延胡索去甲乌药碱合成酶基因CyNCS1克隆及其表达模式分析  

Cloning and expression analysis of norcoclaurine synthase(NCS)gene in Corydalis yanhusuo

作　　者：冯飞雪 吕瑞华 李依民[3] 高静[3] 王凯 周嘉迪 颜永刚[3] 张岗[3] FENG Feixue;L Ruihua;LI Yimin;GAO Jing;WANG Kai;ZHOU Jiadi;YAN Yonggang;ZHANG Gang(Affiliated Hospital,College of Pharmacy,Shaanxi University of Chinese Medicine,Xianyang,Shaanxi 712046,China;College of Medical Technology,College of Pharmacy,Shaanxi University of Chinese Medicine,Xianyang,Shaanxi 712046,China;Key Laboratory for Research and Development of“Qin Medicine”of Shaanxi Administration of Chinese Medicine,College of Pharmacy,Shaanxi University of Chinese Medicine,Xianyang,Shaanxi 712046,China)

机构地区：[1]陕西中医药大学附属医院,陕西咸阳712046 [2]陕西中医药大学医学技术学院,陕西咸阳712046 [3]陕西中医药大学药学院/陕西省中医药管理局“秦药”研发重点实验室,陕西咸阳712046

出　　处：《西北农林科技大学学报（自然科学版）》2024年第3期146-154,共9页Journal of Northwest A&F University(Natural Science Edition)

基　　金：陕西省重点产业创新链(群)项目(2022ZDLSF05-03);陕西省中医药管理局专项(2021-QYZL-02);陕西省科技厅一般项目(青年)(2023-JC-QN-0944,S2023-JC-QN-0932)。

摘　　要：【目的】克隆延胡索去甲乌药碱合成酶(norcoclaurine synthase, NCS)基因,分析其在延胡索异喹啉类生物碱合成中的关键作用。【方法】基于转录组数据库,利用逆转录PCR克隆延胡索CyNCS1基因及其启动子区域序列,采用生物信息学方法分析其编码蛋白的理化性质、结构特征及其启动子区域顺式作用元件,同时进行多序列比对和系统进化树分析,确定其进化关系;利用实时荧光定量PCR对其根、茎、叶及发育早、中、晚期块茎的组织表达模式进行分析;对茉莉酸甲酯(MeJA)、脱落酸(ABA)处理0,4,8,12 h的叶片进行表达谱分析,以体积分数75%酒精溶剂处理为对照(CK),确定该基因的表达特征。【结果】CyNCS1基因开放读码框长873 bp,编码291个氨基酸,编码蛋白分子量为33.09 ku,等电点为6.90,具有去甲乌药碱合成酶保守的催化结构域(GDGGVGTV/IL、YKEKF和MIEGGHLDMG),且不含信号肽,预测定位于细胞核中。多序列比对结果显示,CyNCS1与石生黄堇、罂粟的NCS蛋白同源性较高,分别为83.6%和82.1%;系统进化树结果显示,CyNCS1与石生黄堇CsNCS蛋白聚为一支,说明二者亲缘关系较近。该基因启动子区长2 238 bp,包含多种植物激素及低温、热胁迫等环境因子的顺式作用元件。实时荧光定量PCR结果显示,随着发育推进块茎中CyNCS1的表达量显著增高,且受到MeJA和ABA的诱导,MeJA处理8 h时表达量为0 h的3.10倍,ABA处理8 h为0 h的3.16倍;而对照CyNCS1表达量随时间推移无明显变化。【结论】克隆并鉴定了延胡索NCS酶基因CyNCS1,明确了其在发育进程中的表达模式,并确定该基因表达受MeJA和ABA的诱导。【Objective】The norcoclaurine synthase(NCS)gene from Corydalis yanhusuo was cloned to identify its key role in the synthesis of corydalis alkaloids.【Method】Based on the transcriptome database,the ORF region of CyNCS1 gene was cloned by quantitative real-time PCR technology and bioinformatics methods were used to analyze its physical and chemical properties and structural feature of the encoded protein.Multiple sequence alignment and phylogenetic tree analysis were performed.Finally,the expression pattern of CyNCS1 in different tissues including root,stem,leaf and tubers in development stages of early tuber,middle tuber and later tuber,as well as its expression in leaves treated by methyl jasmonate(MeJA)and abscisic acid(ABA)for 0,4,8 and 12 hours were analyzed by quantitative real-time PCR.【Result】The ORF of CyNCS gene was 873 bp in length,encoding 291 amino acids,with a molecular weight of 33.09 ku and an isoelectric point of 6.90.The encoded protein had a conserved catalytic domain of NCS(GDGGVGTV/IL,YKEKF and MIEGGHLDMG)without signal peptide and it was located in the nucleus.The multiple sequence alignment showed that CyNCS1 had high homology of 83.6%and 82.1%with NCS proteins of Corydalis saxicola and Papaver somniferum,respectively.The phylogenetic tree showed that CyNCS1 and CsNCS proteins were clustered together.The promoter region contained cis-acting elements that responded to environment and plant hormones.Expression pattern analysis showed that the expression of CyNCS1 increased with tuber development and was significantly induced by MeJA and ABA(P<0.05).The expression level of CyNCS1 in the MeJA treatment at 8 h was 3.10 times of that at 0 h,and the expression level of CyNCS1 in the ABA treatment at 8 h was 3.16 times of that at 0 h.The CyNCS1 expression levels in the control showed no significant changes over time.【Conclusion】CyNCS1 gene was cloned,its expression pattern during development was verified,and the induction by MeJA and ABA was confirmed.

关 键 词：延胡索 去甲乌药碱合成酶 异喹啉生物碱 启动子 顺式作用元件 

分 类 号：S567.239[农业科学—中草药栽培]