Sequence and Expression Characterization of Two Genes Encoding Δ-12 Fatty Acid Desaturases from Camellia oleifera

LIN Ping; ZHOU Chang-fu; YAO Xiao-hua; CAO Yong-qing

Volume 29 Issue 5

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Sequence and Expression Characterization of Two Genes Encoding Δ-12 Fatty Acid Desaturases from Camellia oleifera

1.
Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, Zhejiang, China
2.
Institute of Horticulture, Hunan Academy of Agricultural Sciences, Changsha 410125, Hunan, China

Received Date: 2016-03-08

Abstract

[Objective] Exploring the fatty acids contents regulation mechanism is needed. [Method] Two Δ-12 fatty acid desaturase gene sequences were got from Camellia oleifera developing seeds by transcriptome sequencing method, which named Cofad6 and Cofad2-2. The traits of the two genes and encoding proteins were compared, and the correlation of gene expression level and fatty acids contents were analyzed. [Result] Cofad6 has an open reading frame (ORF) of 1 347 bp and encodes a 448 amino acid (AA) protein; Cofad2-2 has an 1 152 bp ORF and encodes 383 AA protein. According to the alignment results, the CoFAD6 has the 65.7%83.68% amino acid sequence similarity with the FAD6 from other species, CoFAD2-2 amino acid sequence exhibits the highest similarity with C. chekiangoleosa (99.22%), and has the 78.59%~81.72% amino acid sequence similarity with FAD2 from other species. CoFAD6 and CoFAD2-2 have a fatty acid desaturase domain both, which means they belong to acyl-CoA desaturase family. CoFAD6 and CoFAD2-2 are transmembrane proteins and the CoFAD2-2 has a microbody C-terminal targeting signal motif. Real-time PCR analysis shows that Cofad6 and Cofad2-2 are both expressed in the developing seed of C. oleifera ‘Changlin No.4’. The expression level of Cofad2-2 is on the decline with the developing of seed, the variation trend is similar with that of linoleic acid content and opposite with that of oleic acid content. [Conclusion] It is indicated that Cofad2-2 gene might be one of the key genes to regulate oil quality in C. oleifera seed. This research provides scientific basis for oil quality improvement and molecular breeding in C. oleifera.
- Camellia oleifera,
- Δ-12 fatty acid desaturase gene,
- expression characterization,
- real-time PCR

