花生油酸脱氢酶基因AhFAD2A和AhFAD2B的时空表达特征
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摘要
花生Arachis hypogaea油酸脱氢酶AhFAD2是调控花生种子中油酸与亚油酸比值(oleic acid/linoleic acid, O/L)的关键酶,已确定存在2个编码AhFAD2的基因:AhFAD2A和AhFAD2B,但两者在花生中的时空表达特征尚不清楚。选取2个有代表性O/L的花生品种‘山花15’‘Shanhua 15’(O/L为1)和高油酸花生突变体(O/L大于20)为材料,根据AhFAD2A和AhFAD2B基因3′-UTR核苷酸序列的差异,设计了新型、简便的区分两者的特异性引物,并利用实时荧光定量聚合酶链式反应(qRT-PCR)技术,对2个花生品种的7个不同组织(根,茎,叶,花,开花后20, 40, 60 d种子)中AhFAD2A和AhFAD2B的表达特征进行了分析。结果表明:AhFAD2A和AhFAD2B在‘山花15’和高油酸花生突变体7个组织中都有表达;其中,在2个花生品种3个不同发育时期的种子中, AhFAD2A和AhFAD2B在开花后40 d的种子中表达量最高,推测在开花至花后40 d这一阶段种子中FAD2的表达量可能对花生最终O/L起主要的调控作用。另外,‘山花15’种子中AhFAD2B的表达量显著高于AhFAD2A,而在高油酸花生突变体种子中则相反,推测花生中AhFAD2B在催化油酸去饱和生成亚油酸的过程中比AhFAD2A可能起更重要的调控作用。
The fatty acid desaturase 2(FAD2) in peanut(Arachis hypogaea), a crucial enzyme for controlling the ratio of oleic acid to linoleic acid(O/L) in the seed, with presently two FAD2-encoding genes(AhFAD2A and AhFAD2B) has unknown spatial and temporal expression patterns. To determine the expression patterns of AhFAD2A and AhFAD2B in seven different tissues(root, stem, leaf, flower, and seeds at 20, 40, and 60 d after pollination) of two distinct cultivars, ‘Shanhua 15' with an O/L of approximately 1.0 and a high-oleate peanut mutant, were analyzed. In doing so, real-time polymerase chain reaction(q RT-PCR) was performed using different pairs of specific primers, suitable for distinguishing AhFAD2A from AhFAD2B, which were designed based on the difference in the 3′-untranslated region(UTR) sequences of these two genes. Results showed that AhFAD2A and AhFAD2B were expressed in all the tissues tested. Among the three developmental stages of the peanut pods for the two cultivars, the highest expression of AhFAD2A and AhFAD2B occurred 40 d after pollination. In addition, the expression of AhFAD2B was shown to be much higher than that of AhFAD2A in the pods of ‘Shanhua 15' than the high-oleate peanut mutant. Thus, the AhFAD2B may play a more important regulatory role in the conversion of oleic acid to linoleic acid in peanut seed compared to AhFAD2A,and possibly the expression level of the FAD2genes at the 40 d stage may exert a large effect on the final ratio of O/L in peanut seed oil.
The fatty acid desaturase 2(FAD2) in peanut(Arachis hypogaea), a crucial enzyme for controlling the ratio of oleic acid to linoleic acid(O/L) in the seed, with presently two FAD2-encoding genes(AhFAD2A and AhFAD2B) has unknown spatial and temporal expression patterns. To determine the expression patterns of AhFAD2A and AhFAD2B in seven different tissues(root, stem, leaf, flower, and seeds at 20, 40, and 60 d after pollination) of two distinct cultivars, ‘Shanhua 15' with an O/L of approximately 1.0 and a high-oleate peanut mutant, were analyzed. In doing so, real-time polymerase chain reaction(q RT-PCR) was performed using different pairs of specific primers, suitable for distinguishing AhFAD2A from AhFAD2B, which were designed based on the difference in the 3′-untranslated region(UTR) sequences of these two genes. Results showed that AhFAD2A and AhFAD2B were expressed in all the tissues tested. Among the three developmental stages of the peanut pods for the two cultivars, the highest expression of AhFAD2A and AhFAD2B occurred 40 d after pollination. In addition, the expression of AhFAD2B was shown to be much higher than that of AhFAD2A in the pods of ‘Shanhua 15' than the high-oleate peanut mutant. Thus, the AhFAD2B may play a more important regulatory role in the conversion of oleic acid to linoleic acid in peanut seed compared to AhFAD2A,and possibly the expression level of the FAD2genes at the 40 d stage may exert a large effect on the final ratio of O/L in peanut seed oil.
