文章利用生物信息学方法对ω-6和ω-3脂肪酸脱氢酶基因家族的氨基酸序列特征、系统进化及功能分化进行分析。结果表明, ω-6和ω-3脂肪酸脱氢酶氨基酸序列均含有3个保守的组氨酸基序(Hisbox)。质体类ω-6和ω-3脂肪酸脱氢酶氨基酸N-端序列均有数目不等的信号肽区域, 并且在信号肽区中部发现1个由10个氨基酸残基组成的相对保守的疏水区, 推测为该类酶信号肽的功能区域。多数植物微体ω-6和ω-3脂肪酸脱氢酶氨基酸C-端均有KKXX-like motif内质网滞留信号, 而红花CtFAD2-3、CtFAD2-4、CtFAD2-5、CtFAD2-6和CtFAD2-7中没有检测到该滞留信号, 但C-端序列富含芳香族氨基酸, 同样具有内质网滞留信号的作用。系统进化分析可将所有序列分为4大类, 结果显示ω-3脂肪酸脱氢酶在原核生物中由ω-6脂肪酸脱氢酶基因进化而来; 并且植物质体和微体类ω-3脂肪酸脱氢酶均可再划分为单子叶和双子叶植物2个亚类, 表明植物质体和微体类ω-3脂肪酸脱氢酶功能的分化早在单子叶和双子叶植物分化之前就已经形成; 而植物微体类ω-6脂肪酸脱氢酶可细分为种子特异表达型和组成性表达型两类, 是在双子叶植物形成之后才开始分化的。各亚群间的功能分化位点分析表明, 除植物 FAD3/植物 FAD2外, 在所有存在功能分化的亚群间, 均存在后验概率值超过0.80的氨基酸位点, 而这些位点主要分布在Hisbox I的前后两端以及Hisbox II的前端, 表明这些位点变化对蛋白质功能域的大小或构象有着重要影响, 对于亚群间功能分化起着非常重要的作用。
The deduced amino acid sequences characteristics, phylogeny, and functional diverge of ω-6 and ω-3 fatty acid desaturase families were analyzed by using Bioinformatics methods. The results showed that all the deduced amino acid sequences shared three highly conserved histidine rich motifs (Hisbox). All the plastidial ω-6 and ω-3 fatty acid desaturases possessed putative N-terminal signal peptide with different amino acids. A relatively conserved hydrophobic region composed of 10 amino-acid residues was found in the middle of signal peptides, which is presumed to be the functional region of the signal peptide of these enzymes. Most of the plant microsomal ω-6 and ω-3 fatty acid desaturases (FAD2 and FAD3) contained a KKXX-like motif of endoplasmic reticulum (ER) retention signal at the C-terminus. However, no such motif was detected in safflower CtFAD2-3, CtFAD2-4, CtFAD2-5, CtFAD2-6, and CtFAD2-7, while an aromatic aa enriched signal (YKNK) was found at their C-terminus which has been reported to be both necessary and sufficient for maintaining localization of the enzymes in the ER. All the amino acid sequences were divided into four categories through phylogenetic analysis. It was suggested that ω-3 fatty acid desaturase originates in a prokaryotic lineage from ω-6 fatty acid desaturase. Both plastidial and microsomal ω-3 fatty acid desaturases could be divided into dicotyledonous and monocotyledonous subgroups, which inferred that functional differentiation of plastidial and microsomal ω-3 fatty acid desaturases had been formed before the divergence of dicotyledonous and monocotyledonous plants. Seed type and housekeeping type FAD2 diverged after the formation of dicotyledonous plants. Except for plant FAD3/plant FAD2, posterior probability values over 0.80 amino acid sites were identified among the functional differentiation subsets, which were mainly distributed at the front and back end of Hisbox I, and the front end of Hisbox II. This indicated that the variations of these amino acid sites played an important role in the size and conformation of protein functional domains and subfamily functional divergence.
[1] Los DA, Norio M. Structure and expression of fatty acid desaturases. Biochim Biophys Acta, 1998, 1394(1): 3-15.
[2] Iba K. Acclimative response to temperature stress in higher plants: approaches of gene engineering for tem-perature tolerance. Ann Rev Plant Biol, 2002, 53: 225-245.
