长穗偃麦草HKT1;4基因的克隆及其对烟草遗传转化的研究

High Affinity K+ Transporter(HKT1;4) is mainly responsible for Na+ unloading from xylem sap in root and sheath to maintain low Na+ concentration and high K+/Na+ ratio in shoot. The SSR (simple sequence repeat) marker specific loci Xgwm044,which closely links to salt tolerant gene, has been selected from Elytrigia elongata germplasm, and the nucleotide sequence of the loci Xgwm044 shared high homology (83%) with TmHKT7-A2 (TmHKT1;4) gene from Triticum monococcum in our previous studies. However,the contribution of HKT1;4 to salt tolerance of E. elongata still remains unclear. In this study, EeHKT1;4 gene would be cloned from E. elongata germplasm with strong salt tolerance, and the expression levels of EeHKT1;4 and the patterns of Na+ and K+ accumulation would be quantitatively analyzed in order to elucidate the role of EeHKT1;4 in Na+ transport mechanism of E. elongata. And then, EeHKT1;4 gene would be transformed into tobacco (Nicotiana tabacum) using Agrobacterium-mediated leaf disc transformation methods in order to analyze the K+, Na+ concentrations in root, stem and leaf of transgenic tobacco exposed to different salt treatments, and to clarify the role of EeHKT1;4 in salt tolerance of tobacco. The research results would have the very important application value in the aspects of the genetic improvement of salt tolerence in wheat, corn and other crops, and forages with high quality.

高亲和K+转运载体类蛋白（HKT1;4）主要负责从植株叶鞘和根木质部汁液中卸载Na+，以维持其地上部较低Na+浓度和较高K+/Na+比。该团队已从长穗偃麦草中筛选到与耐盐基因连锁的SSR标记特异位点Xgwm044，经测序其核苷酸序列与一粒小麦TmHKT7-A2(TmHKT1;4)基因同源性达83%，但HKT1;4在长穗偃麦草中的耐盐作用机制尚不清楚。本项目拟从长穗偃麦草中克隆EeHKT1;4基因，定量分析不同盐处理下其根和地上部的EeHKT1;4基因表达及K+、Na+积累模式，解析EeHKT1;4在长穗偃麦草Na+转运中的作用机制。通过根癌农杆菌介导的叶盘转化法将EeHKT1;4基因导入烟草中，分析盐处理下转基因烟草植株根、茎和叶中的K+、Na+浓度变化，以探明EeHKT1;4在烟草耐盐中的作用。该研究结果必将在小麦、玉米等农作物和优良牧草的耐盐遗传改良方面具有重要的应用价值。

从长穗偃麦草中克隆到基因3个，其中耐盐基因EeHKT1;4和EeSKOR各1个，内参基因EeACT 1个，在GenBank中注册登记基因2个即EeHKT1;4和EeACT（登记号分别为KF956112和KF981729）。EeHKT1;4基因全长cDNA为1977 bp，ORF 1722 bp，可编码573个氨基酸，推测分子量为62.92 kDa，等电点为9.73。完成了EeHKT1;4的生物信息学特征分析，系统进化树结果揭示了“长穗偃麦草EeHKT1;4与单子叶植物高亲和K+转运蛋白HKT1;4亲缘关系较近”的科学结论。对不同浓度KCl和NaCl处理下的EeHKT1;4表达与K+、Na+积累模式变化规律的分析结果表明：长穗偃麦草EeHKT1;4主要在根中表达，并不受外界K+浓度的影响，说明EeHKT1;4是一个特异的Na+转运蛋白，从叶鞘和根木质部汁液中卸载Na+，以维持地上部K+、Na+的稳态作用，解析了EeHKT1;4在长穗偃麦草Na+转运中的作用机制。建立了烟草无菌苗植株再生体系，即基础培养基MS，分化培养基MS+1 mg/L 6-BA，生根培养基1/2 MS；成功构建了长穗偃麦草EeHKT1;4基因的单价植物表达载体pBI121-35S-EeHKT1;4-Nos。通过农杆菌介导法获得了转EeHKT1;4烟草阳性植株，阳性植株转化率达32%，证明EeHKT1;4在转基因烟草中能够成功表达。对不同浓度NaCl处理下野生型与转基因烟草的根、茎、叶中K+、Na+积累模式及变化规律的分析结果表明，高NaCl处理下，烟草EeHKT1;4的过表达导致了其根和茎Na+浓度的聚集，且维持了叶片中的低Na+和高K+含量，从而揭示出EeHKT1;4在降低烟草叶片Na+浓度与提高其耐盐性方面发挥着重要调节作用。通过项目研究，发表SCI 2篇，中文核心4篇（刊出3篇，接受待刊1篇）；申请发明专利1项，授权实用新型专利2项；获中国草业科技奖一等奖1项。