引文
[1] WANG Yun, ZHANG Xinguo, ZHAO Yongli, et al. Insights into the novel members of the FAD2 gene family involved in high-oleate fluxes in peanut[J]. Genome, 2015, 58(8):375-383.
[2] WANG M L, CHEN C Y, DAVIS J, et al. Assessment of oil content and fatty acid composition variability in different peanut subspecies and botanical varieties[J]. Plant Genet Resour, 2009, 8(1):71-73.
[3] NAWADE B, BOSAMIA T C, THANKAPPAN R, et al. Insights into the Indian peanut genotypes for AhFAD2 gene polymorphism regulating its oleic and linoleic acid fluxes[J]. Front Plant Sci, 2016, 7:1271. doi:10.3389/fpls. 2016.01277.
[4] WANG M L, KHERA P, PANDEY M K, et al. Genetic mapping of QTLs controlling fatty acids provided insights into the genetic control of fatty acid synthesis pathway in peanut(Arachis hypogaea L.)[J]. PLoS One, 2015, 10(4):e0119454. doi:10.1371/journal. pone. 0119454.
[5]雷永.花生高油酸的分子遗传机制及其高效遗传改良体系构建[D].北京:中国农业科学院, 2010.LEI Yong. Molecular Mechanism and Genetic Enhancement Technology for High Oleic Acid in Peanut(Arachis hypogaea L.)[D]. Beijing:Chinese Academy of Agricultural Sciences, 2010.
[6] LOPEZ Y, NADAF H L, SMITH O D, et al. Genetic factors influencing high oleic acid content in Spanish market type peanut cultivars[J]. Crop Sci, 2000, 41:51-56.
[7] CHI Xiaoyuan, YANG Qingli, PAN Lijuan, et al. Isolation and characterization of fatty acid desaturase genes from peanut(Arachis hypogaea L.)[J]. Plant Cell Rep, 2011, 30(8):1393-1404.
[8] YIN Dongmei, CUI Dangqun, JIA Bin. Construction of a high-efficient expression vector of Deltal 2 fatty acid desaturase in peanut and its prokaryotical expression[J]. Genet Genom, 2007, 34(1):81-88.
[9]周丽侠,唐桂英,陈高,等.花生AhFAD2基因的多态性及其与籽粒油酸/亚油酸比值间的相关性[J].作物学报, 2011, 37(3):415-423.ZHOU Lixia, TANG Guiying, CHEN Gao, et al. Correlation between AhFAD2 polymorphism and oleic acid/linoleic acid ratio in peanut seeds[J]. Acta Agron Sin, 2011, 37(3):415-423.
[10] YU Shanlin, CHEN Mingna, YANG Qingli, et al. Cloning, characterization and expression analysis of a stearoyl-ACP desaturase gene from Arachis hypogaea L.[R]//IEEE. Proceedings of the 2nd International Conference on Biomedical Engineering and Informatics, Tianjin, 2009:1-6.
[11]殷冬梅,胡晓峰,张幸果,等.不同油酸亚油酸比值花生品种油酸脱氢酶基因的时空表达特征[J].中国油料作物学报, 2013, 35(2):137-141.YIN Dongmei, HU Xiaofeng, ZHANG Xingguo, et al. Temporal and spatial expression of oleate desaturase gene with different O/L values in peanut varieties[J]. Chin J Oil Crop Sci, 2013, 35(2):137-141.
[12] LOPEZ Y, NADAF H L, SMITH O D, et al. Isolation and characterization of theΔ12-fatty acid desaturase in peanut(Arachis hypogaea L.)and search for polymorphisms for the high oleate trait in Spanish market-type lines[J]. Theor Appl Genet, 2000, 101(7):1131-1138.
[13] BARKLEY N A, ISIEIB T G, WANG M L, et al. Genotypic effect of AhFAD2 on fatty acid profiles in six segregating peanut(Arachis hypogaea L.)populations[J]. Bmc Genet, 2013, 14(1):62.
[14] FANG Chaoqi, WANG Chuantang, WANG Piwu, et al. Identification of a novel mutation in FAD2B from a peanut EMS mutant with elevated oleate content[J]. J Oleo Sci, 2012, 61(3):143-148.
[15] JUNG S, POWELL G, MOORE K, et al. The high oleate trait in the cultivated peanut(Arachis hypogaea L.)(Ⅱ)Molecular basis and genetics of the trait[J]. Mol Gen Genet, 2000, 263(5):806-811.
[16] CHEN Zhenbang, WANG Mingli, BARKLEY N A, et al. A simple allele-specific PCR assay for detecting FAD2 Alleles in both A and B genomes of the cultivated peanut for high-oleate trait selection[J]. Plant Mol Biol Rep, 2010,28(3):542-548.
