过量表达星星草(Puccinellia tenuiflora)液泡型H^+-ATP酶c亚基基因提高转基因酵母的耐盐性

Overexpression of A V-ATPase Subunit c Gene from Puccinellia tenuiflora(PutVHA-c) Enhances Tolerance to Salt Stress in Yeast

植物液泡型H+-ATP酶(V-ATPase)在次级转运与应对多种逆境胁迫中具有重要作用。本研究克隆并解析了能够生长在盐碱地上的野生星星草V-ATPase c亚基基因(Put VHA-c)的功能。本研究利用荧光蛋白(GFP)标记结合内涵体染色的方法研究了Put VHA-c蛋白在酵母细胞中的定位,并利用转基因酵母分析了Put VHA-c基因在盐胁迫中的作用。共定位实验显示Put VHA-c-GFP融合蛋白主要定位在酵母细胞的内涵体上。Northern杂交和实时定量PCR显示在星星草悬浮细胞中Put VHA-c基因的表达能够被盐胁迫所诱导。研究结果结合过表达Put VHA-c的转基因酵母具有更高的耐盐性的发现,说明Put VHA-c基因参与着盐胁迫的应答。

Plant vacuolar-type H＋-ATPase（V-ATPase） plays an important role in secondary transport as well as in response to different adverse environmental conditions.In the present study,we cloned and characterized a V-ATPase c subunit gene（Put VHA-c） from Puccinellia tenuiflora,which can grow in saline,alkali soil.We examined the subcellular targeting of Put VHA-c in yeast cells by combining the GFP marker and endosomal marker dye FM4-64 staining.Further,we characterized the biological function of the Put VHA-c gene in salt stress by using the transgenic yeast.Co-localization experiments showed that Put VHA-c-GFP was preferentially localized to the endosome in yeast cells.Northern blotting and quantitative PCR indicated that Put VHA-c expression was induced by salt stress in P.tenuiflora suspension cells.These data,plus the finding that transgenic yeast overexpressing Put VHA-c had increased salt tolerance,suggested that Put VHA-c might be involved in the salt stress response.