转GsPPCK1基因苜蓿植株的获得及其耐碱性分析  

Transgenic Alfalfa with GsPPCK1 and Its Alkaline Tolerance Analysis

作　　者：魏正巍[1] 朱延明[1] 化烨[1] 才华[1] 纪巍[1] 柏锡[1] 王臻昱[1] 文益东[1] 

机构地区：[1]东北农业大学生命科学学院,黑龙江哈尔滨150030

出　　处：《作物学报》2013年第1期68-75,共8页Acta Agronomica Sinica

基　　金：国家自然科学基金项目(31171578);国家转基因生物新品种培育科技重大专项(2011ZX08004002-002);黑龙江省高校科技创新团队建设计划项目(2011TD005);黑龙江省博士后基金项目(LBH-Z12353)资助

摘　　要：磷酸烯醇式丙酮酸羧化酶激酶(phosphoenolpyruvate carboxylase kinase,PPCK)是一种钙不依赖的丝氨酸/苏氨酸(Ser/Thr)类蛋白激酶,参与碳氮代谢等多个生物学过程,然而其在碱胁迫反应中的作用尚未见报道。本研究在前期野生大豆碱胁迫基因表达谱数据基础上,采用同源克隆的方法分离野生大豆(Glycine soja)PPCK1基因,该基因编码的氨基酸序列与大豆(Glycine max)PPCK1蛋白(AAQ83695.1)具有99%的相似性,被命名为GsPPCK1。在50mmolL–1NaHCO3胁迫处理3h内,根和叶中GsPPCK1基因上调表达,属碱胁迫早期应答基因。通过农杆菌介导法对肇东苜蓿进行遗传转化,并对RT-PCR阳性的超表达转基因株系进行耐碱性分析,在100mmolL–1NaHCO3处理15d后转基因株系生长状态良好,而非转基因对照株系明显萎蔫、失绿、甚至死亡;转基因株系的丙二醛含量和相对质膜透性显著低于非转基因株系(P<0.05),而叶绿素含量和根系活力显著高于非转基因对照(P<0.05),说明超量表达GsPPCK1基因增强了苜蓿的耐碱能力。以上结果表明,GsPPCK1参于植物耐碱胁迫反应过程,在碱胁迫基因工程研究领域具有良好的理论和实际应用意义。Environmental stresses, such as drought, high salty and alkali, adversely affect plant growth and productivity. Plants adapt to these environmental stresses by inducing numerous genes at the transcriptional level and by protein phosphorylation. Phosphoenolpyruvate carboxylase kinase （PPCK） is a Ca2＋ independent kinase in response to a range of signals in different plant tissues which plays a key role in the control of plant metabolism. As an important extension of our earlier studies summarized above on global transcriptome profiling of wild soybean under NaHCO3 treatment, an alkaline （NaHCO3） related gene GsPPCK1 was identified and subsequently cloned from Glycine soja, which has 99% similarity with PPCK1 of Glycine max （AAQ83695.1）, named as GsPPCK1. Expression of GsPPCK1 mRNA was induced by NaHCO3 stress in roots and leaves. GsPPCK1 transcripts increased during 3 hour exposures to NaHCO3 stress. These results indicated that wild soybean PPCK1 was an early responded gene to alkaline stress. We transformed GsPPCK1 gene into alfalfa using a developed method, and transgenic alfalfa showed observably enhanced tolerance to NaHCO3 stress compared with wild-type plants. Transgenic alfalfa grew well in the conditions of 100 mmol L1 NaHCO3, while wild type plants exhibited discoloration and stunted growth, or even death. There were significantly changes in malondialdehyde content and relative membrane permeability caused by saline-alkaline stress in non-transgenic lines compared to transgenic lines （P〈0.05）. Moreover, compared with non-transgenic, transgenic alfalfa had higher levels of chlorophyll content and root activity under alkali stress conditions. The result indicated that over-expression of GsPPCK1 in alfalfa could enhance alkaline tolerance. All results showed that GsPPCK1 gene could improve the tolerance of transgenic alfalfa to alkali stress; therefore, the study on this field is of significance not only in theory but also in practice.

关 键 词：苜蓿 GsPPCK1 转基因株系 耐碱性分析 

分 类 号：S541.9[农业科学—作物学]