[17] CHU Y, RAMOS L, HOLBROOK C C, et al. Frequency of a loss-of-function mutation in oleoyl-PC desaturase(AhFAD2A)in the mini-core of the US peanut germplasm collection[J]. Crop Sci, 2007, 47(6):2372-2378.
[18] NOELLEAB B, KELLYDCHENAULT C, WANG M L, et al. Development of a real-time PCR genotyping assay to identify high oleic acid peanuts(Arachis hypogaea L.)[J]. Mol Breed, 2010, 25(3):541-548.
[19] CHI Xiaoyuan, HU Ruibo, YANG Qingli, et al. Validation of reference genes for gene expression studies in peanut by quantitative real-time RT-PCR[J]. Mol Genet Genomics, 2012, 287(2):167-176.
[20] QUEHENBERGER O, ARMANDO A M, DENNIS E A. High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry[J]. Biochim Et Biophysic Acta, 2011, 1811(11):648-656.
[21] VONGSVIVUT J, HERAUD P, ZHANG Wei, et al. Quantitative determination of fatty acid compositions in micro-encapsulated fish-oil supplements using Fourier transform infrared(FTIR)spectroscopy[J]. Food Chem, 2012, 135(2):603-609.
[22]罗伟强.气相色谱法测定葵花籽油的脂肪酸[J].食品工业科技, 2003, 24(6):79-80.LUO Weiqiang. Fatty acids of sunflower seed oil determined by gas chromatography[J]. Sci Technol Food Ind, 2003,24(6):79-80.
[23] OHLROGGE J, BROWSE J. Lipid biosynthesis[J]. Plant Cell, 1995, 7(7):957-970.
[24]陈四龙,李玉荣,徐桂真,等.不同高油花生油分积累性的模拟研究[J].花生学报, 2016, 45(2):33-37.CHEN Silong, LI Yurong, XU Guizhen, et al. Simulation on oil accumulation characteristics in different high-oil peanut varieties[J]. J Peanut Sci, 2016, 45(2):33-37.
[25]迟晓元,郝翠翠,潘丽娟,等.不同花生品种脂肪酸组成及积累规律的研究[J].花生学报, 2016, 45(3):32-36.CHI Xiaoyuan, HAO Cuicui, PAN Liyuan, et al. Fatty acid accumulation pattern in different types of peanut[J]. J Peanut Sci, 2016, 45(3):32-36.
[2] WANG M L, CHEN C Y, DAVIS J, et al. Assessment of oil content and fatty acid composition variability in different peanut subspecies and botanical varieties[J]. Plant Genet Resour, 2009, 8(1):71-73.
[3] NAWADE B, BOSAMIA T C, THANKAPPAN R, et al. Insights into the Indian peanut genotypes for AhFAD2 gene polymorphism regulating its oleic and linoleic acid fluxes[J]. Front Plant Sci, 2016, 7:1271. doi:10.3389/fpls. 2016.01277.
[4] WANG M L, KHERA P, PANDEY M K, et al. Genetic mapping of QTLs controlling fatty acids provided insights into the genetic control of fatty acid synthesis pathway in peanut(Arachis hypogaea L.)[J]. PLoS One, 2015, 10(4):e0119454. doi:10.1371/journal. pone. 0119454.
[5]雷永.花生高油酸的分子遗传机制及其高效遗传改良体系构建[D].北京:中国农业科学院, 2010.LEI Yong. Molecular Mechanism and Genetic Enhancement Technology for High Oleic Acid in Peanut(Arachis hypogaea L.)[D]. Beijing:Chinese Academy of Agricultural Sciences, 2010.
[6] LOPEZ Y, NADAF H L, SMITH O D, et al. Genetic factors influencing high oleic acid content in Spanish market type peanut cultivars[J]. Crop Sci, 2000, 41:51-56.
[7] CHI Xiaoyuan, YANG Qingli, PAN Lijuan, et al. Isolation and characterization of fatty acid desaturase genes from peanut(Arachis hypogaea L.)[J]. Plant Cell Rep, 2011, 30(8):1393-1404.
[8] YIN Dongmei, CUI Dangqun, JIA Bin. Construction of a high-efficient expression vector of Deltal 2 fatty acid desaturase in peanut and its prokaryotical expression[J]. Genet Genom, 2007, 34(1):81-88.
[9]周丽侠,唐桂英,陈高,等.花生AhFAD2基因的多态性及其与籽粒油酸/亚油酸比值间的相关性[J].作物学报, 2011, 37(3):415-423.ZHOU Lixia, TANG Guiying, CHEN Gao, et al. Correlation between AhFAD2 polymorphism and oleic acid/linoleic acid ratio in peanut seeds[J]. Acta Agron Sin, 2011, 37(3):415-423.