References

[1]	Alonso D L, Maroto F G, Ruiz J R, et al. Evolution of the membrane-bound fatty acid desaturases[J]. Biochemical Systematics and Ecology, 2003, 31(10):1111-1124.
[2]	Liu Q, Brubaker C L, Green A G, et al. Evolution of the FAD2-1 fatty acid desaturase 5'UTR intron and the molecular systematics of Gossypium (Malvaceae)[J]. American Journal of Botany, 2001, 88(1):92-102.
[3]	Takeno S, Sakuradani E, Tomi A, et al. Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with Δ12-desaturase gene expression[J]. Applied and Environmental Microbiology, 2005, 71(9):5124-5128.
[4]	张琦,李明春,孙红妍, 等. Δ6-脂肪酸脱氢酶的分子生物学研究进展[J].生物工程学报, 2004, 20(3):319-324.
[5]	Jung S, Swift D, Sengoku E, et al. The high oleate trait in the cultivated peanut (Arachis hypogaea L.).I. Isolation and characterization of two genes encoding microsomal oleoyl-PC desaturases[J]. Molecular and General Genetics, 2000, 263(5):796-805.
[6]	Sperling P, Schmidt H, Heinz E. A cytochrome-b5-containing fusion protein similar to plant acyl-lipid desaturases[J]. European Journal of Biochemistry, 1995, 232(3):798-805.
[7]	Sayamrat P, Dmitry A L, Norio M. Biochemical characterization of a acyl-lipid desaturase after overexpression of the enzyme in Escherichia coli[J]. Biochimicaet Biophysica Acta, 1998, 1390(3):323-332.
[8]	庄瑞林.中国油茶[M].2版.北京:中国林业出版社, 2008.
[9]	姚小华, 王开良, 任华东, 等.油茶资源与科学利用研究[M].北京:科学出版社, 2012.
[10]	林萍,曹永庆,姚小华, 等. 普通油茶种子4个发育时期的转录组分析[J].分子植物育种, 2011,9(4):498-505.
[11]	Zhou C F, Lin P, Yao X H, et al. Selection of reference genes for quantitative real-time PCR in six oil-tea camellia based on RNA-seq[J]. Molecular Biology, 2013, 47(6):836-851.
[12]	周长富,姚小华,林萍,等. 油茶种子发育过程组分含量动态研究[J].中国油料作物学报, 2013,35(6):680-685.
[13]	Smith M A, Cross A R, Jones O T G, et al. Electron-transport components of the 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons[J]. The Biochemical Journal, 1990, 272(1):23-29.
[14]	戴晓峰,肖玲,武玉花,等. 植物脂肪酸去饱和酶及其编码基因研究进展[J].植物学通报, 2007, 24(1):105-113.
[15]	Liu Q, Singh S P, Brubaker C L, et al. Molecular cloning and expression of a cDNA encoding a microsomal ω-6 fatty acid desaturase from cotton (Gossypium hirsutum)[J]. Austrialian Journal of Plant Physiology,1999, 26:101-106.
[16]	Pirtle I L, Kongcharoensuntorn W, Nampaisansuk M, et al. Molecular cloning and functional expression of the gene for a cotton Δ-12 fatty acid desaturase (FAD2)[J]. Biochimica et Biophysica Acta, 2001,1522(2):122-129.
[17]	Mccartney A W, Dyer J M, Dhanoa P K, et al. Membrane-bound fatty acid desaturases are inserted co-translationally into the ER and contain different ER retrieval motifs at their carboxy termini[J]. The Plant Journal, 2004, 37(2):156-173.
[18]	林萍, 汪阳东, 齐力旺, 等.中间锦鸡儿fad2基因克隆及序列分析[J].分子植物育种, 2008,6(1):148-154.
[19]	Martinez-rivas J M, Sperling P, Luhs W, et al. Spatial and temporal regulation of three different microsomal oleate desaturase genes (FAD2) from normal-type and high-oleic varieties of sunflower (Helianthus annuus L.)[J]. Molecular Breeding, 2001,8:159-168.
[20]	Mikkilineni V, Rocheford T R. Sequence variation and genomic organization of fatty acid desaturase-2(fad2) and fatty acid desaturase-6(fad6) cDNAs in maize[J]. Theoretical and Applied Genetics, 2003,106(7):1326-1332.
[21]	Heppard E P, Kinney A J, Stecca K L,et al. Developmental and growth temperature regulation of two different microsomal ω-6 desaturase genes in soybeans[J]. Plant Physiology, 1996,110(1):311-319.
[22]	Schlueter J A, Vasylenko-sanders I F, Deshpande S, et al. The FAD2 gene family of soybean:Insights into the structural and functional divergence of a paleopolyploid genome[J]. Crop Science, 2007, 47(suppl 1):14-26.
[23]	Pham A T, Shannon G J, Bilyeu K D. Combinations of mutant FAD2 and FAD3 genes to produce high oleic acid and low linolenic acid soybean oil[J]. Theoretical and Applied Genetics, 2012,125(3):503-515.
[24]	Hernandez M L, Mancha M, Martinez-rivas J M. Molecular cloning and characterization of genes encoding two microsomal oleate desaturases(FAD2)from olive[J].Phytochemistry, 2005, 66(12):1417-1426.
[25]	林萍, 汪阳东, 齐力旺, 等. 中间锦鸡儿FAD2基因拷贝数检测及组织表达谱分析[J].西北农林科技大学学报:自然科学版, 2010,38(1):119-124.
[26]	肖钢, 张宏军, 彭琪, 等. 甘蓝型油菜油酸脱氢酶基因(fad2)多个拷贝的发现及分析[J].作物学报, 2008,34(9):1563-1568.
[27]	刘永红, 张丽静, 张洪荣,等.Δ-12脂肪酸脱氢酶及其编码基因研究进展[J].草业学报, 2011,20(3):256-267.
[28]	谭晓风, 陈鸿鹏, 张党权, 等. 油茶FAD2基因全长cDNA的克隆和序列分析[J].林业科学, 2008, 4(3):70-75.
[29]	Scheffler J A, Sharpe A G, Schmidt H, et al. Desaturase multigene families of Brassica napus arose through genome duplication[J]. Theoretical and Applied Genetics,1997, 94(5):583-591.
[30]	胡姣.油茶AACT基因和FAD6基因的全长cDNA克隆及原核表达[D]. 长沙:中南林业科技大学, 2010
[31]	Okuley J, Lightner J, Feldmann K, et al. Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis[J]. Plant Cell, 1996, 6(1):l47-l58.
[32]	Ohlrogge J, Browse J. Lipid biosynthesis[J]. Plant Cell, 1995, 7(7):957-970.
[33]	Ohlrogge J, Jaworski J G. Regulation of fatty acid synthesis[J]. Plant Molecular Biology, 1997, 48(48):109-136.
[34]	肖钢, 张振乾, 邬贤梦.六个甘蓝型油菜油酸脱氢酶(fad2)假基因的克隆与分析[J].作物学报, 2010,36(3):435-441.
[35]	Schierholt A, Becker H C, Ecke W. Mapping a high oleic acid mutation in winter oilseed rape (Brassica napus L.)[J]. Theoretical and Applied Genetics, 2000, 101(5):897-901.

