盐胁迫应答基因GmWRKY6的克隆及转基因百脉根的抗盐分析  

Cloning of salt-stressed responsive gene GmWRKY6 and salt resistance analysis of transgenic Lotus japonicus

作　　者：柯丹霞 彭昆鹏 夏远君 朱玉莹 张丹丹 KE Dan-xia;PENG Kun-peng;XIA Yuan-jun;ZHU Yu-ying;ZHANG Dan-dan(College of Life Sciences,Xinyang Normal University,Institute for Conservation and Utilization of Agro-bioresources in Dabie Mountains,Xinyang 464000,Chin)

机构地区：[1]信阳师范学院生命科学学院,大别山农业生物资源保护与利用研究院,河南信阳464000

出　　处：《草业学报》2018年第8期95-106,共12页Acta Prataculturae Sinica

基　　金：国家自然科学基金项目(31400213); 河南省科技攻关计划项目(182102110448); 信阳师范学院青年骨干教师资助计划(2015)和信阳师范学院“南湖学者奖励计划”青年项目(2016)资助

摘　　要：根据大豆盐胁迫RNA-Seq数据,筛选并克隆得到1个大豆WRKY类转录因子GmWRKY6基因。序列分析表明,该基因含有1个1674bp的开放阅读框,编码557个AA,编码蛋白属于IIb类WRKY家族成员,含有1个WRKY保守结构域和1个C2H2锌指结构基序。基因表达模式分析表明,GmWRKY6基因在大豆根中表达量较高,并且该基因受盐胁迫诱导表达。构建GmWRKY6基因的植物超表达载体,获得7株T0代阳性转GmWRKY6基因百脉根。进一步对T1代转基因株系进行抗盐分析发现,在200mmol·L-1 NaCl处理14d后转基因株系生长状态良好,而野生型对照生长矮小,叶片干枯。此外,对相关生理指标的测定发现,转基因株系叶片中脯氨酸和叶绿素含量显著高于野生型对照,而丙二醛含量和相对质膜透性显著低于野生型对照。此外,盐胁迫下转基因百脉根叶片中钠离子含量显著低于对照,而钾离子含量显著高于对照。以上结果表明大豆GmWRKY6基因的超表达能够显著增强百脉根的抗盐能力。AGmWRKY6 gene from WRKY transcription factor family members from soybean was cloned from salt stress RNA-Seq data.Sequence analysis showed that the gene contained an open reading frame（ORF）of1674 bp and 557 encoding amino acids.The GmWRKY6 protein contained a conserved WRKY domain and a C2 H2 zinc finger motif,belonged to group IIb WRKY transcription factor.Analysis of gene expression pattern showed that the expression level of GmWRKY6 gene is highest in the root and was induced by salt stress.Plant overexpression vector of the GmWRKY6 gene was constructed and 7 positive T0 transgenic Lotus japonicus plants were obtained.The salt resistance of T1 transgenic lines was further analyzed,after exposure to 200 mmol·L^-1 NaCl for 14 days;transgenic lines grew well while the wild type were stunted with dry foliage.Proline and chlorophyll content in transgenic lines were significantly higher than wild plants,while the MDA content and the relative permeability of plasma membrane were significantly lower than that of wild plants.In addition,the sodium ion content in transgenic lines was significantly lower than wild plants under salt stress,while the potassium content was significantly higher than that of wild plants.These results showed that over expression of the soybean GmWRKY6 gene can significantly enhance the salt resistance of L.japonicus.

关 键 词：大豆 WRKY转录因子 子叶节转化 百脉根 耐盐性 

分 类 号：Q943.2[生物学—植物学]