【作者】 杜淑媛；

【导师】 张大鹏；

【作者基本信息】 清华大学 ， 生物学， 2012， 博士

【摘要】 镁螯合酶是光合细菌和植物中普遍存在的一种特属于叶绿素合成的关键酶，能催化Mg2+和原卟啉IX形成镁卟啉IX。镁螯合酶由四个组分组成，分别是H亚基（CHLH/ABAR）、I亚基（CHLI）、D亚基（CHLD）和GUN4蛋白。在叶绿素生物合成过程中，这四个组分相互协调，共同调节镁螯合酶的活性。本实验室前期研究表明，H亚基是植物激素脱落酸受体。然而，目前还不清楚镁螯合酶的其它组分是否也参与ABA信号转导，且镁螯合酶催化合成镁卟啉IX与ABA信号转导之间是否有所区分。本论文通过生化、生理和遗传实验来揭示这一问题。本论文利用表面等离子共振技术（SPR），检测到只有镁螯合酶的CHLH/ABAR能够结合ABA，而其它三个组分CHLI、CHLD和GUN4不能结合ABA。目前报道了拟南芥CHLH基因的一个新的突变等位基因rtl1，本论文对其突变体的ABA相关表型分析表明，其在气孔运动和种子萌发中都表现出对ABA脱敏的表型，这与CHLH是ABA信号转导的正调节子的结论一致。通过遗传转化技术在拟南芥中过量表达CHLI1基因，突变体在种子萌发中表现出对ABA超敏的表型；而下调CHLI的植株在气孔运动方面表现出脱敏的表型，这说明CHLI在种子萌发和气孔运动中都正向调控ABA信号转导。利用烟草脆裂病毒所介导的病毒诱导基因沉默系统在烟草（Nicotiana benthamiana）中分别沉默CHLH、CHLI1和CHLD，结果显示CHLH和CHLI正向调控烟草气孔对ABA的敏感性，而CHLD并不参与其中。在拟南芥中，上调CHLD表达量的植株，以及上调和下调GUN4表达量的植株，在ABA主要的三个反应中都表现出与野生型一致的表型，这表明CHLD和GUN4并不参与ABA信号转导。以上结果证明镁螯合酶CHLH/ABAR和CHLI是ABA信号转导中重要的正调节因子，而另两个组分CHLD和GUN4不参与ABA信号转导。本论文的实验结果能够清晰而直接地证明镁螯合酶催化镁卟啉IX合成与ABA信号转导是相互独立的两个过程，也为在分子水平了解其机制提供了重要信息。更多还原

【Abstract】 In plants and photosynthetic bacteria, Mg-chelatase catalyzes the insertionof Mg2+into protoporphyrin IX. This is the first committed step in chlorophyllbiosynthesis. It has been well established that Mg-chelatase functions incatalyzing Mg-Proto IX production as a hetero-tetramer, which is composed ofMg-chelatase subunits H, I (CHLI), D (CHLD), and a supplementary essentialand component GUN4(Genomes Uncoupled4). We previously reported that themagnesium-protoporphyrin IX (Mg-Proto IX) chelatase large subunit(Mg-chelatase H subunit CHLH/putative ABA receptor ABAR), achloroplast/plastid protein, binds ABA and functions in ABA signaling.However, it remains essentially unknown whether Mg-chelatase heterotetramercomplex or only CHLH function in ABA signaling, and why the Mg-Proto IXproduction process may differ from the CHLH-mediated ABA signaling. In thethesis, we designed biochemical, physiological and genetic experiments toexplore the mechanism.Here we report that, using a newly-developed surface plasmon resonancesystem, ABA-binding to CHLH, but not to the other Mg-chelatasecomponents/subunits CHLI, CHLD and GUN4, was detected.A new rtl1mutant allele of the CHLH gene in Arabidopsis thaliana showedABA-insensitive phenotypes in both stomatal movement and seed germination,which is consistent with the positive role of CHLH in ABA signaling.Upregulation of CHLI1resulted in ABA hypersensitivity in seed germination,while downregulation of CHLI conferred ABA insensitivity in stomatal responsein Arabidopsis, suggesting that CHLI positively regulates the major ABAresponses. Using a tobacco rattle virus (TRV) based virus-induced genesilencing (VIGS) system, we showed that CHLH and CHLI, but not CHLD,regulate stomatal sensitivity to ABA in tobacco (Nicotiana benthamiana).Consistently, the overexpression lines of the CHLD gene showed wild-typeABA sensitivity in Arabidopsis. Both the GUN4-RNA interference andoverexpression lines of Arabidopsis showed wild-type phenotypes in the major ABA responses, demonstrating that GUN4is not an ABA signaling regulator.These findings provide clear and direct evidence that the Mg-chelatasecatalyzed Mg-Proto IX production is distinct from ABA signaling, givinginformation to understand the mechanism by which the two cellular processesdiffers at the molecular level.更多还原