柑橘突变体青瓯柑果实采后滞绿的机理研究

Staygreen mutants are important resources to investigate the mechanisms of chlorophyll degredation and senescences of mature fruit.Qingougan mandarin (Citrus suavissima Tanaka),a bud mutant of Ougan mandarine (Citrus suavissima Tanaka), is a rare economiclly important staygreen mutant in citrus family,which fruit flavedo remains green colour throught the ripening and storage phases.Preliminary results from the studies in the past three years demonstrated that Qingougan showed better storability than that of wild type Ourgan, moreover, Qingougan can not be degreened by exogenous ethylene, and the contents of total chlorophyll, chlorophyll a, chlorophyll b, chlorophyllide a, and pheophorbide a are remarkably higher than that in the wild type Ougan. All the accumulated components located at the upstream of pheophorbide a oxygenase (PAO) in high plant chlorophyll metabolic pathway,which indicated that PAO played an important role in this staygreen phenotype. When mature Qingougan fruit were treated by exogenous ethylene, and the expression profile was detected by Affymetrix GeneChip Citrus Genome Arrays (Affymetrix, Santa Clara, CA, USA), only PAO gene among the genes of chlorophyll metobolic pathway was differentially expressed, which demonstrated that PAO was one of the core genes in chlorophyll degredation and simultaneously response ethylene signal. Based on previous studies, the following work will be carryed out in present project. To illustrate the mechanisms of the staygreen and insensative to ethylene of Qingougan, combining with bioinformatics,the structures and functions,upstream regulation elements, expression mode as well as the posttranslation modification of PAO gene will be conducted. This research is important to both theories and practices in citrus postharvest industry.

滞绿突变体是研究成熟果实叶绿素降解和衰老调控机理的珍贵资源。青瓯柑是瓯柑的芽变，是迄今极少数具有重要经济价值的柑橘滞绿突变体，成熟及贮藏期间果皮均不转色。过去三年的研究表明，青瓯柑果实贮藏性能明显优于普通瓯柑，而且无法用外源乙烯对其脱绿，成熟果实黄皮层总叶绿素、叶绿素a、叶绿素b、叶绿素酯a、脱镁叶绿甲酯酸a等成分均极显著高于普通瓯柑，这些组分均位于叶绿素降解关键酶脱镁叶绿甲酯酸a加氧酶（PAO）的上游，推测该酶与青瓯柑滞绿有关。表达谱芯片分析发现，外源乙烯处理后与叶绿素代谢相关的酶中仅有PAO基因显著差异表达，表明PAO是青瓯柑果皮叶绿素代谢途径中响应乙烯信号的关键基因之一。本项目拟在前期研究基础上，结合生物信息学分析，从基因结构与功能、上游调控元件、基因表达模式及蛋白修饰等水平解析PAO对青瓯柑果皮滞绿及对外源乙烯不敏感的机理。本研究对指导柑橘贮藏保鲜具有重要的理论及实践意义。

果面色泽是构成鲜食柑橘商品性能的重要组成部分，但柑橘果面着色与调控的生物学基础研究还不多。本研究以瓯柑和青瓯柑为材料，对比研究了柑橘果实成熟期果面色泽的变化规律，从形态、生理和分子水平开展了系统的研究工作，取得以下主发进展：1.青瓯柑滞绿表型是由于叶绿素降解延缓及类胡萝卜素合成及转化受阻而导致。从花后210 d起， 青瓯柑外表皮叶绿素 a、叶绿素 b、脱植基叶绿素 a、脱镁叶绿素 a均显著高于瓯柑外表皮。在瓯柑果实成熟期，其果皮类胡萝卜素以叶黄素类为主而青瓯柑的色素组成是典型的叶绿体类型的色素组成，即还含有较高的α-胡萝卜素和β-胡萝卜素。作为类胡萝卜素下游产物，相比瓯柑，ABA在青瓯柑外果皮中积累明显偏少。2.通过数字基因表达谱分析发现叶绿素合成降解和类胡萝卜素及ABA代谢相关基因表达差异明显。转录组数据表明滞绿蛋白（CcSGR1）、叶绿素b还原酶（CcNYC1）、脱镁叶绿素脱镁叶绿（甲酯-）酸水解酶（CcPPH）及类胡萝卜素合成的控速酶编码基因（CcPSY）、类胡萝卜素裂解酶（CcCCD4b）和9-顺式紫黄质单加氧酶（CcNCED5）等蛋白的编码基因在花后210 d及采后30 d的青瓯柑有色层中均下调表达，并发现CcMYB68等转录因子在青瓯柑中持续上调表达。3. CcMYB68转录因子潜在转录调控分析。通过PCR-Assisted DNA Binding Site Selection、EMSA和双荧光素酶等实验证明CcMYB68能直接结合到CcBCH2和CcNCED5的启动子上并能显著抑制其转录活性。本研究对揭示植物类胡萝卜素生物合成及柑橘果实着色机制等方面均获得了新的认识，具有重要的理论意义和潜在的实践价值。在本项目资助下，本课题组已在Plant Physiology，Journal of Experimental Botany等国内外学术期刊发表11篇论文。