不同胡麻品种TAG合成途径关键基因表达与含油量、脂肪酸组分的相关性分析

doi:10.11686/cyxb2018321

摘要/Abstract

摘要： 胡麻是一种富含不饱和脂肪酸尤其是α-亚麻酸的油料作物,明晰TAG合成途径中与胡麻含油量和脂肪酸组分相关的基因具有重要的理论意义。以3个含油量和脂肪酸组分有显著差异的胡麻品种(系)为材料,分析了不同品种(系)、不同组织和不同发育阶段胡麻油脂和脂肪酸组分的动态积累模式和TAG合成途径中7个关键基因的动态表达模式,以及含油量和不饱和脂肪酸与7个关键基因表达的相关性。结果表明,胡麻开花后10~20 d,是种子油脂和亚麻酸的快速积累期,且不同胡麻品种(系)油脂和亚麻酸的动态积累模式差异显著。TAG合成途径中的7个关键基因(GPAT9、DGAT1、DGAT2、PDAT1、PDAT2、FAD2A 和 FAD3A)在胡麻不同发育阶段的不同组织中均有表达,在不同品种(系)间的表达模式各不相同。其中PDAT1、DGAT1和DGAT2基因的动态表达模式与含油量的动态积累模式显著正相关,PDAT1的动态表达模式与亚麻酸的动态积累模式显著正相关,且在高油高亚麻酸材料的种子动态发育阶段中PDAT1基因的累积表达量也显著高于低油低亚麻酸材料。因此,PDAT1可能是影响胡麻不同品种(系)中含油量和亚麻酸含量的关键基因。

关键词: 胡麻, 含油量, 亚麻酸, 基因表达分析, 相关性分析

Abstract: Flax (Linum usitatissimum) is an oil crop containing abundant unsaturated fatty acids, especially α-linolenic acid. To better understand biosynthesis of these compounds, it is highly relevant to explore functions of genes in the TAG (triacylglycerol) synthesis pathway, which are associated with flax oil content and levels of various fatty acid components. In this study, the dynamic accumulation patterns of oil and fatty acid components in flax and the corresponding expression patterns of 7 key genes in the TAG synthesis pathway were determined in three varieties (lines) of flax identified as having significant differences in oil content or fatty acid composition. The correlations between expression patterns of 7 key genes, oil contents and unsaturated fatty acid levels were analyzed. It was found that seed oil and linolenic acid accumulate rapidly between 10 and 20 days after flowering in flax. However, the time course of the accumulation patterns of oil and linolenic acid differed significantly in the three varieties studied. Meanwhile, in the TAG synthesis pathway the 7 key genes (GPAT9, DGAT1, DGAT2, PDAT1, PDAT2, FAD2A, and FAD3A) were found to be expressed in different flax tissues at different stages of development, with the patterns of expression also differing among the three lines. The expression patterns of PDAT1, DGAT1, and DGAT2 were positively correlated with the dynamic accumulation pattern of oil content, and the pattern of PDAT1 was significantly positively correlated with the accumulation pattern of linolenic acid. In addition, the cumulative expression of PDAT1 in high oil and high-linolenic-acid flax germplasm is also significantly higher than that in low-oil and low-linolenic-acid material during seed development. Therefore, PDAT1 appears to be a key gene in the regulation of oil and linolenic acid content in different varieties (lines) of flax.

Key words: flax, oil content, linolenic acid, gene expression analysis, correlation analysis

李闻娟, 齐燕妮, 王利民, 党照, 赵利, 赵玮, 谢亚萍, 王斌, 张建平, 李淑洁. 不同胡麻品种TAG合成途径关键基因表达与含油量、脂肪酸组分的相关性分析[J]. 草业学报, 2019, 28(1): 138-149.

LI Wen-juan, QI Yan-ni, WANG Li-min, DANG Zhao, ZHAO Li, ZHAO Wei, XIE Ya-ping, WANG Bin, ZHANG Jian-ping, LI Shu-jie. Correlation between oil content or fatty acid composition and expression levels of genes involved in TAG biosynthesis in flax[J]. Acta Prataculturae Sinica, 2019, 28(1): 138-149.

