羊草LcZIP1的铁转运功能鉴定

Functional identification of iron transport of LcZIP1 in Leymus chinensis

羊草在我国内蒙古草原广泛分布,是重要的乡土牧草,然而关于羊草矿质营养吸收的分子机制尚未受到广泛关注。关于ZIP家族在植物吸收和转运必需微量元素和重金属过程中的作用,在模式植物和农作物中研究较多。本研究从羊草转录组数据库中筛选到一个与ZIP同源的基因Lc206852,发现其与拟南芥ZIP家族的Zn^(2+)转运蛋白AtZIP1亲缘关系较近,因此将其命名为LcZIP1。利用TMHMM Server v.2.0进行跨膜域分析发现,LcZIP1是一种跨膜蛋白,有9个跨膜域,与禾本科短柄草属植物ZIP亲缘关系最近。将LcZIP1-GFP瞬时转染烟草叶表皮细胞和羊草叶原生质体进行亚细胞定位,发现LcZIP1定位于内质网。通过实时荧光定量PCR分析发现,缺铁、高铁和高锌环境可诱导LcZIP1表达,表明LcZIP1参与环境中Zn和Fe营养水平的响应。最后通过酵母功能互补试验证明,LcZIP1在低铁条件下能够使低铁敏感酵母突变体(△fet3/△fet4)恢复生长,暗示LcZIP1具有Fe^(2+)转运功能。以上结果对日后开发和利用微量元素强化农作物品质具有一定的参考价值。

Leymus chinensis is an important native forage grass,which is widely distributed in the Inner Mongolian steppe grasslands of China.However,the molecular mechanism of mineral uptake in L.chinensis is not yet well understood,although it is known from systematic study in many model plants and crops that zinc-regulated transporter/iron-regulated transporter-like proteins(ZIP transporters)are involved in the uptake and allocation of essential trace elements and heavy metal ions.In this study we found that the Lc206852 gene screened from the L.chinensis transcriptome database was homologous with Zn^(2+) transporter AtZIP1 in Arabidopsis thaliana.Transmembrane domain analysis using TMHMM Server v.2.0 indicated that Lc206852,referred to hereafter as LcZIP1,had 9 transmembrane domains,which were closely related to ZIP proteins of plants of the genus Brachypodium.Expression of LcZIP1-GFP in leaf epidermal cells of tobacco and leaf protoplasts of L.chinensis showed that LcZIP1 resided in the endoplasmic reticulum.Real time PCR analysis found that LcZIP1 was responsive to zinc and iron nutrition,and that excess Zn or Fe and Fe deficiency induced LcZIP1 expression in roots at transcription level.The yeast(Saccharomyces cerevisiae)complementation test showed that expression of LcZIP1 stimulated growth of the‘iron uptake-deficient’yeast mutantΔfet3/Δfet4 under Fe deficient conditions.Functional characterization of LcZIP1 will provide an opportunity for crop improvement by mineral nutrition enhancement in the future.