竹子叶黄素循环分子调节机制研究

In long-term evolutionary process, higher plants have developed effective photoprotection mechanisms to adapt the varying light conditions. An important protective mechanism that plants use to deal with excessive radiant energy by non-photochemical fluorescence quenching is known as xanthophyll cycle, which involves the interconversions between the carotenoids violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z) catalyzed by violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP). Bamboo is one of the important forest resources distributed widely in tropical and subtropical regions of the world, with a wealth of species and ecological types. However, its photoprotective mechanisms are still blank. In this project, the cDNAs encoding VDE and ZEP will be isolated, and the expression profiles of VDE and ZEP will be carried out using real time PCR and Western blot, respectively. The overexpression vectors of VDE and ZEP will be constructed and transformed into mutations of Arabidopsis respectively to make functional complementation. The constituent variation rule of xanthophyll cycle in the leaves of bamboo and trangenics under different light treatments will be detected by using pigment analysis with high performance liquid chromatography, the chlorophyll fluorescence kinetics parameters and electrical impedance spectroscopy parameters of the leaves will be measured respectively. The genes will be heterologously expressed in Escherichia coli respectively and the biological activity of recombinant proteins will be identified. Comprehensive analysis will be carried out to reveal the internal relationship between the physiological & biochemical characteristics and the molecular regulation mechanism of xanthophyll cycle in bamboo, which will not only has important theoretical value for enriching the light protection mechanisms of plants but also has practical guiding significance for stress tolerance of bamboo breeding.

高等植物在长期进化中形成了一套完善的光保护机制，其中依赖于叶黄素循环的非光化学荧光淬灭是植物应对光胁迫的重要保护机制之一，该循环涉及紫黄质脱环氧化酶和玉米黄质环氧化酶催化的叶黄素循环组分之间的转化。竹子是重要的森林资源，广泛分布于热带、亚热带地区，具有丰富的种类和生态类型，但对其光保护机制的研究尚属空白。本研究从叶黄素循环入手，分离竹子紫黄质脱环氧化酶基因和玉米黄质环氧化酶基因，利用实时定量PCR、Western blot技术研究基因的表达模式；构建基因过量表达载体，转化拟南芥突变体，进行功能互补实验；测定不同光照条件下竹子和转基因植株叶片中叶黄素循环组分的变化规律、叶绿素荧光动力学参数和电阻抗图谱参数；通过基因在大肠杆菌中表达获得重组蛋白，鉴定其生物活性。综合分析，揭示竹子叶黄素循环分子调节与生理生化之间的内在机制，不但有助于丰富光保护机制理论，而且对指导竹子抗逆育种具有重要的现实意义。

竹子广泛分布于热带和亚热带地区，其生长的自然光照环境复杂多变。为揭示竹子适应强光环境的光保护机制，本课题对竹子依赖于叶黄素循环的光保护分子机制开展了研究。研究内容主要包括不同竹子的叶绿素荧光参数特征，黄素循环关键酶基因的克隆、表达特征以及功能鉴定。结果表明，毛竹枝条上的第三片叶是最具代表性的叶片，能够较好的反映竹子叶片的实际光化学效率Y(Ⅱ)和PSⅡ反应中心的关闭程度；Fv/Fm是反映植物是否受到光抑制的重要指标，通测定Fv/Fm表明黄秆金竹和菲黄竹比金镶玉竹更适合在北京地区生长。克隆了毛竹紫黄质脱环氧化酶(VDE)基因、玉米黄质环氧化酶(ZEP)基因的cDNA全长，PeVDE为1690 bp (JQ347804)，编码451个氨基酸。Western blot分析和半定量分析表明，PeVDE均在叶片中的表达丰度最高，叶鞘次之，茎中最低，而根中没有检测到表达。在大肠杆菌中表达PeVDE，获得了分子量约为65 kDa的可溶性重组蛋白，以紫黄质为反应底物进行重组蛋白的体外酶促反应，HPLC检测结果表明重组蛋白能够将紫黄质单脱环氧化依次形成花药黄质和玉米黄质。过量表达PeVDE转基因植株比野生型拟南芥(Col-0)表现出较高的非光化学淬灭(NPQ)能力，另外PeVDE可以恢复拟南芥突变体npq1的NPQ能力，减少强光下拟南芥PSⅡ的光抑制。PeZEP为2532 bp (KP274885)，编码670个氨基酸。PeZEP基因主要在毛竹的叶片和叶鞘中表达，在根和茎中也检测到了少量表达。PeZEP的表达量随着光照强度升高而持续上升。在强光(1200 μmol·m-2·s-1) 条件下，其表达量随处理时间持续上升，在1 h表达量达到最高，随后维持在较高的水平。与野生型拟南芥(Col-0)植株相比，过表达PeZEP转基因植株的抗干旱能力提高。另外，过量表达PePsbS1和PePsbS2可以提高野生型拟南芥的NPQ，减少拟南芥光抑制程度，提高光保护能力。总之，在高温、强光环境条件下，依赖于叶黄素循环的非光化学荧光猝灭在竹子光保护中发挥着重要作用。