甘薯交替氧化酶(AOX)对采后低温胁迫响应及其与耐冷性的关系

Alternating oxidase (AOX) play an important role in plants' resistance to cold stress. We found by transcriptome analysis that the AOX gene was up-regulated in response to low-temperature conditioning in sweetpotato root, suggesting that AOX is involved in the regulation of low-temperature adaptive metabolism. This project will screen high-cold sweet potato varieties, clone sweet potato AOX gene family members and analyze the relationship between cold tolerance and AOX gene differences. We will explore the expression pattern of sweetpotato AOX gene to low temperature and cold resistance pathway by exogenous physiological regulation, transcriptome sequencing and qPCR techniques, and screen key genes of sweetpotato AOX gene family. The RNAi and overexpression vectors for AOX will be constructed and transformed into tobacco and sweetpotato by Agrobacterium to obtain transgenic plants and verify the cold resistance function of AOX. The degree of chilling injury of transgenic sweetpotato roots at low temperature was investigated. The effects of AOX gene on the chilling injury, active oxygen metabolism, energy metabolism, antioxidant metabolism and other metabolic pathways of sweetpotato were compared at gene expression and protein level. The results of this project further validated and confirmed the relationship between AOX and cold tolerance of postharvest sweetpotato, providing a new approach for preventing chilling injury of postharvest sweetpotato toot and breeding cold-tolerant sweetpotato varieties through regulating AOX gene.

交替氧化酶(AOX)在植物抵抗低温胁迫中发挥重要的作用。我们通过转录组分析发现AOX基因在采后甘薯冷锻炼过程中具有应答性的表达上调，暗示AOX参与调控甘薯块根的低温适应性代谢。本项目将选择高耐冷性甘薯品种，克隆甘薯AOX基因家族成员并分析耐冷性与AOX基因差异的关系。通过外源生理调控、转录组测序和qPCR技术分析甘薯AOX基因对低温胁迫的应答模式及抗冷代谢通路，确定甘薯AOX基因家族响应低温胁迫的关键基因。构建甘薯AOX的RNAi和过表达植物表达载体，并通过农杆菌将其转入烟草和甘薯获得转基因植株来验证AOX的抗冷功能。考察转基因甘薯块根在低温下的冷害发生程度，在基因表达和蛋白水平比较调控AOX基因对甘薯冷害、渗透调节物、活性氧代谢、能量代谢和抗氧化等代谢通路的影响。本项目研究结果进一步验证和明确AOX与甘薯采后耐冷性的关系，为通过调控AOX基因预防甘薯采后冷害和选育耐冷甘薯品种提供新途径。

项目的背景 低温是制约甘薯采后安全贮藏的关键要因素之一，我国每年约有30%的甘薯在贮藏过程中由于受到低温胁迫而导致利用率下降，甚至失去利用价值，所以研究甘薯对低温胁迫的响应及调控机制意义重大。.主要研究内容 本项目筛选高耐冷性甘薯品种，克隆甘薯AOX基因家族成员并分析耐冷性与AOX基因的关系。通过外源生理调控、转录组分析和qPCR技术分析甘薯AOX基因对低温胁迫的应答模式及抗冷代谢通路，确定甘薯AOX基因家族响应低温胁迫的关键基因。构建了甘薯AOX的RNAi和过表达植物表达载体，并通过农杆菌将其转入甘薯获得转基因植株来验证了AOX的抗冷功能。考察转基因甘薯块根在低温下的冷害发生程度，在基因表达和蛋白水平比较调控AOX基因对甘薯冷害、渗透调节物、活性氧代谢、能量代谢和抗氧化等代谢通路的影响。.重要结果 筛选了“徐32”、“广87”和“心香”三个抗冷性较好品种，为后续抗冷生理调控和基因筛选提供实验材料，能为生产上针对性的选择耐冷甘薯品种提供参考；克隆了IbAOX1a、IbAOX1b和IbAOX2IbAOX1a三个AOX家族成员基因，通过不同组织、低温、干旱和盐胁迫的表达水平确定IbAOX1a可以作为甘薯响应低温胁迫的关键 AOX 成员基因；通过IbAOX1a过表达和 RNAi 沉默转基因以及IbAOX1a基因瞬时沉默证明了IbAOX1a在甘薯抗冷性中的作用，为后续抗冷品种选育提供了靶点；黄体酮（PROG）有效地提高了IbAOX1的转录水平和AOX酶活性，抑制了甘薯冷害的形成，降低了细胞膜透性、丙二醛含量和活性氧产生，PROG还通过维持较高的抗氧化酶活性和抗坏血酸和总酚类物质含量来增强抗氧化保护系统；低温胁迫下IbAOX1a和IbAOX1b表达量迅速增加，而IbAOX2基本无响应，冷锻炼（LTC）处理可以显著提高AOX蛋白的表达，降低冷害指数，抑制了相对电导率、丙二醛、超氧阴离子产生速率及过氧化氢含量，提高了抗氧化酶（SOD、CAT、APX、GR）活性，增加了游离脯氨酸含量，还原型谷胱甘肽含量及ATP含量，保持较高的还原型辅酶的含量。经过SHAM处理后反转了LTC的积极影响。因此，AOX参与了冷锻炼提高甘薯耐冷性。.科学意义 .本项目明确了甘薯AOX基因在冷锻炼、短时低温处理改善甘薯品质和耐冷性中功能和作用机理，能够用来指导甘薯采后贮藏和加工中改善甘薯食用品质。