[10] YU Shanlin, CHEN Mingna, YANG Qingli, et al. Cloning, characterization and expression analysis of a stearoyl-ACP desaturase gene from Arachis hypogaea L.[R]//IEEE. Proceedings of the 2nd International Conference on Biomedical Engineering and Informatics, Tianjin, 2009:1-6.
[11]殷冬梅,胡晓峰,张幸果,等.不同油酸亚油酸比值花生品种油酸脱氢酶基因的时空表达特征[J].中国油料作物学报, 2013, 35(2):137-141.YIN Dongmei, HU Xiaofeng, ZHANG Xingguo, et al. Temporal and spatial expression of oleate desaturase gene with different O/L values in peanut varieties[J]. Chin J Oil Crop Sci, 2013, 35(2):137-141.
[12] LOPEZ Y, NADAF H L, SMITH O D, et al. Isolation and characterization of theΔ12-fatty acid desaturase in peanut(Arachis hypogaea L.)and search for polymorphisms for the high oleate trait in Spanish market-type lines[J]. Theor Appl Genet, 2000, 101(7):1131-1138.
[13] BARKLEY N A, ISIEIB T G, WANG M L, et al. Genotypic effect of AhFAD2 on fatty acid profiles in six segregating peanut(Arachis hypogaea L.)populations[J]. Bmc Genet, 2013, 14(1):62.
[14] FANG Chaoqi, WANG Chuantang, WANG Piwu, et al. Identification of a novel mutation in FAD2B from a peanut EMS mutant with elevated oleate content[J]. J Oleo Sci, 2012, 61(3):143-148.
[15] JUNG S, POWELL G, MOORE K, et al. The high oleate trait in the cultivated peanut(Arachis hypogaea L.)(Ⅱ)Molecular basis and genetics of the trait[J]. Mol Gen Genet, 2000, 263(5):806-811.
[16] CHEN Zhenbang, WANG Mingli, BARKLEY N A, et al. A simple allele-specific PCR assay for detecting FAD2 Alleles in both A and B genomes of the cultivated peanut for high-oleate trait selection[J]. Plant Mol Biol Rep, 2010,28(3):542-548.
[17] CHU Y, RAMOS L, HOLBROOK C C, et al. Frequency of a loss-of-function mutation in oleoyl-PC desaturase(AhFAD2A)in the mini-core of the US peanut germplasm collection[J]. Crop Sci, 2007, 47(6):2372-2378.
[18] NOELLEAB B, KELLYDCHENAULT C, WANG M L, et al. Development of a real-time PCR genotyping assay to identify high oleic acid peanuts(Arachis hypogaea L.)[J]. Mol Breed, 2010, 25(3):541-548.
[19] CHI Xiaoyuan, HU Ruibo, YANG Qingli, et al. Validation of reference genes for gene expression studies in peanut by quantitative real-time RT-PCR[J]. Mol Genet Genomics, 2012, 287(2):167-176.
[20] QUEHENBERGER O, ARMANDO A M, DENNIS E A. High sensitivity quantitative lipidomics analysis of fatty acids in biological samples by gas chromatography-mass spectrometry[J]. Biochim Et Biophysic Acta, 2011, 1811(11):648-656.
[21] VONGSVIVUT J, HERAUD P, ZHANG Wei, et al. Quantitative determination of fatty acid compositions in micro-encapsulated fish-oil supplements using Fourier transform infrared(FTIR)spectroscopy[J]. Food Chem, 2012, 135(2):603-609.
[22]罗伟强.气相色谱法测定葵花籽油的脂肪酸[J].食品工业科技, 2003, 24(6):79-80.LUO Weiqiang. Fatty acids of sunflower seed oil determined by gas chromatography[J]. Sci Technol Food Ind, 2003,24(6):79-80.
[23] OHLROGGE J, BROWSE J. Lipid biosynthesis[J]. Plant Cell, 1995, 7(7):957-970.
[24]陈四龙,李玉荣,徐桂真,等.不同高油花生油分积累性的模拟研究[J].花生学报, 2016, 45(2):33-37.CHEN Silong, LI Yurong, XU Guizhen, et al. Simulation on oil accumulation characteristics in different high-oil peanut varieties[J]. J Peanut Sci, 2016, 45(2):33-37.
[25]迟晓元,郝翠翠,潘丽娟,等.不同花生品种脂肪酸组成及积累规律的研究[J].花生学报, 2016, 45(3):32-36.CHI Xiaoyuan, HAO Cuicui, PAN Liyuan, et al. Fatty acid accumulation pattern in different types of peanut[J]. J Peanut Sci, 2016, 45(3):32-36.