参考文献

[1] Thambugala D, Duguid S, Loewen E, et al. Genetic variation of six desaturase genes in flax and their impact on fatty acid composition. Theoretical & Applied Genetics, 2013, 126(10): 2627-2641.
[2] Huis R, Hawkins S, Neutelings G.Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biology, 2010, 10(1): 1-14.
[3] Green A G.Genetic control of polyunsaturated fatty acid biosynthesis in flax (Linum usitatissimum) seed oil. Tag. theoretical & Applied Genetics. theoretische Und Angewandte Genetik, 1986, 72(5): 654-661.
[4] Westcott N D, Muir A D.Flax seed lignan in disease prevention and health promotion. Phytochemistry Reviews, 2003, 2(3): 401-417.
[5] Daun J, Declercq D.Sixty years of Canadian flaxseed quality surveys at the grain research laboratory. Flax Institute of the United States, 1994.
[6] Mozaffarian D.Does alpha-linolenic acid intake reduce the risk of coronary heart disease? A review of the evidence. Alternative Therapies in Health & Medicine, 2005, 11(3): 24-30.
[7] Simopoulos A P.Essential fatty acids in health and chronic disease. American Journal of Clinical Nutrition, 1999, 70(3): 560-569.
[8] Williams D, Verghese M, Walker L T, et al. Flax seed oil and flax seed meal reduce the formation of aberrant crypt foci (ACF) in azoxymethane-induced colon cancer in Fisher 344 male rats. Food & Chemical Toxicology an International Journal Published for the British Industrial Biological Research Association, 2007, 45(1): 153-159.
[9] Huang A H C. Oil bodies and oleosins in seeds. Annual Review of Plant Biology, 1992, 43(4): 177-200.
[10] Pan X, Siloto R M, Wickramarathna A D, et al. Identification of a pair of phospholipid: Diacylglycerol acyltransferases from developing flax (Linum usitatissimum L.) seed catalyzing the selective production of trilinolenin. Journal of Biological Chemistry, 2013, 288(33): 24173-24188.
[11] Vigeolas H, Geigenberger P.Increased levels of glycerol-3-phosphate lead to a stimulation of flux into triacylglycerol synthesis after supplying glycerol to developing seeds of Brassica napus L. in planta. Planta, 2004, 219(5): 827-835.
[12] Jain R K, Coffey M, Lai K, et al. Enhancement of seed oil content by expression of glycerol-3-phosphate acyltransferase genes. Biochemical Society Transactions, 2000, 28(6): 958-961.
[13] Cao J, Li J L, Li D, et al. Molecular identification of microsomal acyl-CoA: Glycerol-3-phosphate acyltransferase, a key enzyme in de novo triacylglycerol synthesis. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(52): 19695-19700.
[14] Lewin T M, Wang P, Coleman R A.Analysis of amino acid motifs diagnostic for the sn-glycerol-3-phosphate acyltransferase reaction. Biochemistry, 1999, 38(18): 5764-5771.
[15] Weselake R J, Shah S, Tang M, et al. Metabolic control analysis is helpful for informed genetic manipulation of oilseed rape (Brassica napus) to increase seed oil content. Journal of Experimental Botany, 2008, 59(13): 3543-3549.
[16] Li R, Yu K D.DGAT1, DGAT2 and PDAT expression in seeds and other tissues of epoxy and hydroxy fatty acid accumulating plants. Lipids, 2010, 45(2): 145-157.
[17] Perry H J, Harwood J L.Changes in the lipid content of developing seeds of Brassica napus. Phytochemistry, 1993, 32(6): 1411-1415.
[18] Turchetto-Zolet A C, Maraschin F S, Morais G L D, et al. Evolutionary view of acyl-CoA diacylglycerol acyltransferase (DGAT), a key enzyme in neutral lipid biosynthesis. BMC Evolutionary Biology, 2011, 11(1): 1-14.
