环境的进化规律提供有力证据。

松叶猪毛菜是典型的荒漠C3-C4中间型植物，具有区别于C3/C4植物特有的结构功能。前期研究表明它通过增强C4途径来应对干旱胁迫，但光合酶中仅NADP-苹果酸酶（NADP-ME）活性在不同程度干旱胁迫下均显著升高，暗示该酶基因在响应干旱胁迫中具有重要作用。本项目基于松叶猪毛菜的根、茎和叶混库建立的转录组数据，鉴定其NADP-ME基因家族共有3个成员，命名为Salchl-NADP-ME（Salchl），Salcyt-NADP-ME1和Salcyt-NADP-ME2。它们编码区序列长度分别为1941、1755和1758bp，编码646、584和585个氨基酸。通过拟南芥原生质体亚细胞定位研究，明确Salchl编码的蛋白定位于叶绿体，其余2个基因编码的蛋白均定位于细胞质。在转录水平上，它们均没有明显的组织特异性表达；在强光、干旱和盐胁迫下，松叶猪毛菜幼苗根和叶中仅Salchl的转录表达被这3种处理显著诱导。在此基础上将Salchl稳定转化至拟南芥进行功能验证。①过表达Salchl能显著提高转基因植株的光合能力。与WT相比，转基因植株的光系统和捕光色素相关基因的表达量、光合电子传递速率、实际光能转化效率以及叶绿素含量都有明显的增加；②对WT和转基因拟南芥植株进行强光、干旱和盐胁迫下的对比研究。种子萌发阶段，两者并无明显差异；与WT相比，幼苗阶段过表达Salchl能显著提高植株的根长、鲜重和侧根数目；在成苗阶段，上述胁迫下转基因植株的光能转化速率及电子传递能力均增加；并且转基因拟南芥的H2O2含量减少，脯氨酸含量增加，表明过表达Salchl能显著提高植株的光合和抗逆能力。盐和强光胁迫、干旱胁迫下转基因植株分别通过提高酶促抗氧化系统和非酶促抗氧化系统来减轻伤害。因此，本项目获得的多个方面证据均表明松叶猪毛菜Salchl-NADP-ME在抵御环境逆境中发挥作用。