碱胁迫应答基因GsARHP的克隆及转基因紫花苜蓿的耐碱性分析

摘要

本研究基于实验室前期野生大豆碱胁迫转录组数据,筛选出一个碱胁迫下上调表达的假定蛋白基因,暂命名为GsARHP(alkali stress related hypothetical protein gene).首先利用Real-time PCR方法验证了GsARHP基因受碱胁迫诱导表达.生物信息学分析表明,该基因编码一个含有130个氨基酸的亲水蛋白,含有信号肽但无跨膜结构域;构建了GsARHP植物超量表达载体,利用农杆菌介导的子叶节侵染法转化肇东紫花苜蓿,通过PCR,Southern Blot和RT-PCR方法检测获得了3个超量表达GsARHP基因的转基因株系,并对其耐碱性进行了分析.结果表明,在0,100和150 mmol/L NaHCO3处理14 d后,非转基因株系明显萎蔫、黄化甚至死亡,而转基因株系则长势良好;进一步分析其生理指标显示,相对质膜透性与丙二醛含量均显著低于非转基因株系(P＜0.01),而叶绿素含量与CAT活性显著高于非转基因株系(P＜o.01),说明GsARHP基因的超量表达可以增强紫花苜蓿的耐碱能力.%GsARHP,which encodes hypothetical protein up-regulated by alkaline stress,was identified from RNA-seq data of wild soybean (Glycine soja) at the early stage of the salt stress response.Real-time PCR analyses showed that GsARHP was induced by alkali stress.A bioinformatics analysis showed that the gene encodes a hydrophilic protein consisting of 130 amino acids,with a signal peptide but no transmembrane domain.A plant overexpression vector was constructed and transformed into alfalfa by Agrobacterium tumefaciens-mediated cotyledonary node infection.Three transgenic lines were identified by PCR,Southern blotting,and RT-PCR analyses.The non-transgenic lines became wilted and yellow or even died under 0,100,and 150 mmol/L NaHCO3 treatment for 14 days,while the transgenic lines grew well under the same conditions.Fur-ther analyses showed that the transgenic lines had significantly lower relative plasma membrane permeabilityand malondialdehyde (MDA) content than did non-transgenic lines (P＜0.01),and significantly higher chloro-phyll content and CAT activity (P＜0.01).These results indicated that overexpression of GsARHP could en-hance the alkali resistance of alfalfa.