蔗糖转化酶在机械损伤诱导的果蔬酚类物质合成中的作用机制研究

When mechanical injury occurs, fruits and vegetables usually synthesize and accumulate secondary metabolites like phenolic compounds, lignin and suberin, which has an adverse impact on its overall appearance, flavor, texture and nutrition. Previously, we found that wounding induced the decomposition of sucrose in fruits and vegetables into glucose and fructose, the increasing content of reducing sugar had a close relationship with the biosynthesis of phenolic compounds. In this study, based on the theory that wounding stress stimulated invertase and thus enhanced the cleavage of sucrose to supply raw materials for biosynthesis of phenolic compounds, the effect of different wounding intensities on the activity of invertase, the content of soluble sugar and the biosynthesis of phenolic compounds in carrot was evaluated. Furthermore, both inhibitor and accelerator of invertase were used to elucidate the interrelationship between the cleavage of sucrose and the biosynthesis of phenolic compounds in wounded carrot. In addition, the transcriptomics was used to screen the differently expressed genes (DEGs) encoding the invertase and the bioinformatics analysis of these DGGs was performed, and the acid invertase from whole and wounded carrot was separated and purified to analyze its variation on aecondary structure, tertiary structure and molecular morphology to reveal molecular mechanism of invertase on the wound-induced biosynthesis of phenolic compounds in fruits and vegetables. These results will help to provide new theoretical values for controlling secondary metabolic reactions and enhancing quality of fresh-cut fruits and vegetables.

果蔬在受到切割损伤后，常常会合成积累多酚类、木质素、木栓质等次生代谢产物，对鲜切产品的外观、风味、质地、营养产生不利影响。前期研究发现机械损伤能诱导果蔬蔗糖分解成为葡萄糖和果糖，还原糖含量的变化与酚类物质的生物合成密切相关。基于此，本项目以蔗糖转化酶响应损伤胁迫进而加速蔗糖分解从而为酚类物质合成提供原料为切入点，研究不同机械损伤强度对胡萝卜蔗糖转化酶活性、可溶性糖含量及酚类物质合成的影响；采用蔗糖转化酶抑制剂和促进剂分别处理损伤后的胡萝卜，阐明蔗糖分解过程与酚类物质合成的关系；通过转录组学方法筛选蔗糖转化酶差异表达基因并进行生物信息学分析，通过分离、纯化损伤前后胡萝卜酸性蔗糖转化酶并分析其二级结构、三级结构和分子形态变化，从转录水平和蛋白水平揭示蔗糖转化酶对机械损伤的响应机制。研究成果可为控制鲜切果蔬的次生代谢反应、提升鲜切果蔬的品质提供新的理论依据。

鲜切果蔬因其新鲜、方便、卫生和快捷等优点受到消费者的青睐，但当果蔬受到机械损伤后，常常会在伤口及邻近部位合成、积累多酚类物质，对鲜切产品的外观、风味、质地、营养产生不利影响。本项目以蔗糖转化酶响应损伤胁迫进而加速蔗糖分解从而为酚类物质合成提供原料为切入点，对蔗糖转化酶在机械损伤诱导的果蔬多酚类物质合成中的作用机制进行了研究，结果发现：（1）酸性蔗糖转化酶（AI）和中性蔗糖转化酶（NI）活性在切割后的贮藏期间均有明显上升，温度越高，切割强度越大，上升越明显。这也与总酚含量和抗氧化能力的变化趋势相一致，说明损伤后的胡萝卜蔗糖转化酶活性的变化与多酚类物质的合成呈正相关。（2）蔗糖转化酶抑制剂硝酸银（AgNO3）和碘代乙酰胺处理能显著降低鲜切胡萝卜AI和NI的活性，减缓了糖类物质的消耗，同时也抑制了总酚含量和抗氧化能力的上升；与之相反，蔗糖转化酶促进剂蔗糖转化酶、伴刀豆蛋白辅酶A（ConA）能显著提高鲜切胡萝卜AI和NI的活性，加速了糖类物质的消耗，同时也促进了总酚含量和抗氧化能力的上升。这些数据证实了蔗糖转化酶可以通过调控糖类物质的分解过程，从而调控鲜切果蔬多酚类物质的合成进程。（3）通过转录组学筛选到5个能快速响应伤信号的编码蔗糖转化酶合成的基因，通过功能比对，发现Loc108204362、Loc108218454、Loc108218455编码细胞壁蔗糖转化酶（CWIN），Loc108218494、Loc108222817编码细胞质蔗糖转化酶（CIN）。（4）对上述5个基因进行序列分析和蛋白结构预测，三级结构分析表明，CWIN-1（Loc108204362）和CWIN-3（Loc108218455）的空间结构类似，CIN-1（Loc108218494）和CIN-2（Loc108222817）的空间结构类似，推测其蛋白功能具有相似性。以上研究结果表明蔗糖转化酶在在机械损伤诱导的果蔬多酚类物质合成中发挥了重要作用，为控制鲜切果蔬的次生代谢反应、提升鲜切果蔬的品质提供了新的思路。