珊瑚菜盐胁迫相关GlRTH基因的克隆和表达量分析  

Cloning and expression quantity analysis of GlRTH gene related to salt stress in Glehnia littoralis

作　　者：徐瑶 任宏伟 张馨方 王芳 朱丹[1] 谭玲玲[1] 袁涛 高婷[1] XU Yao;REN Hong-wei;ZHANG Xin-fang;WANG Fang;ZHU Dan;TAN Ling-ling;YUAN Tao;GAO Ting(Shandong Provincial University Key Laboratory of Plant Biotechnology,College of Life Sciences,Qingdao Agricultural University,Qingdao 266109,China;Collegeof Civil Engineering&Architecture,Qingdao Agricultural University,Qingdao 266109,China)

机构地区：[1]青岛农业大学生命科学学院山东省高校植物生物技术重点实验室,山东青岛266109 [2]青岛农业大学建筑工程学院,山东青岛266109

出　　处：《中草药》2023年第11期3639-3646,共8页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金委员会青年基金项目(81903748);山东省教育厅山东省高校科技计划项目(2019KJE003)。

摘　　要：目的筛选得到珊瑚菜Glehnia littoralis抗盐相关基因GlRTH,并进行开放阅读框(open reading frame,ORF)全长克隆以及生物信息学和表达量分析。方法从珊瑚菜转录组数据中筛选到GlRTH基因,通过反转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)以及c DNA末端快速扩增(rapid amplification of cDNA ends,RACE)获得该基因全长ORF序列,利用生物信息学分析方法分析其所编码蛋白质的理化性质,预测该蛋白二、三级结构、保守结构域,建立系统发育进化树;利用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)方法检测该基因组织表达特异性及其在NaCl和1-氨基环丙烷羧酸(1-aminocyclopropanecarboxylic acid,ACC)处理下的表达差异。结果GlRTH基因序列全长ORF为690bp,共编码229个氨基酸。系统进化分析表明,珊瑚菜的RTH蛋白与黄胡萝卜Daucuscarotasubsp.sativus的RTH蛋白亲缘关系最近,相似度达88.65%;qRT-PCR结果显示,GlRTH基因在珊瑚菜的根、叶、花中均有表达,在花中表达量较高,叶和根中无显著差异;200 mmol/L的NaCl处理3、6、12、24、36 h,GlRTH基因表达量明显上调,且处理36 h后GlRTH基因表达量上调最为显著;100 mmol/L ACC处理,GlRTH基因的表达量呈先上升再下降的趋势,在3 h时GlRTH基因表达量显著上调,在6 h和12 h时GlRTH基因表达量下调。结论GlRTH蛋白是乙烯响应因子,并且与珊瑚菜的耐盐性相关。为探究乙烯信号通路和药用植物抗盐反应的关系及改良珊瑚菜的耐盐能力奠定了基础。Objective The salt resistance related gene GlRTH of Glehnia littoralis was screened,and ORF(open reading frame)full-length cloning,bioinformatics and expression analysis were carried out.Methods The GlRTH gene was screened from the transcriptome data of G.littoralis,and the full-length ORF sequence of the gene was obtained by RT-PCR(reverse transcription-polymerase chain reaction)and RACE(rapid-amplification of cDNA ends).Thebioinformatics analysis method was used to analyze the physicochemical properties of the encoded protein,the protein secondary and tertiary structure,conserved domains were predicted,and a phylogenetic evolutionary tree was established.The tissue or organ expression specificity of this gene and the difference in expression under NaCl and ACC(1-aminocyclopropanecarboxylic acid)treatment were detected by real-time fluorescence quantitative PCR(RT-qPCR).Results The full length ORF of GlRTH sequence was 690 bp,encoding 229 amino acids.Phylogenetic analysis showed that the GlRTH was closely related to DcRTH of Daucus carota subsp.sativus with a similarity of 88.65%.RT-qPCR results showed that GlRTH gene was expressed in roots,leaves and flowers,with a high expression in flowers,but there was no significant difference between leaves and roots.GlRTH gene expression was significantly increased at 3,6,12,24,36 h after 200 mmol/L NaCl treatment,and the most significant increase was observed at 36 h after treatment.The expression of GlRTH gene increased first and then decreased after 100 mmol/L ACC treatment,and increased significantly at 3 h,while the expression of GlRTH gene was down-regulated at 6 and 12 h.Conclusion GlRTH is an ethylene responsive factor and related to the salt tolerance of G.littoralis.This study laid a foundation for exploring the relationship between ethylene signaling pathway and salt tolerance of medicinal plants and improving the salt tolerance of G.littoralis.

关 键 词：珊瑚菜 GlRTH基因 表达量分析 QRT-PCR 生物信息学分析 

分 类 号：R286.12[医药卫生—中药学]