颠茄精氨酸脱羧酶基因的克隆与表达分析  

Cloning and expression analysis on arginine decarboxylase genes in Atropa belladonna

作　　者：王中平[1,2] 秦白富[3] 强玮[1,2] 邱飞[1,2] 侯艳玲[1,2] 陈敏[4] 兰小中[5] 廖志华[1,2] 

机构地区：[1]西南大学生命科学学院三峡库区教育部生态环境教育部重点实验室,重庆400715 [2]西南大学西藏农牧学院药用植物联合研发中心,重庆400715 [3]中山大学生命科学学院,广东广州510275 [4]西南大学药学学院,重庆400715 [5]西藏大学农牧学院,西藏林芝850000

出　　处：《中草药》2016年第15期2734-2740,共7页Chinese Traditional and Herbal Drugs

基　　金：教育部新世纪人才支持计划(NCET-12-0930);国家自然科学基金项目(31370333);重庆市科技攻关项目(CSTC2012GGYYJS80013);西南大学中央高校基本科研业务费专项(XDJK2013A024)

摘　　要：目的克隆颠茄Atropa belladonna精氨酸脱羧酶(arginine decarboxylase,ADC)基因并进行生物信息学、组织表达谱和诱导谱分析。方法以颠茄总RNA反转录的cDNA为模板,采用RACE技术克隆颠茄ADC基因的全长cDNA序列,利用BLAST和PBIL等在线工具进行生物信息学分析,采用实时定量PCR(q RT-PCR)技术对ADC基因进行组织表达谱和诱导谱分析。结果克隆到2个ADC基因,分别命名为AbADC1(登录号KT802752)和AbADC2(登录号KT802751)。AbADC1基因cDNA全长为2 817 bp,编码712个氨基酸残基;AbADC2基因cDNA全长为2 992 bp,编码715个氨基酸残基。蛋白质序列比对表明,AbADC1与马铃薯Solanum tuberosum ADC的相似性较高,为89%;AbADC2与曼陀罗Datura stramonium ADC的相似性较高,为90%。q RT-PCR检测结果表明,AbADC1在须根中的表达量最高,而AbADC2在主根中的表达量最高;AbADC1和AbADC2均不受盐胁迫响应,AbADC2受低温胁迫响应。结论首次克隆并获得了2条颠茄ADC基因全长序列,为进一步研究颠茄托品烷类生物碱的代谢合成奠定了基础。Objective To clone the full-length cDNAs encoding arginine decarboxylase （ADC） from Atropa belladonna, and to characterize the genes at the bioinforrnatics and expression levels. Methods cDNAs were used as templates, the full-length cDNAs of ADC in A, belladonna was cloned through rapid amplification of cDNA ends （RACE） technique; Bioinforrnatics analysis of AbADC genes was performed by BLAST and PBIL on line. The expression levels of the two AbADC genes in different tissues as well as under two different types of stresses were detected based on qPCR analysis. Results Two ADC genes, namely AbADC 1 and AbADC2, were cloned from A. belladonna. AbADC 1 was 2 817 bp in length that encoded 712 amino acids with the highest identity of 89% with ADC from Solanum tuberosum; The full-length cDNA of AbADC2 was 2 992 bp in length that encoded 715 amino acids with the highest identity of 90% with ADC from Datura stramonium. The expression level of AbADC1 was significantly higher in secondary roots than that in any other organ, while the AbADC2 expression level was higher in main roots than that in other detected organs. The transcriptional levels of both AbADC1 and AbADC2 were not affected under salinity stress; AbADC2 expression decreased under cold stress, while AbADC 1 did not. Conclusion The cloning and characterization of the cDNAs encoding ADC from A. belladonna are reported for the first time, which provides the new candidate genes for engineering biosynthetic pathway of tropane alkaloids.

关 键 词：颠茄 精氨酸脱羧酶 基因表达 托品烷类生物碱 总RNA CDNA 

分 类 号：R282.12[医药卫生—中药学]