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
发布时间：2018-08-08 15:53
【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(P0.01),而叶绿素含量与CAT活性显著高于非转基因株系(P0.01),说明GsARHP基因的超量表达可以增强紫花苜蓿的耐碱能力。
[Abstract]:In this study, based on the data of alkaline stress transcriptional group in early laboratory of wild soybean, a hypothesis protein gene up regulation under alkali stress was screened and named GsARHP (alkali stress related hypothetical protein gene). First, Real-time PCR method was used to verify the expression of GsARHP based on alkali stress. Bioinformatics analysis table This gene encodes a hydrophilic protein containing 130 amino acids, containing a signal peptide but without a transmembrane domain, and constructs a GsARHP plant overexpression vector, which uses Agrobacterium mediated cotyledon node infection to convert alfalfa in Zhaodong, and 3 overexpressed GsARHP genes are detected by PCR, Southern Blot and RT-PCR method. The results showed that after 0100 and 150mmol/L NaHCO3 treatment of 14d, the non transgenic lines were obviously wilted, yellow and even died, while the transgenic lines had good growth. Further analysis of the physiological indexes showed that the relative plasma membrane permeability and the content of prop two aldehyde were significantly lower than those of the non transgenic lines (P0.01). The content of green pigment and CAT activity were significantly higher than those of non-transgenic lines (P 0.01), indicating that overexpression of GsARHP gene could enhance alkali tolerance of alfalfa.
【作者单位】： 东北农业大学农业生物功能基因重点实验室;
【基金】：国家自然科学基金项目(31171578) 黑龙江省高校科技创新团队建设计划项目(2011TD005) 东北农业大学学科团队建设项目—团队1资助
【分类号】：Q943.2;S541.9
，
本文编号：2172277
[Abstract]:In this study, based on the data of alkaline stress transcriptional group in early laboratory of wild soybean, a hypothesis protein gene up regulation under alkali stress was screened and named GsARHP (alkali stress related hypothetical protein gene). First, Real-time PCR method was used to verify the expression of GsARHP based on alkali stress. Bioinformatics analysis table This gene encodes a hydrophilic protein containing 130 amino acids, containing a signal peptide but without a transmembrane domain, and constructs a GsARHP plant overexpression vector, which uses Agrobacterium mediated cotyledon node infection to convert alfalfa in Zhaodong, and 3 overexpressed GsARHP genes are detected by PCR, Southern Blot and RT-PCR method. The results showed that after 0100 and 150mmol/L NaHCO3 treatment of 14d, the non transgenic lines were obviously wilted, yellow and even died, while the transgenic lines had good growth. Further analysis of the physiological indexes showed that the relative plasma membrane permeability and the content of prop two aldehyde were significantly lower than those of the non transgenic lines (P0.01). The content of green pigment and CAT activity were significantly higher than those of non-transgenic lines (P 0.01), indicating that overexpression of GsARHP gene could enhance alkali tolerance of alfalfa.
【作者单位】： 东北农业大学农业生物功能基因重点实验室;
【基金】：国家自然科学基金项目(31171578) 黑龙江省高校科技创新团队建设计划项目(2011TD005) 东北农业大学学科团队建设项目—团队1资助
【分类号】：Q943.2;S541.9
，
本文编号：2172277
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