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Gene Cloning and Functional Analysis of PfSAD3 and PfSAD4 in Perilla frutescens |
YIN Miao1,2, WANG Yao1,2, WANG Zhuang-Lin1,2, HU Ting1,2, CHEN Shu-Wei1,2, ZHOU Ya-Li1,2, XING Zhi1,2, WANG Ji-Ping1,2,*, LI Run-Zhi1,2 |
1 College of Agronomy, Shanxi Agricultural University, Taigu 030801, China; 2 Shanxi Engineering Research Center for Genetics and Metabolism of Specific Crops, Taigu 030801, China |
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Abstract Δ9-stearoyl-ACP dehydrogenase (SAD) is a key enzyme involved in the synthesis and accumulation of unsaturated fatty acids in plants. In this study, to explore the role of the SAD enzyme in the fatty acid biosynthesis of Perilla frutescens, PfSAD3 (GENE_023515) and PfSAD4 (GENE_042129) genes were cloned and bioinformatic analysis and functional verification on them were conducted. Using the seeds of 'Jinzisu 1' as the experiment material, two full-length sequences of PfSAD3 and PfSAD4 were obtained, with the ORF lengths of 1 173 and 1 116 bp, respectively. MEGA 11.0 was used to construct evolutionary trees for the protein sequences of PfSAD3 and PfSAD4, as well as the SAD sequences of Arabidopsis thaliana and other species, it was found that PfSAD3 was closely related to LcSAD of Leucas cephalotes, and PfSAD4 was closely related to RcSAD of Ricinus communis. After constructing the yeast (Saccharomyces cerevisiae) expression vectors pYES2.0-PfSAD3 and pYES2.0-PfSAD4, PfSAD3 and PfSAD4 were transformed into the wild-type yeast strain INVSc1 and the defective yeast strain BY4389, respectively. The results showed that overexpression of PfSAD3 and PfSAD4 significantly increased the total fatty acid content and unsaturated fatty acid content in yeast (P<0.05), and restored the ability of the defective yeast to synthesize palmitoleic acid (C16:1) and oleic acid (C18:1). The substrate specificity of the PfSAD3 and PfSAD4 proteins was analyzed by adding exogenous fatty acids. The result showed that compared to PfSAD3, PfSAD4 had a more substantial substrate preference ratio for C18:0, and exhibited stronger enzyme activity. The functions of PfSAD3 and PfSAD4 were verified by overexpressing them in wild tobacco (Nicotiana tabacum). The result showed that the total fatty acid content and unsaturated fatty acid content in transgenic tobacco leaves were significantly increased (P<0.05), which was consistent with the results of yeast experiments. This study provides scientific bases and molecular targets for further elucidating the molecular regulatory mechanism of unsaturated fatty acid synthesis in oil crops such as P. frutescens, and for cultivating new oil crop varieties rich in oils through genetic engineering methods.
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Received: 12 January 2024
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Corresponding Authors:
*sxndwjp@163.com
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[1] 程宏, 王慧杰, 冯瑞云, 等. 2018. 山西省紫苏产业的发展现状及对策[J]. 山西农业科学, 46(04): 648-650, 655. (Cheng H, Wang H J, Feng R Y, et al.2018. Development status and countermeasures of Perilla industry in Shanxi province[J]. Shanxi Agricultural Sciences, 46(04): 648-650, 655.) [2] 李腾. 2022. 特色油料作物油莎豆硬脂酰-ACP脱氢酶基因CeSAD的鉴定及功能分析[D]. 硕士学位论文, 山西农业大学, 导师: 李润植, pp. 40-49. (Li T.2022. Identification and functional analysis of CeSAD, a stearoyl-ACP dehydrogenase gene from the specialty oilseed crop oil salsa bean[D]. Thesis for M.S., Shanxi Agricultural University, Supervisor: Li R Z, pp. 40-49.) [3] 邢志, 董书言, 王超, 等. 2022. 紫苏硬脂酰-ACP脱氢酶基因家族鉴定及表达分析[J]. 西北植物学报, 42(01): 57-65. (Xing Z, Dong S Y, Wang C, et al.2022. Identification and expression analysis of the stearoyl-ACP dehydrogenase gene family in Perilla frutescens[J]. Acta Botanica Sinica of Northwest China, 42(01): 57-65.) [4] 许林, 向琮琳, 陈宝林, 等. 2021. 山茶CjSAD1的克隆及其在耐寒中的作用[J]. 植物生理学报, 57(03): 589-596. (Xu L, Xiang C L, Chen B L, et al.2021. Cloning of Camellia sinensis CjSAD1 and its role in cold tolerance[J]. Journal of Plant Physiology, 57(03): 589-596.) [5] Broadwater J A, Ai J, Loehr T M, et al.1998. Peroxodiferric intermediate of stearoyl-acyl carrier protein delta 9 desaturase: Oxidase reactivity during single turnover and implications for the mechanism of desaturation[J]. Biochemistry, 37(42): 14664-14671. [6] Chen J, Gao J, Zhang L, et al.2023. Tung tree stearoyl-acyl carrier protein Δ9 desaturase improves oil content and cold resistance of Arabidopsis and Saccharomyces cerevisiae[J]. Frontiers in Plant Science, 14: 1144853. [7] Ding Z T, Shen J Z, Pan L L, et al.2016. CsSAD: A fatty acid desaturase gene involved in abiotic resistance in Camellia sinensis (L.)