人参PgWRKY22基因的克隆及在磷胁迫下的表达分析  

Molecular cloning of PgWRKY22 from Panax ginseng and its expression in response to external phosphorus supply

作　　者：梁浩 孙海[1] 邵财[1] 钱佳奇 张亚玉[1,2] LIANG Hao;SUN Hai;SHAO Cai;QIAN Jia-qi;ZHANG Ya-yu(Institute of Special Animal and Plant Sciences,Chinese Academy of Agricultural Sciences,Changchun 130112,China;College of Pharmacy and Biological Engineering,Chengdu University,Chengdu 610106,China)

机构地区：[1]中国农业科学院特产研究所,吉林长春130112 [2]成都大学药学与生物工程学院,四川成都610106

出　　处：《中草药》2023年第13期4286-4294,共9页Chinese Traditional and Herbal Drugs

基　　金：财政部和农业农村部国家现代农业产业技术体系(CARS-21);中国农业科学院科技创新工程协同创新任务(CAAS-XTCX20190025-6);国家重点研发计划项目子课题(2021YFD1600902);非林地老参地再利用研究与示范项目(LNFYZBDL-JL18-246C)。

摘　　要：目的克隆人参Panax ginseng转录因子基因PgWRKY22,进行生物信息学、亚细胞定位及外源磷胁迫表达分析。方法根据人参转录组数据库设计特异性引物,PCR扩增PgWRKY22全长cDNA序列,命名为PgWRKY22,并对其进行生物信息分析;构建绿色荧光融合表达载体PgWRKY22-eGFP,利用农杆菌侵染烟草叶片的瞬时表达法分析亚细胞定位;实时荧光定量PCR(qRT-PCR)检测PgWRKY22在人参组织中的表达特异性以及在外源磷作用下的表达模式。结果人参转录因子基因PgWRKY22的cDNA序列为975bp,编码324个氨基酸;PgWRKY22蛋白属于WRKY超家族蛋白,具有典型的WRKY结合域,该蛋白不稳定,属于亲水性蛋白,无规则卷曲为主要组成部分;PgWRKY22蛋白与丹参SmWRKY、葡萄VvWRKY22、长春花CrWRKY22、独脚金SaWRKY27蛋白具有较高的同源性,与西洋参PqWRKY1、PqWRKY2,人参PgWRKY1、PgWRKY3、PgWRKY4具有较近的系统发育关系;亚细胞定位显示PgWRKY22定位于细胞核上;qRT-PCR表明PgWRKY22在人参的侧根和主根中表达较高,在叶和茎中次之;在外源磷浓度为2 mmol/L下的表达显著高于其他磷浓度下的表达。结论克隆获得PgWRKY22的cDNA序列及表达信息,可为后续进行基因功能鉴定和人参栽培的磷营养调控提供参考。Objective To clone a transcription factor gene PgWRKY22 from Panax ginseng followed by bioinformatics,subcellular localization and expression in response to external phosphorus supply were performed.Methods According to Panax ginseng transcriptome database to design the specific primer,by PCR to amplify the cDNA sequence of PgWRKY22,which was named as PgWRKY22,and its bioinformatics was analyzed.Enhanced green fluorescent protein(eGFP)fused expression vector PgWRKY22-eGFP was constructed and subcellular localization of PgWRKY22 was observed by Agrobacterium tumefaciens transient expression method in tobacco.Quantitative real-time PCR(qRT-PCR)was used to detect the expression specificity of PgWRKY22 in plant tissues and its expression in response to external phosphorus supply.Results The cDNA sequence of the Panax ginseng transcription factor gene PgWRKY22 was 975bp,encoding 324 amino acids;PgWRKY22 protein was a WRKY superfamily protein with a typical WRKY binding domain.It was an unstable hydrophilic protein with random coils as its main component;PgWRKY22 protein has high homology with Salvia miltiorrhiza SmWRKY,Vitis vinifera VvWRKY22,Catharanthus roseus CrWRKY22,Striga asiatica SaWRKY27 protein,and has a close phylogenetic relationship with Panax quinquefolius PqWRKY1,PqWRKY2,Panax ginseng PgWRKY1,PgWRKY3,PgWRKY4;subcellular localization shows that PgWRKY22 was localizted to the nuclei;real-time fluorescence quantitative PCR showed that PgWRKY22 was highly expressed in the lateral roots and main roots of Panax ginseng,followed by the leaves and stems;the expression at exogenous phosphorus concentration of 2 mmol/L was significantly higher than that at other phosphorus concentrations.Conclusion The cDNA sequence and expression information of PgWRKY22 were obtained by cloning,which could provide reference for gene function identification and phosphorus nutrition regulation in Panax ginseng cultivation.

关 键 词：人参 转录因子 生物信息学 亚细胞定位 表达分析 

分 类 号：R286.12[医药卫生—中药学]