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150mmol/L NaHCO3处理14d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<0.01),说明GsARHP基因的超量表达可以增强紫花苜蓿的耐碱能力。
The enzyme myo-inositol-1-phosphate synthase（MIPS EC 5.5.1.4） catalyzes the first step of myo-inositol biosynthesis, a product that plays crucial roles in plants as an osmoprotectant, transduction molecule, cell wall constituent and production of stress related molecule. Previous reports highlighted an important role of MIPS family genes in abiotic stresses particularly under salt stress tolerance in several plant species; however, little is known about the cellular and physiological functions of MIPS2 genes under abiotic conditions. In this study, a novel salt stress responsive gene designated Gs MIPS2 from wild soybean Glycine soja 07256 was functionally characterized contained an open reading frame（ORF） of 1 533 bp coding a peptide sequence of 510 amino acids along with mass of 56 445 ku. Multiple sequence alignment analysis revealed its 92%-99% similarity with other MIPS family members in legume proteins. Quantitative real-time PCR results demonstrated that Gs MIPS2 was induced by salt stress and expressed in roots of soybean. The positive function of Gs MIPS2 under salt response at different growth stages of transgenic Arabidopsis was also elucidated. The results showed that Gs MIPS2 transgenic lines displayed increased tolerance as compared to WT and atmips2 mutant lines under salt stress. Furthermore, the expression levels of some salt stress responsive marker genes, including KIN1, RD29 A, RD29 B, P5 Cs and COR47 were significantly up-regulated in Gs MIPS2 overexpression lines than wild type and atmips2 mutant. Collectively, these results suggested that Gs MIPS2 gene was a positive regulator of plant tolerance to salt stress. This was the first report to demonstrate that overexpression of Gs MIPS2 gene from wild soybean improved salt tolerance in transgenic Arabidopsis.
