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| Cloning and Expression Analysis of 4-coumaric Acid Coenzyme A Ligase Gene Af4CL in Allium fistulosum |
| LI Yi1,2, XU Huan-Huan1,2, ZHANG Yu-Chen1,2, WANG Yong-Qin2,*, LIU Le-Cheng1,* |
1 School of horticulture, Yangtze University, Jingzhou 434025, China;
2 Vegetable Research Center/Key Laboratory of Horticultural Crop Biology and Germplasm Creation in North China, Ministry of Agriculture and Rural Areas, Beijing Academy of Agriculture and Forestry, Beijing 100097, China |
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Abstract 4-coumaric acid coenzyme A ligase (4CL) is a key enzyme in the flavonoid synthesis pathway, catalyzing the hydroxycoumaric acid to the formation of coumaroyl-CoA, which is a key enzyme in plant flavonoid biosynthesis at the starting substrate. In this research, the welsh onion (Allium fistulosum) was used as the material to clone the Af4CL gene, which encoding the welsh onion 4-coumaric acid-CoA ligase, and the bioinformatics analysis were also carried out. At the same time, the localization and prokaryotic expression system of Af4CL gene were analyzed. The results showed that the full-length coding region of Af4CL was 1 644 bp and it encoded 547 amino acids. The bioinformatics analysis showed that there were 2 conserved structural regions: BoxⅠ and BoxⅡ; Evolutionary analysis results indicated that Af4CL was most closely related to As4CL of garlic (Allium sativum) . The 35S:Af4CL-GFP vector was constructed, and the localization of Af4CL was analyzed by Agrobacterium-mediated transient expression system in tobacco (Nicotiana tabacum) leaves. The results showed that Af4CL was localized in the cell membrane; and the target protein was successfully induced and purified by the prokaryotic expression system. In conclusion, Af4CL gene was successfully cloned and the bioinformation analysis were carried out. At the same time, the target protein was successfully induced. These studies provide reference for the study of flavonoid metabolism and in vitro synthesis of flavonoids in A. fistulosum.
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Received: 24 March 2022
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Corresponding Authors:
*wangyqly@163.com;lchliu18@yangtzeu.edu.cn
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