灰毡毛忍冬LmCCoAOMT基因的全长克隆及功能初步分析

Cloning and Functional Analysis of LmCCoAOMT Gene from Lonicera macranthoides Hand-Mazz

绿原酸是灰毡毛忍冬生长发育过程中产生的重要的次级代谢产物,而CCoAOMT是绿原酸合成过程中的关键基因。为进一步揭示灰毡毛忍冬LmCCoAOMT基因的功能,本研究利用RACE技术克隆LmCCoAOMT全长基因,通过生物信息学进行分析,并在大肠杆菌中表达该蛋白。此外,通过RT-qPCR和HPLC的方法研究CCoAOMT基因表达量和绿原酸的含量之间的相关性。结果显示LmCCoAOMT基因全长1 096 bp(GenBank:MN103348),ORF 735 bp,编码245个氨基酸。系统进化树分析发现LmCCoAOMT与藤本植物牵牛花的亲缘关系最近。生物信息学分析表明LmCCoAOMT的氨基酸序列不具有跨膜结构,其蛋白三级结构主要以α-螺旋和无规卷曲的形式存在。在大肠杆菌BL21 (DE3)中表达结果显示,其分子量为31.36 kD。RT-qPCR及HPLC结果表明,CCoAOMT基因在灰毡毛忍冬和金银花早期花中表达量最高,在末期表达量最低,且表达量与绿原酸含量有相关性。本研究为深入研究LmCCoAOMT基因功能及阐明灰毡毛忍冬绿原酸合成的分子机制提供了科学依据。

Chlorogenic acid(CGA) is an important secondary metabolite produced during the growth and development of Lonicera macranthoides Hand-Mazz, and CCoAOMT is a key gene in the synthesis of CGA. In order to further reveal the function CCoAOMT gene of L. macranthoides Hand-Mazz, RACE technology was used to clone the full length of LmCCoAOMT gene. The gene was analysed through bioinformatics, and expressed in Escherichia coli. In addition, the correlation between CCoAOMT gene expression and CGA content was studied by RT-qPCR and HPLC. The results showed that the full-length of LmCCoAOMT gene was 1 096 bp(GenBank: MN103348), the ORF was 735 bp, encoding 245 amino acid. The phylogenetic analysis revealed that LmCCoAOMT was the closest homolog of the Ipomoea nil CCoAOMT. Bioinformatics analysis showed that the amino acid sequence of LmCCoAOMT had no transmembrane structure, its tertiary protein structure was mainly in the form of α-helix and random coil. The expression results in E. coli BL21(DE3) showed that its molecular weight was 31.36 kD. RT-qPCR and HPLC results showed that the expression level of CCoAOMT gene was the highest in the early flowers and the lowest in the old flowers of L. macranthoides Hand-Mazz and Lonicera japonica Thunb, Meanwhile, the CCoAOMT gene expression level was correlated with the content of CGA. This paper provides a strong basis for future functional studies of LmCCoAOMT gene and elucidating the molecular mechanism of the synthesis of L.macranthoides Hand-Mazz CGA.