【作者】 金松恒；

【导师】 蒋德安；

【作者基本信息】 浙江大学 ， 植物学， 2006， 博士

【摘要】 Rubisco作为催化光合碳循环和光呼吸的第一步反应的酶，在调节光合速率中发挥关键作用，该酶需要进行氨甲酰化并与Mg²⁺结合才具有催化活力。目前公认的看法是该酶在体内的活化需要Rubisco活化酶（RCA）的促进，RCA通过释放Rubisco活化位点上的磷酸糖抑制物，促进Rubisco的氨甲酰化。为了进一步研究RCA在调节Rubisco和光合作用中的作用，我们通过反义RNA技术获得反义rca水稻，并研究RCA含量的降低对光合作用、能量耗散、Rubisco和RCA的细胞定位的影响，结果摘要如下： 利用pGEM-T easy、pBluescript KS+获得了RCA基因两端可以引入与双元表达载体pCAMBIA1301多克隆位点相匹配的单酶切位点，将该片段连接到pCAMBIA1301载体，构建了RCA基因的反义植物表达载体pCAMR02。通过电激法将该载体导入根癌农杆菌EHA105菌株，以根癌农杆菌介导的方法，将反义载体pCAMR02转化粳稻品种中花11，获得了抗潮霉素的再生植株，经过GUS组织化学检测和PCR鉴定，明确目的基因已经整合到这些转基因水稻基因组中。表型测定显示，有大部分反义植株在大气CO₂浓度下很难生长或不能生长，部分与野生型的长势基本相同，部分存活下来转化株生长缓慢。 研究表明，只有RCA含量低于野生型的35％的植株才能引起稳态光合速率的明显下降。因此，我们选择RCA含量为野生型15—30％的反义植株进行研究，这些反义水稻能在大气CO₂浓度下生长，但相对于野生型水稻，生长比较缓慢。气体交换特性测定发现与野生型水稻相比，反义植株的净光合速率（Pn）明显下降，但气孔导度（g_s）和蒸腾速率（E）不变，胞间CO₂浓度（C_i）明显高于野生型水稻，这说明反义水稻Pn的下降不是由于气孔导度引起的。另外，这些转基因植株的Rubisco初始活力和最大羧化速率(V_cmax)明显下降，但Rubisco含量和总活力却显著上升。因此，RCA含量的降低影响了Rubisco氨甲酰作用是光合速率下降的主要原因，而Rubisco含量的上升可能是对RCA含量下降，导致Rubisco氨甲酰作用下降引起酶活性降低的一种补偿反应。 反义rca水稻的叶肉导度大大低于野生型水稻，但是Rubisco羧化位点的CO₂浓度（C_c）却明显高于野生型水稻，表明其光合速率和V_cmax)的下降不是由更多还原

【Abstract】 Rubisco catalyzes the first step of both the photosynthetic carbon assimilation and photorespiratory carbon oxidation, and is a key regulatory enzyme of photosynthesis. The catalytic sites of Rubsico need to be activated by the carbamylation of a lysine residue with the sites follwed by binding of magnesium. The activation of Rubsico in vivo is regulated by Rubisco activase （RCA）. The enzyme is a nuclear-encoded chloroplast protein that modifies the conformation of Rubisco, releases inhibitors from the active sites, and increases enzymatic activity. In this study, we regenerated transgenic rice plants with an antisense gene for RCA, and investigated the effects of the reduction of RCA content on photosynthesis, energy dissipation and cellular localization of Rubisco and RCA of rice leaves. The results are as follows:RCA gene （rca） was amplified using specific primers and inserted into pGEM T-easy vector, and then cut with EcoRI after confirming by sequencing. The fragment was subcloned into pBluescript KS+, digested with the enzyme BamHI and inserted into the binary expression vector pCAMBIA1301, and the resulting construction with antisense rca was named pCAMR02. The pCAMR02 vector was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation and transformed to embryos of rice （Oryza. Sativa L.ssp.japonica） cultivar Zhonghuall via Agrobacterium tumefaciens system. Plantlets were regenerated in vitro by resistance selection on medium containing various concentrations of hygromycin. Both GUS histochemical assays and PCR amplification demonstrated that antisense rca was integrated into T₀ genomes and inherited to T₁. The measurement of phenotypes of transgenic rice plants with antisense rca showed that most of them could hardly survive at ambient CO₂ concentration even could not grow. The antisense plants that survived under natural condition were dwarf and grew slower than the wild-type controls.The photosynthetic rate was largely unaffected by RCA concentration until it更多还原