[3] Zhang M, Barg R, Yin MA, Gueta-Dahan Y, Leikin- Frenkel A, Salts Y, Shabtai S, Ben-Hayyim G. Modulated fatty acid desaturation via overexpression of two distinct ω-3 desaturases differentially alters tolerance to various abiotic stresses in transgenic tobacco cells and plants. Plant J, 2005, 44(3): 361-371.
[4] Zhang JT, Liu H, Sun J, Li B, Zhu Q, Chen SL, Zhang HX. Arabidopsis fatty acid desaturase FAD2 is required for salt tolerance during seed germination and early seedling growth. PLoS One, 2012, 7(1): e30355.
[5] Zhang JT, Zhu JQ, Zhu Q, Liu H, Gao XS, Zhang HX. Fatty acid desaturase-6 (Fad6) is required for salt tolerance in Arabidopsis thaliana. Biochem Biophys Res Commun, 2009, 390(3): 469-474.
[6] Arondel V, Lemieux B, Hwang I, Gibson S, Goodman HM, Somerville CR. Map-based cloning of a gene controlling omega-3 fatty acid desaturation in Arabidopsis. Sci-ence, 1992, 258(5086): 1353-1355.
[7] Okuley J, Lightner J, Feldmann K, Yadav N, Lark E, Browse J. Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell, 1994, 6(1): 147-158.
[8] Iba K, Gibson S, Nishiuchi T, Fuse T, Nishimura M, Arondel V, Hugly S, Somerville C. A gene encoding a chloroplast omega-3 fatty acid desaturase complements alterations in fatty acid desaturation and chloroplast copy number of the fad7 mutant of Arabidopsis thaliana. J Biol Chem, 1993, 15(32): 24099-240105.
[9] Gibson S, Arondel V, Iba K, and Somerville C. Cloning of a temperature-regulated gene encoding a chloroplast ω-3 desaturase from Arabidopsis thaliana. Plant Physiol, 1994, 106(4): 1615-1621.
[10] Pham AT, Lee JD, Shannon JG, Bilyeu KD. Mutant alleles of FAD2-1A and FAD2-1B combine to produce soybeans with the high oleic acid seed oil trait. BMC Plant Biol, 2010, 10: 195, doi: 10.1186/1471-2229-10-195.
[11] Pham AT, Lee JD, Shannon JG, Bilyeu KD. A novel FAD2-1 A allele in a soybean plant introduction offers an alternate means to produce soybean seed oil with 85% oleic acid content. Theor Appl Genet, 2011, 123(5): 793-802.
[12] Pham AT, Shannon JG, Bilyeu KD. Combinations of mu-tant FAD2 and FAD3 genes to produce high oleic acid and low linolenic acid soybean oil. Theor Appl Genet, 2012, 125(3): 503-515.
[13] 戴晓峰, 肖玲, 武玉花, 吴刚, 卢长明. 植物脂肪酸去饱和酶及其编码基因研究进展. 植物学通报, 2007, 24(1): 105-113.
[14] 李冠, 杜钰, 黄琼, 李金玉. 脂肪酸脱氢酶研究进展. 食品与生物技术学报, 2007, 26(2): 121-126.
[15] 刘训言, 孟庆伟, 李滨. 植物ω-3脂肪酸去饱和酶研究进展. 细胞生物学杂志, 2004, 26(1): 34-38.
[16] Mártínez-Rivas JM, Sperling P, Lühs W, Heinz E. Spatial and temporal regulation of three different microsomal oleate desaturase genes (FAD2) from normal-type and high-oleic varieties of sunflower (Helianthus an-nuus L.). Mol Breed, 2001, 8(2): 159-168.
[17] Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, GibsonTJ, Higgins DG. Clustal W and Clustal X version 2.0. Bioinformatics, 2007, 23(21): 2947-2948.
[18] Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecu-lar evolutionary genetics analysis (MEGA) software ver-sion 4.0. Mol Bio Evol, 2007, 24(8): 1596-1599.
[19] Gu X. Statistical methods for testing functional divergence after gene duplication. Mol Biol Evol, 1999, 16(12): 1664-1674.
[20] Gu X. Maximum-likelihood approach for gene family evolution under functional divergence. Mole Biol and Evol, 2001, 18(4): 453-464.
[21] López Alonso D, García-Maroto F, Rodríguez-
