菘蓝中咖啡酰辅酶A氧甲基转移酶基因克隆与表达特征  

Cloning and Expression Characteristics of Caffeoyl-CoA Oxygen Methyltransferase Gene from Isatis indigotica Fort.

作　　者：李元玉 杨颖博 张国宁 李姝诺 马雪祺 陈万生[1,2,3] 肖莹 Li Yuanyu;Yang Yingbo;Zhang Guoning;Li Shunuo;MaXueqi;Chen Wansheng;XiaoYing(School of Pharmacy,Fujian University of Traditional Chinese Medicine,Fuzhou,350122;Research and Development Center of Chinese Medicine Resources and Biotechnology,Shanghai University of Traditional Chinese Medicine,Shanghai,201203;Medical Guarantee Center,Changzheng Hospital,Naval Medical University,Shanghai,200003;Shanghai Tufeng Pharmaceutical Technology Co.,Ltd.,Shanghai,201203)

机构地区：[1]福建中医药大学药学院,福州350122 [2]上海中医药大学中药资源与生物技术中心,上海201203 [3]海军军医大学附属长征医院医学保障中心,上海200003 [4]上海图锋医药科技有限公司,上海201203

出　　处：《分子植物育种》2022年第2期349-356,共8页Molecular Plant Breeding

基　　金：国家自然科学基金项目(81874335;31872665);上海市青年科技启明星计划项目(18QB1402700);上海市中央引导地方科技发展资金项目(YDZX20203100002948);上海中医药大学预算内项目(2020LK013)共同资助。

摘　　要：咖啡酰辅酶A氧甲基转移酶是植物中参与木脂素前体松柏醇生物合成的关键酶,为了研究菘蓝中CCoAOMT的表达特征,采用c DNA末端快速扩增(RACE)、PCR克隆IiCCoAOMT的全长cDNA及其基因组DNA序列;通过RT-qPCR检测比较IiCCoAOMT在四倍体菘蓝和二倍体菘蓝各器官中的表达以及不同胁迫刺激下的表达情况。IiCCoAOMT的c DNA序列全长1 098 bp (GenBank登录号:DQ115904),含774 bp的开放阅读框(ORF),编码257个氨基酸残基,对应的DNA序列含有5个外显子和4个内含子。多重序列比对结果显示Ii CCoAOMT与拟南芥At CCoAOMT同源性最高,且含有植物氧甲基转移酶-I的三个特征元件:motif A、motif B、motif C。在两种倍性菘蓝的根和叶中,四倍体表达量高于二倍体,而在两种倍性菘蓝的茎中表达量接近相同。冷处理(4℃)、盐处理(NaCl)、茉莉酸甲脂(MeJA)、赤霉素(GA_(3))、脱落酸(ABA)和水杨酸(SA)均能诱导IiCCoAOMT表达水平的上调。首次从菘蓝中克隆得到IiCCoAOMT并进行了表达特征研究,为进一步阐明菘蓝多倍性优势的分子机制提供理论基础,也为通过基因工程技术提高菘蓝中活性木脂素的含量提供了条件。Caffeoyl-CoA oxygen methyltransferase is a key enzyme involved in the biosynthesis of lignan precursor coniferyl alcohol in plants. To explore the expression characteristics of CCoAOMT in Isatis indigotica, the full-length cDNA and its genomic DNA sequence of IiCCoAOMT were cloned by rapid amplification of cDNA ends(RACE) and PCR. The expression levels of IiCCoAOMT in different organs of tetraploid and diploid I. indigotica and under different stress stimuli were detected by RT-qPCR. The cDNA sequence of IiCCoAOMT has a total length of 1 098 bp(GenBank accession No.: DQ115904), containing open reading frame(ORF) of 774 bp,encoding a polypeptide of 257 amino acid residues, and the corresponding DNA sequence contained 5 exons and 4 introns. Multiple sequence alignment showed that Ii CCoAOMT had the highest homology and closest genetic distance with At CCoAOMT from Arabidopsis and contained three characteristic elements of plant oxygen methyltransferase-I: motif A, motif B, and motif C. In the roots and leaves of the two ploidy I. indigotica, the expression level of tetraploid I. indigotica is higher than that of diploid, while the expression level in the stems of the two ploidy I. indigotica is close to the same. Cold treatment(4 ℃), salt treatment(NaCl), methyl jasmonate(MeJA), gibberellin(GA_(3)), abscisic acid(ABA) and salicylic acid(SA) treatments could up-regulate the expression level of IiCCoAOMT. The Caffeoyl-CoA oxygen methyltransferase gene(IiCCoAOMT) was cloned from I. indigotica for the first time and the expression characteristics were investigated, which provide the theoretical basis for further eluci-dating the molecular mechanism of polyploidy advantage of I. indigotica, and also prompted the possibility to increase the content of active lignans in I. indigotica through genetic engineering technology.

关 键 词：菘蓝(Lsatis indigotica) 咖啡酰辅酶A氧甲基转移酶 基因克隆 表达分析 RACE 

分 类 号：S567.2[农业科学—中草药栽培]