Comparative Transcriptomics Analysis for Gene Mining and Identification of a Cinnamyl Alcohol Dehydrogenase Involved in Methyleugenol Biosynthesis from Asarum sieboldii Miq.

Jinjie Liu,Zhong Liu,Fawang Liu,Dongming Ma,Honglei Zhang,Chong Xu

doi:10.3390/molecules23123184

Abstract

Asarum sieboldii Miq., one of the three original plants of TCM ASARI RADIX ET RHIZOMA, is a perennial herb distributed in central and eastern China, the Korean Peninsula, and Japan. Methyleugenol has been considered as the most important constituent of Asarum volatile oil, meanwhile asarinin is also employed as the quality control standard of ASARI RADIX ET RHIZOMA in Chinese Pharmacopeia. They both have shown wide range of biological activities. However, little was known about genes involved in biosynthesis pathways of either methyleugenol or asarinin in Asarum plants. In the present study, we performed de novo transcriptome analysis of plant tissues (e.g., roots, rhizomes, and leaves) at different developmental stages. The sequence assembly resulted in 311,597 transcripts from these plant materials, among which 925 transcripts participated in ‘secondary metabolism’ with particularly up to 20.22% of them falling into phenylpropanoid biosynthesis pathway. The corresponding enzymes belong to seven families potentially encoding phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-monooxygenase (C4H), p-coumarate 3-hydroxylase (C3H), caffeoyl-CoA O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), and eugenol synthase (EGS). Moreover, 5 unigenes of DIR (dirigent protein) and 11 unigenes of CYP719A (719A subfamily of cytochrome P450 oxygenases) were speculated to be involved in asarinin pathway. Of the 15 candidate CADs, four unigenes that possessed high FPKM (fragments per transcript kilobase per million fragments mapped) value in roots were cloned and characterized. Only the recombinant AsCAD5 protein efficiently converted p-coumaryl, coniferyl, and sinapyl aldehydes to their corresponding alcohols, which are key intermediates employed not only in biosynthesis of lignin but also in that of methyleugenol and asarinin. qRT-PCR revealed that AsCAD5 had a high expression level in roots at three developmental stages. Our study will provide insight into the potential application of molecular breeding and metabolic engineering for improving the quality of TCM ASARI RADIX ET RHIZOMA.

Highlights

The genus Asarum, containing about 100 species which have conventionally been used as herbal medicines, are distributed in East Asia (65 species), North America (15 species), and EuropeMolecules 2018, 23, 3184; doi:10.3390/molecules23123184 www.mdpi.com/journal/molecules (1 species) [1,2]
The recombinant AsCAD5 protein efficiently converted p-coumaryl, coniferyl, and sinapyl aldehydes to their corresponding alcohols, which are key intermediates employed in biosynthesis of lignin and in that of methyleugenol and asarinin. qRT-PCR revealed that AsCAD5 had a high expression level in roots at three developmental stages
A. heterotropoides var. mandshuricum and A. sieboldii var. seoulense are both distributed in mountainous areas around Changbaishan within Northeast China

Summary

Introduction

The genus Asarum, containing about 100 species which have conventionally been used as herbal medicines, are distributed in East Asia (65 species), North America (15 species), and EuropeMolecules 2018, 23, 3184; doi:10.3390/molecules23123184 www.mdpi.com/journal/molecules (1 species) [1,2]. The Chinese Pharmacopeia has all along enlisted ASARI RADIX ET RHIZOMA, which is stipulated to be dried roots and rhizomes of three different taxa, that is, Asarum heterotropoides. (called Bei Xixin in Chinese), A. sieboldii Miq. var. Seoulense Nakai (called Hancheng Xixin in Chinese), or A. sieboldii Miq. (called Hua Xixin in Chinese) [3]. Seoulense are both distributed in mountainous areas around Changbaishan within Northeast China. For the similar habitat and overlapped distribution, A. heterotropoides var. Seoulense usually tend to be mixed together so that they are collectively called Liao Xixin in Chinese. A. sieboldii grows in central and eastern China with the Qinling–Dabashan mountainous areas as the center of its distribution, clearly separating it from the two former varieties on the basis of occupation [1]

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