[19] Zhang M, Fan J, Taylor D C, et al. DGAT1 and PDAT1 acyltransferases have overlapping functions in Arabidopsis triacylglycerol biosynthesis and are essential for normal pollen and seed development. Plant Cell, 2009, 21(12): 3885-3901.
[20] Xu J, Francis T, Mietkiewska E, et al. Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content. Plant Biotechnology Journal, 2008, 6(8): 799-818.
[21] Kroon J T, Wei W, Simon W J, et al. Identification and functional expression of a type 2 acyl-CoA: Diacylglycerol acyltransferase (DGAT2) in developing castor bean seeds which has high homology to the major triglyceride biosynthetic enzyme of fungi and animals. Phytochemistry, 2006, 67(23): 2541-2549.
[22] Li R, Yu K, Hatanaka T, et al. Vernonia DGATs increase accumulation of epoxy fatty acids in oil. Plant Biotechnology Journal, 2010, 8(2): 184-195.
[23] Weiss S B, Kennedy E P, Kiyasu J Y.The enzymatic synthesis of triglycerides. Journal of Biological Chemistry, 1960, 235(9): 40-44.
[24] Shockey J M, Gidda S K, Chapital D C, et al. Tung tree DGAT1 and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic reticulum. Plant Cell, 2006, 18(9): 2294-2313.
[25] Banas A, Dahlqvist A, Stahl U, et al. The involvement of phospholipid: Diacylglycerol acyltransferases in triacylglycerol production. Biochemical Society Transactions, 2000, 28(6): 703-705.
[26] Stahl U, Carlsson A S, Lenman M, et al. Cloning and functional characterization of a phospholipid: Diacylglycerol acyltransferase from Arabidopsis. Plant Physiology, 2004, 135(3): 1324-1335.
[27] Fofana B, Cloutier S, Duguid S, et al. Gene expression of stearoyl-ACP desaturase and Δ12 fatty acid desaturase 2 is modulated during seed development of flax (Linum usitatissimum). Lipids, 2006, 41(7): 705-712.
[28] Liu Y H, Zhang L J, Zhang H R.Progress of research on Δ12 fatty acid desaturases and their coding genes. Acta Prataculturae Sinica, 2011, 20(3): 256-267.
刘永红, 张丽静, 张洪荣. Δ12-脂肪酸脱氢酶及其编码基因研究进展. 草业学报, 2011, 20(3): 256-267.
[29] Banik M, Duguid S, Cloutier S.Transcript profiling and gene characterization of three fatty acid desaturase genes in high, moderate, and low linolenic acid genotypes of flax (Linum usitatissimum L.) and their role in linolenic acid accumulation. Genome, 2011, 54(6): 471-483.
[30] Covello P S, Reed D W.Functional expression of the extraplastidial Arabidopsis thaliana oleate desaturase gene (FAD2) in Saccharomyces cerevisiae. Plant Physiology, 1996, 111(1): 223-226.
[31] Yadav N S, A Wierzbicki, L Pérez-Grau, et al. Cloning of higher plant ω-3 fatty acid desaturases. Plant Physiology, 1993, 103(2): 467-476.
[32] Schmittgen T D, Livak K J.Analyzing real-time PCR data by the comparative C(T) method. Nature Protocols, 2008, 3(6): 1101-1108.
[33] Rajwade A V, Arora R S, Kadoo N Y, et al. Relatedness of Indian flax genotypes (Linum usitatissimum L.): An inter-simple sequence repeat (ISSR) primer assay. Molecular Biotechnology, 2010, 45(2): 161-170.
[34] Chia T Y, Pike M J, Rawsthorne S.Storage oil breakdown during embryo development of Brassica napus. Journal of Experimental Botany, 2005, 56(415): 1285-1296.
[35] Jako C, Taylor D C.Seed-specific over-expression of an Arabidopsis cDNA encoding a diacylglycerol acyltransferase enhances seed oil content and seed weight. Plant Physiology, 2001, 126(2): 861-874.
[36] Shockey J, Regmi A, Cotton K, et al. Identification of Arabidopsis GPAT9 (At5g60620) as an essential gene involved in triacylglycerol biosynthesis. Plant Physiology, 2016, 170(1): 163.
[37] Kuo T M, Gardner H.Lipid biotechnology. New York: Crc Press, 2002.