论文部分内容阅读
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。
In this study, a hypothetical protein gene up-regulated under alkali stress was screened out based on the data of the wild-type soybean rhizobium stress transcriptome pre-laboratory, tentatively named GsARHP (alkali stress related hypothetical protein gene). Real-time PCR method was used to verify that GsARHP gene was induced by alkali stress. Bioinformatics analysis showed that the gene encoded a 130 amino acid hydrophilic protein containing signal peptide but no transmembrane domain. The GsARHP plant overexpression vector was constructed and transformed by Agrobacterium tumefaciens-mediated infection Zhaodong alfalfa, three transgenic lines overexpressing GsARHP gene were obtained by PCR, Southern Blot and RT-PCR methods, and its alkali resistance was analyzed. The results showed that after treated with 0, 100 and 150 mmol / L NaHCO3 for 14 days, the non-transgenic lines obviously wilted, yellowed and even died, while the transgenic lines grew well. Further analysis of the physiological indicators showed that the relative plasma membrane permeability and malondialdehyde (P <0.01), while chlorophyll content and CAT activity were significantly higher than non-transgenic lines (P <0.01), indicating that overexpression of GsARHP gene can enhance the alkali resistance of alfalfa.