菘蓝中咖啡酸氧甲基转移酶IiCOMT的克隆与表达分析  

Cloning and expression analysis of caffeic acid O-methyltransferase in Isatis indigotica(IiCOMT)

作　　者：张国宁 李元玉 马雪祺 李姝诺 陈万生 肖莹[1] ZHANG Guo-ning;LI Yuan-yu;MA Xue-qi;LI Shu-nuo;CHEN Wan-sheng;XIAO Ying(Research and Development Center of Chinese Medicine Resources and Biotechnology,Institute of Chinese Materia Medica,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Department of Pharmacy,Shanghai Changzheng Hospital,Second Military Medical University,Shanghai 200003,China;Department of TCM Processing,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)

机构地区：[1]上海中医药大学中药研究所中药资源与生物技术中心,上海201203 [2]第二军医大学附属长征医院药学部,上海200003 [3]上海中医药大学中药炮制教研室,上海201203

出　　处：《中草药》2022年第1期214-221,共8页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(81874335);国家自然科学基金资助项目(31872665);上海市青年科技启明星计划(18QB1402700);上海中医药大学项目(A1-GY20-306-02-08)。

摘　　要：目的克隆菘蓝Isatis indigotica中咖啡酸氧甲基转移酶(I. indigotica fortune caffeic acid O-methyltransferase,IiCOMT)编码基因,并进行生物信息学及表达分析。方法采用cDNA末端快速扩增(RACE)技术克隆菘蓝IiCOMT的全长cDNA,进而通过PCR获得其基因组DNA序列。通过qRT-PCR检测IiCOMT在四倍体菘蓝和二倍体菘蓝各器官以及不同胁迫条件下的表达情况。结果 IiCOMT全长cDNA为1376 bp(GenBank登录号DQ115905),包含1个1092 bp的开放阅读框(open readingframe,ORF),编码363个氨基酸残基,其对应的DNA序列含有3个内含子,4个外显子。IiCOMT基因在2种倍性菘蓝的根茎叶均有表达,且四倍体菘蓝中的表达均高于二倍体。胁迫因素盐处理(NaCl)、冷处理(4℃)、水杨酸(salicylicacid,SA)、茉莉酸甲脂(methyl jasmonate,MeJA)、赤霉素(gibberellic acid,GA3)和脱落酸(abscisic acid,ABA)均能诱导IiCOMT表达水平上调。结论 IiCOMT的克隆及表达分析,为进一步阐明与咖啡酸氧甲基转移酶相关的四倍体菘蓝优良性状形成的分子机制奠定了基础。Objective To clone the caffeic acid O-methyltransferase(IiCOMT) coding gene from Isatis indigotica, and perform its bioinformatics and expression analysis. Methods The full-length c DNA of IiCOMT was cloned by rapid amplification of c DNA ends(RACE), and its genomic DNA sequence was obtained by PCR. qRT-PCR was used to detect the expression of IiCOMT in tetraploid and diploid I. indigotica organs and under different stress conditions. Results The full-length c DNA of IiCOMT was 1376 bp(GenBank accession number: DQ115905) in length with an open reading frame(ORF) of 1092 bp, encoding a polypeptide of 363 amino acid residues, and its corresponding DNA sequence contained three introns and four exons. IiCOMT gene was expressed in the roots, stems and leaves of the two kinds of I. indigotica, and the expression level in the tetraploid I. indigotica was higher than that of diploid. Salt(NaCl), cold(4 ℃), salicylic acid(SA), methyl jasmonate(MeJA), gibberellic acid(GA3) and abscisic acid(ABA) treatments could all induce the up-regulation of IiCOMT expression. Conclusion The investigation of gene cloning and expression analysis of IiCOMT laid a foundation for further elucidating the molecular mechanism underlying the superiority of tetraploid I. indigotica related to caffeic acid O-methyltransferase.

关 键 词：菘蓝 咖啡酸氧甲基转移酶 基因克隆 生物信息学 表达分析 

分 类 号：R282.12[医药卫生—中药学]