H2O2调控嫁接黄瓜耐盐性的信号途径研究

H2O2 has been demonstrated as a stress signal in plants in recent years, which can induce expression of many stress tolerance genes and enhance plant tolerance to environmental stresses. We found that salt tolerance of grafted cucumber was mediated by H2O2 signal in previous studies. However, the regulated signal pathway was unclear. Using self- grafted and pumpkin- grafted cucumber as materials, the plants with different H2O2 levels was obtained by chemical inhibitor and exogenous H2O2 application. The H2O2 content of grafted cucumber and role of NADPH oxidase in the production of H2O2 were analyzed through histochemical staining and qRT-PCR techniques. The root Na+ exclusion and antioxidant system of grafted cucumber under salt stress were analyzed. Meanwhile, the MAPK, Ca2+ and ABA signalling pathway of grafted cucumber were analyzed, and were analyzed with salt tolerance related physiological and biochemical parameters. The important signalling pathway and genes involved in H2O2 signalling mediated salt tolerance can be obtained. Based on this evidence, the key signalling pathway and genes involved in H2O2 signalling mediated salt tolerance of grafted cucumber will be confirmed by pharmacology and transgenic approaches. The implementation of this project will provide signal transduction mechanism for the H2O2 mediated salt tolerance of cucumber and enrich the knowledge of rootstock-scion interaction of horticultural crops and plant salt tolerance mechanism, and will promote the cultivation of grafted cucumber under salt stress.

近年来的研究表明，H2O2具有信号分子的作用，通过诱导许多抗逆相关基因表达，以提高植物对环境胁迫的抗性。课题组前期研究发现嫁接黄瓜耐盐性受H2O2信号的调控，但是具体机制尚不明晰。本申请项目拟以自嫁黄瓜和南瓜砧木嫁接黄瓜为材料，通过化学抑制剂以及外施H2O2来构建不同H2O2水平植株。利用组织化学染色、qRT-PCR等技术，分析嫁接黄瓜H2O2含量以及NADPH氧化酶在H2O2产生中的作用；研究嫁接黄瓜H2O2信号相关的MAPK、Ca2+、ABA信号途径及基因表达的变化，与抗氧化和Na+外排等耐盐生理生化参数进行相关分析，获得重要的H2O2信号途径和基因。在此基础上，利用药理学实验、以及转基因技术等，鉴定出H2O2调控嫁接黄瓜耐盐性的关键信号作用途径和基因。本课题完成后可揭示H2O2调控嫁接黄瓜耐盐性的信号转导机制，丰富园艺作物砧穗互作的基本理论和植物的耐盐机理，促进嫁接黄瓜的耐盐栽培。

H2O2是一种重要的信号分子，通过诱导抗逆相关基因表达，提高植物对环境胁迫的抗性。项目组前期研究发现嫁接黄瓜耐盐性受H2O2信号的调控，但是具体机制尚不明晰。本项目以自嫁黄瓜和南瓜砧木嫁接黄瓜为材料，发现盐胁迫（75 mM NaCl）早期（3h）南瓜砧木嫁接黄瓜根系和叶片H2O2含量（信号）快速增加，增加幅度显著高于自嫁黄瓜。利用药理学和非损伤微测等技术，发现依赖于南瓜砧木嫁接黄瓜根系NADPH氧化酶产生的H2O2信号通过激活下游质膜H+-ATPase基因（PMA）表达和酶活性，调控Na+/H+反转运蛋白，进而在Na+外排和耐盐性中发挥作用。盐胁迫下南瓜砧木根源H2O2信号还通过影响地上部黄瓜ABA的合成和敏感性、气孔早期关闭、抗氧化酶活性等生理过程，来调控黄瓜的耐盐性。利用钙通道抑制剂证明Ca2+信号在嫁接南瓜耐盐过程中发挥了重要作用。本项目还利用南瓜和黄瓜自根苗为研究对象，发现南瓜根系依赖于NADPH氧化酶产生的H2O2信号通过调控HAK5的表达和K吸收，在耐盐性中发挥作用，分析了南瓜编码NADPH氧化酶的Rboh基因在盐胁迫下的表达，结合拟南芥异源超量表达结果，发现盐胁迫下RbohD的高量表达是南瓜砧木根系H2O2信号快速产生的重要原因。本项目的完成对于揭示H2O2调控嫁接黄瓜耐盐性的信号转导机制，丰富园艺作物砧穗互作的基本理论和植物的耐盐机理，促进嫁接黄瓜的耐盐栽培均具有重要意义。项目执行期间发表SCI 论文4 篇，其中3篇Journal of Experimental Botany，1篇Frontiers in Plant Science论文，培养博士毕业生1人，硕士毕业生3人。项目主持人别之龙教授牵头申报的“设施蔬菜生长发育调控团队” 2019年获批为湖北省自然科学基金创新群体项目。