腺苷酸激酶差异表达对茭白采后衰老的作用机制研究

Postharvest senescence is the most important factor that cause fast quality deterioration of Zizania latifolia. The understanding of the processes leading to postharvest senescence of Z. latifolia is essential in enhancing commercial value and extending shelf-life of the vegetable. Our previous study found that the glycosyl hydrolases family 17, transketolase, adenosine kinase, cysteine protease and papain-like cysteine proteinase were up-regulated while the enolase, Isocitrate dehydrogenase (NADP+) and diphosphonucleotide phosphatase 1 were down-regulated during postharvest senescence of Z. latifolia. And studies have shown that the release of adenosine kinase from mitochondrial intermembrane space into the cytosol in the process of programmed cell death is a very common phenomenon, which have closely connection with programmed cell death. Therefore, we speculate that the adenosine kinase is probably the key enzyme for postharvest senescence of Z. latifolia, but the mechanism is still unclear. On the basis of previous researches, the relationship between differential expression of adenosine kinase and postharvest senescence of Z. latifolia will be studied by using a combination of two-dimensional fluorescence difference gel electrophoresis (DIGE), MALDI-TOF/TOF, western blotting and molecular biology technology. This study will demonstrate our hypothesis as mentioned before and reveal the mechanism of adenosine kinase differential expression in postharvest senescence of Z. latifolia, and provide a theoretical basis to explore new techniques for storage and transport of postharvest of Z. latifolia.

衰老是导致茭白采后品质快速劣变的重要因素，研究茭白采后衰老机制对提高其商品价值、延长货架寿命具有重要意义。前期研究发现，茭白采后衰老期间糖基水解酶家族17、转酮醇酶、腺苷酸激酶、半胱氨酸蛋白酶和木瓜半胱氨酸蛋白酶上调表达，烯醇化酶、异柠檬酸脱氢酶(NADP+)和二磷酸核苷酸磷酸酶1下调表达。而有研究表明，腺苷酸激酶从线粒体膜间释放到胞浆在细胞程序性死亡过程中是一种非常普遍的现象，因而与细胞程序性死亡有着密切的联系。基于此，我们推测腺苷酸激酶可能是导致茭白采后衰老的关键酶，但作用机制尚不明确。本项目在前期工作的基础上，采用荧光差异双向电泳、质谱鉴定、蛋白质印迹分析和分子生物学相关技术，研究腺苷酸激酶差异表达与茭白采后衰老的关系，从而论证我们的假说，揭示腺苷酸激酶差异表达对茭白采后衰老的作用机制。该研究将深入揭示茭白采后衰老机理，为茭白采后贮运保鲜新方法的建立提供理论依据。

为探明茭白采后衰老的分子生物学机制，应用生理生化检测、电子显微镜观察、双向电泳(2-DE)、同位素标记相对和绝对定量(iTRAQ)标记结合二维液相色谱-串联质谱(2D-LC-MS/MS)技术、平行反应监测(PRM)技术和分子生物学相关技术，研究了茭白采后衰老过程中生理生化、细胞超微结构、线粒体蛋白质表达谱变化规律及其与衰老的关系。研究结果如下：⑴茭白采后常温贮藏期间色差L值、a*值和b*值均逐渐上升，呼吸强度逐渐下降，琥珀酸脱氢酶(SDH)和过氧化物酶(POD)活性显著上升而细胞色素c氧化酶(CCO)活性下降，贮藏6 d后细胞壁有降解现象，线粒体发生衰变、清晰度下降且数量减少，1-甲基环丙烯(1-MCP)处理对茭白上述衰老特征有显著抑制作用，表明茭白采后衰老与呼吸代谢及其伴随的能量代谢、活性氧代谢、细胞结构降解密切相关。⑵蛋白质组学研究进一步证明自由基损伤加剧、碳水化合物和核酸分解代谢加速、能量代谢平衡失调及细胞结构降解可能是导致茭白采后衰老的重要原因。⑶采用3000×g~14000×g差速离心联合20%：24%：40%=2：4：2 percoll不连续密度梯度离心可获得纯度较高的茭白线粒体。应用iTRAQ标记结合2D-LC-MS/MS技术对纯化后的茭白线粒体进行分析，共鉴定到Peptide ≥ 2的可信蛋白数目1908个，与贮藏第0 d相比，对照、1-MCP和ET处理茭白贮藏3 d和6 d后，共有315个蛋白具有2.0倍以上统计学显著差异(P < 0.05)。PRM监测结果与iTRAQ检测结果基本一致。基因本体(GO)分子功能分析和KEGG Pathway分析显示，茭白采后碳水化合物水解加速，磷酸戊糖途径加强而糖酵解途径和氧化磷酸化减弱，导致能量合成减少同时形成氧化胁迫，通过激活CaMK/MAPK、Ca2+/PKC、细胞色素c和茉莉酸(JA)等信号途径，造成初级代谢紊乱和次级代谢产物(如木质素)积累，从而促进细胞凋亡或细胞坏死，最终加速衰老。本项目研究结果为探明茭白采后衰老分子机理提供了直接证据，为茭白采后品质劣变控制提供了理论依据。