艾叶类黄酮O-甲基转移酶基因家族的鉴定及表达分析

Identification and expression analysis of flavonoid O-methyltransferases gene family in Artemisia argyi

艾(Artemisia argyi)以叶片入药,为我国常用大宗中药材,主要活性成分为挥发油、黄酮等化合物,具有多种药理活性。目前鉴定到的艾叶黄酮类物质多为氧甲基化修饰,而类黄酮O-甲基转移酶(flavonoid O-methyltransferase,FOMT)是黄酮类化合物氧甲基化修饰的关键酶。为进一步了解FOMT蛋白功能及特性,本研究对艾中FOMT基因家族进行了全基因组的挖掘和鉴定,并进行系统发育、染色体定位、基因序列特征、亚细胞定位预测、蛋白结构、基因结构分析及表达模式的分析和验证。结果表明,在艾的基因组中,共鉴定得到83个FOMT基因,系统发育树显示FOMT基因分为CCoAOMT(caffeoyl CoA O-methyltransferase)亚家族(32个基因)和COMT(caffeic acid O-methyltransferase)亚家族(51个基因)两个亚类。基因序列特征分析显示FOMT编码氨基酸的数目介于70~734 aa,分子质量为25296.55~34241.3 Da,等电点为4.51~9.99,与32个CCoAOMT亚家族成员相比,51个COMT亚家族成员中,约1/3的FOMT蛋白为疏水性蛋白,2/3蛋白为亲水性蛋白。亚细胞定位预测结果显示超过80%的CCoAOMT亚家族成员定位于细胞质,96%的COMT亚家族成员定位于叶绿体。COMT成员相对CCoAOMT成员motif数较多,且位于N端的motifs变异相对较大,位于C端的motifs相对保守;表达模式分析显示CCoAOMT亚家族成员主要在根中表达,而COMT成员主要在叶片中高量表达;实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)验证了部分FOMT在叶片中优势表达,具有组织表达特异性。本研究对艾叶FOMT基因家族进行鉴定与分析,为进一步深入研究FOMT功能和甲基化黄酮类化合物的生物合成提供理论依据。

Artemisia argyi(A.argyi)is a Chinese herbal medicine in China.The main active components are volatile oils,flavonoids,and other compounds,which have various pharmacological activities.Methoxylated flavonoids are the main active ingredients in A.argyi.Flavonoid O-methyltransferase(FOMT)is a key enzyme in the O-methylation of flavonoids.In order to further understand the function and characteristics of FOMT proteins,this paper carried out the whole genome mining and identification of FOMT genes in A.argyi and performed phylogenetic,chromosomal localization,gene sequence characterization,subcellular localization prediction,protein structure,gene structure analysis,and expression pattern analysis.The results showed that a total of 83 FOMT genes were identified in the genome of A.argyi.The phylogenetic tree shows that FOMT genes are divided into two subgroups,CCoAOMT(caffeoyl CoA O-methyltransferase)subfamily(32 genes)and COMT(caffeic acid O-methyltransferase)subfamily(51 genes).Gene sequence analysis showed that the number of amino acids encoded by FOMT was 70-734 aa,the molecular weight was 25296.55-34241.3 Da,and the isoelectric point was 4.51-9.99.Compared with 32 members of the CCoAOMT subfamily,nearly 1/3 of the 51 members of the COMT subfamily were hydrophobic proteins and 2/3 were hydrophilic proteins.Subcellular localization prediction showed that more than 80%of CCoAOMT subfamily members were located in the cytoplasm,and 96%of COMT subfamily members were located in the chloroplast.COMT subfamily members have more motifs than CCoAOMT subfamily members.The N-terminal motifs of COMT subfamily proteins are relatively variable,while the C-terminal motifs are relatively conserved.Expression pattern analysis showed that CCoAOMT subfamily members were mainly expressed in roots,while COMT members were mainly expressed in leaves.Some FOMTs showed the tissue expression specificity by real-time quantitative PCR analysis,especially in leaves.In this study,we identified and analyzed the FOMT gene family in A.argyi,and provided a theoretical basis for further research on the function of FOMTs and the biosynthesis of methylated flavonoids in A.argyi.