过表达谷子液泡H＋-ATPase E亚基基因在拟南芥中的耐盐性

Overexpression of Vacuole H^+-ATPase E Subunit Gene Si VHA-E from Foxtail Millet Enhances Salt Resistance in Transgenic Arabidopsis thaliana

V-H+-ATPase在植物生长、发育和胁迫响应等生物学过程中发挥重要作用。本研究通过序列比对,从谷子中克隆到V-H+-ATPase E亚基基因Si VHA-E。进化树分析显示,Si VHA-E基因在进化上属于E1/E3亚族,与玉米V-H+-ATPase E亚基基因Zm VHA-EL亲缘关系较近。表达谱分析结果显示,Si VHA-E在高盐、茉莉酸甲酯(Me JA)、水杨酸(SA)和脱落酸(ABA)处理下表达上调,在低温和低氮处理下表达下调。亚细胞定位分析表明Si VHA-E定位于液泡膜上。遗传转化拟南芥的耐盐性鉴定表明,在盐处理条件下,转基因株系的种子萌发率、幼苗主根长、植株鲜重及存活率显著高于野生型的。与野生型拟南芥植株相比,Si VHA-E过表达植株体内Na+含量减少,体内相对含水量提高。此外,ABA萌发试验结果显示,在种子萌发后期,Si VHA-E过表达植株对ABA更加敏感。研究表明,谷子Si VHA-E可以显著提高拟南芥耐盐性,这可能与其正向调控ABA信号途径以及减少植株体内Na+积累和水分散失有关。

The V-H＋-ATPase plays an important role in processes of plant growth, development and response to stresses. In this research,SiVHA-E,a V-H＋-ATPase E subunit gene, was cloned from millet by the Blast analysis against GenBank database. Phy-logenetic tree showed that the gene belongs to E1/E3 subgroup and is close withZmVHA-EL, a V-H＋-ATPase E subunit from maize. The quantitative Real-time PCR （qRT-PCR） analysis revealed that the expression levels ofSiVHA-Ewere up-regulated under treatments of high-salt, exogenous MeJA, SA, and ABA hormones, while down-regulated under stresses of cold and low nitrogen. Protein subcellular localization analysis using protoplast showed that SiVHA-E is located on tonoplast. The results of salt tolerance assay showed that the germination rate ofSiVHA-E transgenic lines was significantly higher than that of wild type plant under salt stress. During seedlings period, the root lengths were significantly longer as well as fresh weight and survival rate were significantly higher in transgenic lines than in wild type plant under salt treatment. Compared with wild type plant, trans-genic plant reduced the content of Na＋and increased the relative water content inside cells. In addition, the results of germination experiment used ABA showed thatSiVHA-E transgenicArabidopsis was more sensitive to ABA than wild type plant during post-germination. In short, overexpressingSiVHA-Ein transgenicArabidopsis lines enhances salt tolerance, which might be relates to positive regulation of ABA signaling pathway or reduction of Na＋ accumulation and water loss in transgenic plants.