桃树根系氰化物代谢缓解连作障碍的分子机制

Replant problem is a major obstacle limiting the sustainable development for peach industry. For perennial Prunus species, replant problem occurs frequently and is mostly caused by autotoxicity. On peach, previous studies proved the toxic cyanide was the leading autotoxic substance, and it was accumulated by the catabolism of cyanogenic glycosides deposited in root residuals. Therefore, it would be a great potential method to alleviate peach replant problem through boosting plants own cyanide metabolism. Evolutionally, to protect themselves against cyanide poison, plants resort to two pathways, the cyanide detoxification pathway or the cyanide assimilation pathway. Based on the two canonical pathways and the related key genes, we intend to improve peach replant problem by modulating the cyanide metabolism at molecular levels. First of all, we will analyze the key enzymes of rhodanese (detoxification) and β-cyanoalanine synthase (assimilation) on peach [Prunus persica (L.) Batsch] seedlings to identify the main cyanide metabolism pathway in roots. Subsequently, the corresponding key genes in the pathway will be retrieved from peach genome and characterized by in vitro enzymatic assay, Arabidopsis mutant complementation and bioinformatical analysis. Afterwards, the characterized key genes will be overexpressed and transformed into Prunus domestica (plum), to study the regulation of cyanide metabolism in planta. A comprehensive evaluation of seedlings growth will be conducted on overexpressed transformants, which are either grown in replant soil, or exogenously treated with cyanide or root extracts solution. Meanwhile, the metabolites levels of cyanide, cyanogenic glycosides and amino acids will be analyzed to elucidate the possible mechanism for the enhanced stress tolerance in overexpressed transgenic plants. Taken together, our work aims to improve the rate of survival after transplant and seedlings growth performance by enhancing the cyanide metabolism, and expectantly the output of the research would potentially provide practical significance for increased peach production.

连作障碍是影响桃产业健康发展的重要问题。自毒作用是引起李属果树连作障碍的主要原因之一，已有研究表明根系残茬中的生氰糖苷降解后生成的氰化物是导致桃连作障碍的主要自毒物质。因此，提高桃树自身代谢氰化物的能力是缓解连作障碍的重要潜在手段。植物在长期进化过程中形成了解毒和同化两条主要途径避免植物遭受氰化物毒害。本项目基于植物体内氰化物的代谢途径及其关键基因，以普通毛桃实生苗和超表达转基因“欧洲李”为材料，探讨氰化物代谢的调控对缓解桃树连作障碍的作用。通过酶活性检测确定桃树氰化物代谢的主要通路。利用转基因技术超表达关键基因，综合评价转基因植物在外源氰化物、连作土和根系浸提液等处理下的生长状况，同时检测转基因植株体内氰化物、生氰糖苷和氨基酸等代谢物的变化并分析其抗性机制。本研究旨在通过增强植物自身代谢氰化物的能力，提高连作条件下桃幼苗定植成活率和促进幼苗生长，以期为桃生产实践提供理论依据。

连作障碍是影响桃产业健康发展的重要产业问题。自毒作用是引起李属果树连作障碍的主要原因之一，已有研究表明根系残茬中的生氰糖苷降解后生成的氰化物是导致桃连作障碍的主要自毒物质。本研究分析了桃树根系及土壤中氰化物的含量，发现CN-主要存在于桃树根系的韧皮部中，且根际土壤中CN-的含量要显著高于根围土，并且随着时间的推移，土壤中CN-会不断增加。通过外源氰化物处理，明确了桃中氰化物代谢的主要途径为β-氰丙氨酸合成途径。研究克隆了关键基因PpCAS1，亚细胞定位分析表明PpCAS1定位于线粒体。在烟草中超表达PpCAS1后显著提高了烟草的抗氰胁迫能力，PpCAS1转基因植株中活性氧积累量更低。同时发现外源NaCl处理后，PpCAS1转基因植株活性氧清除能力增强，SOS途径相关基因表达上调，植株抗盐性显著增加。将成年桃树树皮进行腐解，利用获得的根皮腐解液模拟田间桃树连作障碍发生条件，发现转基因植株对其有较强的抗性，体内活性氧水平较低。项目的开展确定了桃树体内氰化物的主要代谢途径，并鉴定了关键基因的抗性作用及机制，为桃树连作障碍的防治提供了新的思路和研究方法。