Transformation and Characterization of Δ12-Fatty Acid Acetylenase and Δ12-Oleate Desaturase Potentially Involved in the Polyacetylene Biosynthetic Pathway from Bidens pilosa.

Po-Yen Chen,Lie-Fen Shyur,Hsiao-Hang Chung,Mi-Jou Hsieh,Yung-Ting Tsai,Kin-Ying To,Cheng-Han Hsieh

doi:10.3390/plants9111483

Abstract

Bidens pilosa is commonly used as an herbal tea component or traditional medicine for treating several diseases, including diabetes. Polyacetylenes have two or more carbon–carbon triple bonds or alkynyl functional groups and are mainly derived from fatty acid and polyketide precursors. Here, we report the cloning of full-length cDNAs that encode Δ12-fatty acid acetylenase (designated BPFAA) and Δ12-oleate desaturase (designated BPOD) from B. pilosa, which we predicted to play a role in the polyacetylene biosynthetic pathway. Subsequently, expression vectors carrying BPFAA or BPOD were constructed and transformed into B. pilosa via the Agrobacterium-mediated method. Genomic PCR analysis confirmed the presence of transgenes and selection marker genes in the obtained transgenic lines. The copy numbers of transgenes in transgenic lines were determined by Southern blot analysis. Furthermore, 4–5 FAA genes and 2–3 OD genes were detected in wild-type (WT) plants. Quantitative real time-PCR revealed that some transgenic lines had higher expression levels than WT. Western blot analysis revealed OD protein expression in the selected transformants. High-performance liquid chromatography profiling was used to analyze the seven index polyacetylenic compounds, and fluctuation patterns were found.

Highlights

Bidens pilosa L., which belongs to the Asteraceae family, is an erect annual plant
We report the first cloning of full-length cDNAs that encode ∆12-fatty acid acetylenase and ∆12-oleate desaturase, which we predicted to be key genes in the polyacetylene biosynthesis pathway, from the medicinal plant B. pilosa var. radiata
Expression vectors (Supplementary Figure S1) containing two full-length cDNAs that encode ∆12-oleate desaturase and ∆12-fatty acid acetylenase were transformed independently into B. pilosa var. radiata according to the protocol detailed in our previous paper [30]

Summary

Introduction

Bidens pilosa L., which belongs to the Asteraceae family, is an erect annual plant. It is native to South America and is widely distributed in subtropical and tropical regions of the world.B. pilosa is commonly used as an herbal tea component or as traditional medicine in Latin America, Africa, and Asian countries for treating various disorders, such as inflammation, stomach illnesses, malaria, liver disorders, enteritis, dysentery, diabetes, and hypertension [1,2,3]. Bidens pilosa L., which belongs to the Asteraceae family, is an erect annual plant. It is native to South America and is widely distributed in subtropical and tropical regions of the world. B. pilosa is commonly used as an herbal tea component or as traditional medicine in Latin America, Africa, and Asian countries for treating various disorders, such as inflammation, stomach illnesses, malaria, liver disorders, enteritis, dysentery, diabetes, and hypertension [1,2,3]. In Taiwan, three variants (namely, radiata, pilosa, and minor) of B. pilosa are often used as a folk medicine for curing diabetes. Better anti-diabetic properties were observed in the plant extract from the variant radiata as compared with the other two variants [4]. Around 200 secondary metabolites have been identified from B. pilosa, including polyacetylenes, flavonoids, phenylpropanoids, and terpenes; Plants 2020, 9, 1483; doi:10.3390/plants9111483 www.mdpi.com/journal/plants

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