摘要：

为探究大豆转录因子GRAS家族GmGRAS69基因在植物干旱胁迫中的功能和可能的分子机制。通过氨基酸序列比对和构建系统进化树分析大豆GmGRAS69与其他物种GRAS成员的序列保守性和进化关系。利用荧光定量PCR方法检测GmPP2C69基因在PEG处理的大豆根和叶中的表达模式。接着构建GmPP2C69基因过表达载体,进而利用农杆菌介导的浸花法转化野生型拟南芥;在正常培养和干旱处理条件下观察野生型和转基因拟南芥的生长表型,测定单株鲜质量、叶片相对含水量和地上部可溶性糖含量、抗氧化酶(SOD、POD和CAT)活性及对应的抗氧化酶基因表达水平。结果显示,GmGRAS69基因在PEG处理的大豆植株根和叶中都显著上调,并且在根中响应更显著。此外,成功获得GmGRAS69基因过表达的转基因拟南芥株系,且过表达株系的耐旱性相比野生型拟南芥WT明显增强。干旱处理后,过表达植株鲜质量、叶片相对含水量和可溶性糖含量均显著高于WT;抗氧化酶SOD、POD和CAT活性以及对应基因SOD、POD和CAT的表达水平也显著高于WT。结果表明,大豆GmGRAS69基因在干旱胁迫下表达上调,进而通过激活SOD、POD和CAT抗氧化酶编码基因、增强抗氧化酶活性和增加可溶性糖的积累,赋予转基因植株更强的耐旱性。

关键词: 大豆, GRAS家族, GmGRAS69基因, 干旱胁迫, 耐旱性

Abstract:

To investigate the function and possible molecular mechanisms of soybean GmGRAS69 gene,which belongs to the GRAS transcription factor family,in plant drought stress.Sequence conservation and evolutionary relationship between soybean GmGRAS69 and GRAS members of other species were analyzed by amino acid sequence alignment and phylogenetic tree construction.Quantitative Real-time PCR was used to detect the expression pattern of GmPP2C69 in soybean roots and leaves treated with PEG.Then,GmPP2C69 overexpression vector was constructed,and then wild-type Arabidopsis was transformed by Agrobacterium-mediated floral dip method.The growth phenotypes of wild-type and transgenic Arabidopsis were observed under normal culture and drought treatment.The fresh weight per plant,relative water content of leaf,soluble sugar content of shoot,antioxidant enzymes(SOD,POD and CAT)activities and corresponding antioxidant enzyme gene expression levels were measured.The results showed that GmGRAS69 was significantly up-regulated in both roots and leaves of PEG-treated soybean plants,and the response was more significant in roots.In addition,GmGRAS69-overexpressed Arabidopsis was successfully obtained,and the drought tolerance of the GmGRAS69-overexpressed plants was significantly enhanced compared with WT.Under drought treatment,the fresh weight,leaf relative water content and soluble sugar content of GmGRAS69-overexpressed plants were significantly higher than those of WT.The activities of antioxidant enzymes SOD,POD and CAT and the expression levels of corresponding genes SOD,POD and CAT were also significantly higher than those of WT.The results showed that the expression of GmGRAS69 was up-regulated under drought stress,and the transgenic plants were enhanced drought tolerance by activating SOD,POD and CAT antioxidant enzyme encoding genes,enhancing antioxidant enzyme activities and increasing soluble sugar accumulation.

Key words: Soybean, GRAS family, GmGRAS69 gene, Drought stress, Drought tolerance