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
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.
[1] 程龙军, 童再康, 黄华宏, 等. 2010. 光皮桦中4-香豆酸辅酶A连接酶基因Bl4CL的克隆和表达分析[J]. 植物生理学通讯, 46(1): 5-10. (Chen L J, Tong Z K, Huang H H, et al.2010. Cloning and expression analysis of 4-coumarate: CoA ligase gene Bl4CL in Betula luminifera H. Winkl[J]. Plant Physiology Communications, 46(1): 5-10.) [2] 黄满芬. 2014. 桑树C4H、4CL及CHS基因克隆及其在不同桑种质间的表达差异[D]. 硕士学位论文, 江苏科技大学, 导师: 刘利, pp. 37-41. (Huang M F.2014. Molecular cloning of cinnamic acid 4-hydroxylase gene, 4-coumarate: CoA ligase gene and chalcone synthase gene in Mulberry (Morus multicaulis) and expression analysis of them among different germplasm resources[D]. Thesis for M.S., Jiangsu University of Science and Technology, Supervisor: Liu L, pp. 37-41.) [3] 黄胜雄, 胡尚连, 孙霞, 等. 2008. 木质素生物合成酶4CL基因的遗传进化分析[J]. 西北农林科技大学学报(自然科学版), 36(10): 199-206. (Huang S X, Hu S L, Sun X, et al.2008. Genetic and evolutionary analysis of lignin biosynthase 4CL gene[J]. Journal of Northwest A&F University (Natural Science Edition), 36(10): 199-206.) [4] 倪志勇, 王娟, 吕萌, 等. 2010. 棉花4-香豆酸辅酶A连接酶基因克隆及原核表达[J]. 西北植物学报, 30(3): 0429-0436. (Ni Z Y, WANG J, Lv M, et al.2010. Cloning and prokaryotic expression of Gh4CL2 gene from Gossypium hirsutum L.[J] Acta Botanica Boreali-Occidentalia Sinica, 30(3): 0429-0436.) [5] Cao S, Huang C, Luo L, et al.2020. Cell-specific suppression of 4-Coumarate-CoA ligase gene reveals differential effect of lignin on cell physiological function in Populus[J]. Frontiers in Plant Science, 11(17): 1-11. [6] Chen X, Fang X, Zhang Y Y, et al.2019. Overexpression of a soybean 4-coumaric acid: Coenzyme a ligase (GmPI4L) enhances resistance to Phytophthora sojae in soybean[J]. Functional Plant Biology, 46(4): 304-313. [7] Djoukeng J D, Arbona V, Argamasilla R, et al.2008. Flavonoid profiling in leaves of citrus genotypes under different environmental situations[J]. Journal of Agricultural and Food Chemistry, 56(23): 11087-11097. [8] Gui J S, Shen J H, Li L G, et al.2011. Functional characterization of evolutionarily divergent 4-coumarate: Coenzyme a ligases in rice[J]. Plant Physiology, 157(2): 574-586. [9] Hu W J, Kawaoka A, Tsai C J, et al.1998. Compartmentalized expression of two structurally and functionally distinct 4-coumarate: CoA ligase genes in aspen (Populus tremuloides)[J]. Proceedings of the National Academy of Sciences of the USA, 95(9): 5407-5412. [10] Knobloch K H, Hahlbrock K.1977. 4-coumarate: CoA, ligase from cell suspension cultures of Petroselenium hortense Hoffm. Partial purification, substrate specificity, and further properties[J]. Archives of Biochemistry and Biophysics, 184(1): 237-248. [11] Koutaniemi S, Warinowski T, Kärkönen A, et al.2007. Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR[J]. Plant Molecular Biology, 65(3): 311-328. [12] Li Y, Kim J L, Pysh L, et al.2015. Four isoforms of Arabidopsis 4-Coumarate: CoA ligase have overlapping yet distinct roles in phenylpropanoid metabolism[J]. Plant Physiology, 169(4): 2409-2421. [13] Li S S, Chang Y, Li B, et al.2020. Functional analysis of 4-coumarate: CoA ligase from Dryopteris fragrans in transgenic tobacco enhances lignin and flavonoids[J]. Genetics Molecular Biology, 43(2): 1-7. [14] Liu Y, Fang S, Yang W, et al.2018. Light quality affects flavonoid production and related gene expression in Cyclocarya paliurus[J]. Journal of Photochemistry & Photobiology B: Biology, 179: 66-73. [15] Livak K J, Schmittgen T D.2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J]. Methods, 25(4): 402-408. [16] Maritim T K, Masand M, Seth R, et al.2021. Transcriptional analysis reveals key insights into seasonal induced anthocyanin degradation and leaf color transition in purple tea (Camellia sinensis)[J]. Scientific Reports, 11(1): 1244-1258. [17] Rastogil S, Kumar R, Chanotiya C S, et al.2013. 4-Coumarate: CoA ligase partitions metabolites for eugenol biosynthesis[J]. Plant Cell Physiology, 54(8): 1238-1252. [18] Rodriguez-Garcia C, Sanchez-Quesada C.2019. Dietary flavonoids as cancer chemopreventive agents: An updated review of human studies[J]. Antioxidants (Basel), 8(5): 137-160. [19] Soltani B M, Ehlting J, Hamberger B, et al.2006. Multiple cis-regulatory elements regulate distinct and complex patterns of developmental and wound-induced expression of Arabidopsis thaliana 4CL gene family members[J]. Planta, 224(5): 1226-1238. [20] Sun S C, Xiong X P, Zhang X L, et al.2020. Characterization of the Gh4CL gene family reveals a role of Gh4CL7 in drought tolerance[J]. BMC Plant Biology, 20(1): 125-140. [21] Sun H, Guo K, Feng S, et al.2015. Positive selection drives adaptive diversification of the 4-coumarate: CoA ligase (4CL) gene in angiosperms[J]. Ecology and Evolution, 5(16): 3413-3420. [22] Voelker S L, Lachenbruch B, Meinzer F C, et al.2011. Reduced wood stiffness and strength, and altered stem form, in young antisense 4CL transgenic poplars with reduced lignin contents[J]. New Phytologist, 189(4): 1096-1109. [23] Wasson A P, Pellerone F I, Mathesius U, et al.2006. Silencing the flavonoid pathway in Medicago truncatula inhibits root nodule formation and prevents auxin transport regulation by rhizobia[J]. The Plant Cell, 18(7): 1617-1629. [24] Xie D Y, Sharma S. B, Zhang S, et al.2011. Anthocyanin reductases from Medicago truncatula and Arabidoposis thaliana[J]. Archives of Biochemistry and Biophysics,422(1): 91-102. [25] Yang Y N, Li R G, Qi M.2000. In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves[J]. The Plant Journal, 22(6): 543-551. [26] Zhang E H, Guo X F, Meng Z F, et al.2015. Construction, expression, and characterization of Arabidopsis thaliana 4CL and Arachis hypogaea RS fusion gene 4CL::RS in Escherichia coli[J]. World Journal of Microbiology and Biotechnology, 31(9): 1379-1385.
