大豆GmHDL57基因的克隆及抗盐功能鉴定

Cloning and Salt Resistance Function Identification of GmHDL57 Gene from Glycine max

HD-Zip I类转录因子在植物抵御非生物胁迫过程中发挥重要功能,本研究克隆得到1个大豆HD-Zip I类基因Gm HDL57(Glycine max homeodomain-leucine zipper protein 57)。序列分析表明,Gm HDL57基因包含1个1038 bp的开放读码框,编码345个氨基酸,具有HD-Zip类家族蛋白典型的保守结构域。基因时空表达分析表明,大豆Gm HDL57基因在大豆植株的各个不同时期及不同器官中均有表达,在花中表达量最高。采用实时荧光定量PCR技术分析了4种非生物胁迫(脱落酸、Na Cl、PEG、冷)对幼苗期大豆根中Gm HDL57基因表达的影响。结果表明,该基因表达量受高盐胁迫诱导显著升高,在脱落酸及干旱胁迫下上升幅度较小,但在冷胁迫下呈下降趋势。盐胁迫前后Gm HDL57基因在根中的表达量明显高于茎和叶,在盐胁迫48 h时达到峰值,72 h和96 h时表达量缓慢下降。此外,构建Gm HDL57基因的植物超表达载体,利用根癌农杆菌转化法获得转基因百脉根,200 mmol L–1 Na Cl处理条件下,转基因百脉根的株高、根长、叶绿素含量、根系活力以及阳离子K+、Ca2+含量显著高于野生型,而丙二醛含量、相对质膜透性以及Na+的含量明显低于野生型。以上研究结果表明,Gm HDL57基因参与了大豆对非生物胁迫的应答过程,过量表达Gm HDL57基因能够显著提高百脉根的抗盐能力。

The HD-Zip I class transcription factor plays an important role in plant resistance to abiotic stresses.An HD-Zip I class gene GmHDL57(Glycine max homeodomain-leucine zipper protein 57)was cloned from soybean in this study.Sequence analysis showed that GmHDL57 gene contained a 1038 bp ORF,encoding 345 amino acids,and featured with HD-Zip family proteins’typical conserved domain.GmHDL57 was expressed in different organs of soybean plants and the highest expression occurred in flowers.The effects of abiotic stresses(abscisic acid,NaCl,PEG,and cold)on GmHDL57 gene expression in soybean seedling stage were analyzed by real-time quantitative PCR.The expression level of GmHDL57 gene was obviously increased under high salinity stress and less affected by ABA and drought stress but decreased by cold stress.The expression level of GmHDL57 gene in roots was significantly higher than that in stems and leaves before and after salt stress,and reached the peak at 48 h,then decreased slowly at 72 h and 96 h after salt stress.The overexpression vector of GmHDL57 was constructed and transformed into Agrobacterium tumefaciens strain EHA105 to obtain the stable transgenic Lotus japonicus plants.After being treated with 200 mmol L^–1 NaCl for 14 d,the shoot height,root length,chlorophyll content,root activity as well as K^+and Ca^2+content increased significantly in transgenic plants compared with the wild type.The malondialdehyde content,relative membrane permeability and Na^+content were obviously reduced in transgenic plants compared with the wild type.It is hypothesized that the GmHDL57 gene participates in the abiotic stress response of soybean,and over-expression of GmHDL57 gene could enhance resistance to saline in Lotus japonicus.