ClO2介导的活性氧代谢对马铃薯块茎发芽的抑制机理研究

Sprouting is the important cause leading to the loss of potato tubers during storage. Our previous studies showed that the ClO2 treatment could effectively inhibit the sprout of potato tubers during storage, and it was also observed that the active oxygen content was significantly increased in the growth sites of tubers, and H2O2 was significantly increased, but more in-depth mechanism has to be revealed. This project is to "Longshu10" potato tubers as material, colligating physiology and biochemistry, cell biology and molecular biology and other disciplines methods, using fluorescent probe labeling, confocal laser scanning microscopy, RT-PCR and other technical means, systematically study on the effects of ClO2 to the content and chemical positioning of ROS, the activity of enzyme production and scavenging, the content of antioxidant and the degree of membrane lipid peroxidation, further analysis of the differential expression of key enzyme genes in ROS metabolism, To determine the inhibition effect of ClO2 mediated ROS metabolism to tissue cell structure and sprout of bud growth site and shoot tips of potato tuber, to reveal inhibition mechanism of ClO2 mediated ROS metabolic to tuber sprouting, the results of this study will enrich the inhibition sprout mechanism and provide scientific basis for the safe and efficient application of ClO2 in potato storage.

发芽是导致马铃薯块茎贮藏期间损失的重要原因，我们前期研究发现，杀菌消毒剂ClO2处理可有效抑制马铃薯块茎贮藏期间的发芽，同时，还观察到处理块茎芽生长部位的活性氧含量显著升高，H2O2明显积累。但深入的机制还有待揭示。本项目以“陇薯10号”马铃薯块茎为试材，综合生理生化、细胞生物学和分子生物学等多学科方法，采用荧光探针标记、激光共聚焦显微镜和RT-PCR等技术手段，通过系统研究ClO2处理对块茎ROS产生含量及组织化学定位、产生和清除相关酶活性、抗氧化物质含量和膜脂过氧化程度的影响，进一步分析ClO2处理后ROS代谢关键酶家族基因的差异表达，明确其介导的ROS代谢变化对块茎芽生长位点和芽尖组织细胞结构以及发芽的抑制作用，以期揭示ClO2介导后ROS代谢对块茎发芽的抑制机理。研究结果理论上将丰富马铃薯抑芽机理，实践上为ClO2在马铃薯贮藏中的安全高效应用提供科学依据。

发芽是马铃薯块茎的重要采后特性，也是导致马铃薯块茎贮藏期损失的重要原因。我们前期研究发现二氧化氯（ClO2）处理可以抑制马铃薯块茎的发芽，但其机理尚不清楚。本项目以‘大西洋’为试材，采用180mg/L ClO2处理马铃薯块茎，通过生理生化和组织学方法观察ClO2处理对块茎发芽效果的影响，分析活性氧（ROS）产生与清除相关酶及产物的变化规律，并利用转录组学和代谢组学分析ClO2介导的ROS代谢抑制马铃薯块茎发芽的分子机理，为揭示ClO2处理抑制马铃薯块茎发芽的作用机理提供理论依据。结果表明：1）ClO2处理有效推迟了马铃薯块茎发芽的时间，显著降低了块茎的发芽率和发芽指数；组织学观察发现，ClO2处理后的块茎芽生长锥受到抑制，芽原基部位呈现坏死。2）ClO2处理提高了马铃薯块茎芽眼处细胞膜透率和MDA的含量，破坏了细胞膜的完整性。3）ClO2处理提高了马铃薯块茎芽眼处NADPH氧化酶（NOX）和超氧化物歧化酶（SOD）的活性，促进了超氧阴离子（O2·−）和过氧化氢（H2O2）的积累。4）ClO2处理提高了马铃薯块茎芽眼处过氧化氢酶（CAT）和过氧化物酶（POD）的活性；降低了马铃薯块茎芽眼处抗坏血酸过氧化物酶（APX）、谷胱甘肽还原酶（GR）、单脱氢抗坏血酸还原酶（MDHAR）和脱氢抗坏血酸还原酶（DHAR）的活性以及抗坏血酸（AsA）和还原性谷胱甘肽（GSH）的含量。5）转录组测序显示，ClO2处理组和CK组之间共发现3228个差异表达基因，其中1725个上调表达，1503个下调表达；从差异表达基因中筛选到了113个与ROS产生和清除相关的基因，并对其中StNOX1、StSOD2、StAPX2、StDHAR1 等20个与ROS产生（7个）和清除 (13个）相关的基因进行了实时定量PCR表达分析验证。6）代谢组分析显示，ClO2处理组和CK组之间共筛选出85种差异代谢物，其中31种显著上调，54种显著下调。所筛选的差异代谢物共注释到20条差异显著的代谢途径，其中氧化应激代谢、谷胱甘肽代谢、脂肪代谢、辅酶A生物合成、能量代谢5条代谢途径受到显著干扰。综上所述，ClO2对马铃薯块茎发芽的抑制作用与其诱导ROS的积累密切相关，过量的ROS造成氧化胁迫，从而导致块茎芽生长发育代谢途径受阻，进而抑制生长，以此为马铃薯抑芽机理提供了新的见解。