砧用南瓜CmHKT1基因功能分析及其在嫁接黄瓜耐盐中的分子机理研究

In our previous research work, we have found that the utilization of pumpkin as a rootstock improves salt tolerance of grafted cucumber, owing to pumpkin roots have strong Na+ accumulation and unloading capacity. Recently we have also found that CmHKT1 has Na+ transport activity in yeast and also was up-regulated expression in root stelar cell under salt treatment. So we speculated that CmHKT1 participation in the restricted Na+ loading into xylem process and protected cucumber scions leaves from salinity stress, but the exact regulation mechanism of this process is yet unclear. In this project we intends to study the exact tissue and cellular localization of CmHKT1 in root and rootstock stem tissues by utilizing immunohistochemical localization techniques. Then Xenopus laevis Oocytes electrophysiological technique, cucumber genectic transformation technology, and reciprocal grafting combinations strategy will be utilized to clearly understand the functions of CmHKT1. Finally, we will identify the regulatory factors and interaction proteins through the yeast one-hybrid and two-hybrid screening system to resolve its regulatory mechanism to limit Na+ transport in pumpkin grafted cucumber. The implementation of this project will not only reveal the CmHKT1 gene function and the molecular mechanisms of long distance transport of Na+ in grafted plants, but it will also promote salt resistance gene mining of pumpkin rootstock and the cultivation of grafted cucumber.

课题组前期研究发现以耐盐南瓜作为砧木可以提高嫁接黄瓜的耐盐性，主要在于砧木根系具有较强的Na+蓄积和木质部卸载能力，南瓜HKT1基因（CmHKT1）组织定位于根系中柱细胞，受盐胁迫诱导上调表达，具有较强的Na+转运能力，推测其参与限制Na+由南瓜砧木向黄瓜接穗转运过程，但相关分子机制尚不明晰。本项目拟通过免疫组化技术明晰该基因在南瓜砧木根系和茎段具体定位情况；通过爪蟾卵母细胞电生理技术分析CmHKT1基因的离子转运特性；采用黄瓜遗传转化、正反嫁接技术明确CmHKT1基因在嫁接复合体中调控Na+转运的具体功能。在此基础上利用酵母杂交筛选互作蛋白及调控因子，解析CmHKT1基因在砧用南瓜中的调控机制。本课题完成后不仅可以揭示CmHKT1基因的功能及其在黄瓜/南瓜嫁接复合体中限制Na+长距离运输的分子机制，还有利于丰富园艺作物砧穗互作的基本理论，促进砧用南瓜耐盐基因挖掘和黄瓜嫁接耐盐栽培。

以耐盐南瓜作为砧木可以提高嫁接黄瓜的耐盐性，主要在于砧木根系具有较强的Na+蓄积和木质部卸载能力，南瓜HKT1基因（CmHKT1;1）参与限制Na+由南瓜砧木向黄瓜接穗转运过程，但相关分子机制尚不明晰。本课题通过原位杂交技术，发现CmHKT1;1定位于根系中柱细胞，受到盐胁迫诱导上调表达，参与Na+蓄积和木质部卸载。进一步研究发现，CmHKT1;1是一个定位于细胞质膜上的Na+特异性转运蛋白，将其敲除后南瓜对盐胁迫抗性降低，而在黄瓜中超表达CmHKT1;1能提高黄瓜的耐盐性，此外，CmHKT1;1可以一定程度上恢复拟南芥hkt1突变体的耐盐性。通过构建南瓜膜蛋白文库，从中筛选到CmHKT1;1的互作蛋白CmCNIH1，通过酵母双杂点对点和萤火虫荧光素酶互补试验进行验证。我们发现CmCNIH1属于跨膜蛋白，定位于质膜、内质网与高尔基体，爪蟾卵母细胞异源表达实验显示CmCNIH1抑制了CmHKT1;1介导产生的电流。利用课题组建立的南瓜发根体系敲除CmCNIH1基因，发现cmcnih1突变体的耐盐性降低，表明CmCNIH1能通过调控CmHKT1;1进而影响南瓜耐盐性。本项目的完成不仅揭示了CmHKT1;1基因的功能及其互作蛋白CmCNIH1调控黄瓜/南瓜嫁接复合体耐盐性的分子机制，还有利于丰富园艺作物砧穗互作的基本理论，促进砧用南瓜耐盐基因挖掘和黄瓜嫁接耐盐栽培。项目执行期间在Journal of Experimental Botany等刊物上发表SCI论文6篇，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。