[J]. Genetics and Molecular Research, 15(1): 15017512. [8] Dong Y, Ren Y C, Yang M S, et al.2017. Construction of a new type of multi-gene plant transformation vector and genetic transformation of tobacco[J]. Biologia Plantarum, 61(1): 13-23. [9] Du H, Huang M, Hu J, et al.2016. Modification of the fatty acid composition in Arabidopsis and maize seeds using a stearoyl-acyl carrier protein desaturase-1 (ZmSAD1) gene[J]. BMC Plant Biology, 16(1): 137. [10] Kachroo A, Shanklin J, Whittle E, et al.2007. The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the contribution of leaf isoforms to oleic acid synthesis[J]. Plant Molecular Biology, 63(2): 257-271. [11] Kim H U, Lee K R, Shim D, et al.2016. Transcriptome analysis and identification of genes associated with ω-3 fatty acid biosynthesis in Perilla frutescens (L.) var. frutescens[J]. BMC Genomics,17: 474. [12] Li L, Li Y, Wang R, et al.2020. Characterization of the stearoyl-ACP desaturase gene (PoSAD) from woody oil crop Paeonia ostii var. lishizhenii in oleic acid biosynthesis[J]. Phytochemistry, 178: 112480. [13] Li T, Sun Y, Chen Y, et al.2022. Characterisation of two novel genes encoding Δ9 fatty acid desaturases (CeSADs) for oleic acid accumulation in the oil-rich tuber of Cyperus esculentus[J]. Plant Science, 319: 111243. [14] Lindqvist Y, Huang W, Schneider G, et al.1996. Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins[J]. EMBO Journal, 15(16): 4081-4092. [15] Liu B, Sun Y, Hang W, et al.2020. Characterization of a novel Acyl-ACP Δ9 desaturase gene responsible for palmitoleic acid accumulation in a diatom Phaeodactylum tricornutum[J]. Frontiers in Microbiology, 11: 584589. [16] Liu B, Sun Y, Xue J, et al.2019. Stearoyl-ACP Δ9 desaturase 6 and 8 (GhA-SAD6 and GhD-SAD8) are responsible for biosynthesis of palmitoleic acid specifically in developing endosperm of upland cotton seeds[J]. Frontiers in Plant Science, 10: 703. [17] Liu Z J, Yang X H, Fu Y.2009. SAD, a stearoyl-acyl carrier protein desaturase highly expressed in high-oil maize inbred lines[J]. Russian Journal of Plant Physiology, 56: 709-715. [18] Orlova I V, Serebriiskaya T S, Popov V, et al.2003. Transformation of tobacco with a gene for the thermophilic acyl-lipid desaturase enhances the chilling tolerance of plants[J]. Plant & Cell Physiology, 44(4): 447-450. [19] Peng D, Zhou B, Jiang Y, et al.2018. Enhancing freezing tolerance of Brassica napus L. by overexpression of a stearoyl-acyl carrier protein desaturase gene (SAD) from Sapium sebiferum (L.) Roxb[J]. Plant Science, 272: 32-41. [20] Qin J, Kurt E, LBassi T, et al.2023. Biotechnological production of omega-3 fatty acids: Current status and future perspectives[J]. Frontiers in Microbiology, 14: 1280296. [21] Ramesh A M, Kesari V, Rangan L.2014. Characterization of a stearoyl-acyl carrier protein desaturase gene from potential biofuel plant, Pongamia pinnata L[J]. Gene, 542(2): 113-121. [22] Sakai H, Kajiwara S.2003. A stearoyl-CoA-specific delta 9 fatty acid desaturase from the basidiomycete Lentinula edodes[J]. Bioscience Biotechnology and Biochemistry, 67(11): 2431-2437. [23] Tong L, Shu-Ming P, Wu-Yuan D, et al.2006. Characterization of a new stearoyl-acyl carrier protein desaturase gene from Jatropha curcas[J]. Biotechnology Letters, 28(9): 657-662. [24] Troncoso-Ponce M A, Barthole G, Tremblais G, et al.2016. Transcriptional activation of two delta-9 palmitoyl-ACP desaturase genes by MYB115 and MYB118 is critical for biosynthesis of omega-7 monounsaturated fatty acids in the endosperm of Arabidopsis seeds[J]. Plant Cell, 28(10): 2666-2682. [25] Wallis J G, Browse J.2002. Mutants of Arabidopsis reveal many roles for membrane lipids[J]. Progress in Lipid Research, 41(3): 254-278. [26] Wang J, Yu X, Wang K, et al.2023. Reprogramming the fatty acid metabolism of Yarrowia lipolytica to produce the customized omega-6 polyunsaturated fatty acids[J]. Bioresource Technology, 383: 129231. [27] Yu H, Qiu J F, Ma L J, et al.2017. Phytochemical and phytopharmacological review of Perilla frutescens L. (Labiatae), a traditional edible-medicinal herb in China[J]. Food and Chemical Toxicology, 108(Pt B): 375-391. [28] Zaborowska Z, Starzycki M, Femiak I, et al.2002. Yellow lupine gene encoding stearoyl-ACP desaturase-organization, expression and potential application[J]. Acta Biochimica Polonica, 49(1): 29-42. [29] Zhang Y, Shen Q, Leng L, et al.2021. Incipient diploidization of the medicinal plant Perilla within 10,000 years[J]. Nature Communications, 12(1): 5508. |
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