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

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Sequence and Expression Characterization of Two Genes Encoding Δ-12 Fatty Acid Desaturases from Camellia oleifera

1. Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, Zhejiang, China

2. Institute of Horticulture, Hunan Academy of Agricultural Sciences, Changsha 410125, Hunan, China

Received Date: 2016-03-08

Abstract: [Objective] Exploring the fatty acids contents regulation mechanism is needed. [Method] Two Δ-12 fatty acid desaturase gene sequences were got from Camellia oleifera developing seeds by transcriptome sequencing method, which named Cofad6 and Cofad2-2. The traits of the two genes and encoding proteins were compared, and the correlation of gene expression level and fatty acids contents were analyzed. [Result] Cofad6 has an open reading frame (ORF) of 1 347 bp and encodes a 448 amino acid (AA) protein; Cofad2-2 has an 1 152 bp ORF and encodes 383 AA protein. According to the alignment results, the CoFAD6 has the 65.7%83.68% amino acid sequence similarity with the FAD6 from other species, CoFAD2-2 amino acid sequence exhibits the highest similarity with C. chekiangoleosa (99.22%), and has the 78.59%~81.72% amino acid sequence similarity with FAD2 from other species. CoFAD6 and CoFAD2-2 have a fatty acid desaturase domain both, which means they belong to acyl-CoA desaturase family. CoFAD6 and CoFAD2-2 are transmembrane proteins and the CoFAD2-2 has a microbody C-terminal targeting signal motif. Real-time PCR analysis shows that Cofad6 and Cofad2-2 are both expressed in the developing seed of C. oleifera ‘Changlin No.4’. The expression level of Cofad2-2 is on the decline with the developing of seed, the variation trend is similar with that of linoleic acid content and opposite with that of oleic acid content. [Conclusion] It is indicated that Cofad2-2 gene might be one of the key genes to regulate oil quality in C. oleifera seed. This research provides scientific basis for oil quality improvement and molecular breeding in C. oleifera.

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Reference (35)

[1]	Alonso D L, Maroto F G, Ruiz J R, et al. Evolution of the membrane-bound fatty acid desaturases[J]. Biochemical Systematics and Ecology, 2003, 31(10):1111-1124.
[2]	Liu Q, Brubaker C L, Green A G, et al. Evolution of the FAD2-1 fatty acid desaturase 5'UTR intron and the molecular systematics of Gossypium (Malvaceae)[J]. American Journal of Botany, 2001, 88(1):92-102.
[3]	Takeno S, Sakuradani E, Tomi A, et al. Improvement of the fatty acid composition of an oil-producing filamentous fungus, Mortierella alpina 1S-4, through RNA interference with Δ12-desaturase gene expression[J]. Applied and Environmental Microbiology, 2005, 71(9):5124-5128.
[4]	张琦,李明春,孙红妍, 等. Δ6-脂肪酸脱氢酶的分子生物学研究进展[J].生物工程学报, 2004, 20(3):319-324.
[5]	Jung S, Swift D, Sengoku E, et al. The high oleate trait in the cultivated peanut (Arachis hypogaea L.).I. Isolation and characterization of two genes encoding microsomal oleoyl-PC desaturases[J]. Molecular and General Genetics, 2000, 263(5):796-805.
[6]	Sperling P, Schmidt H, Heinz E. A cytochrome-b5-containing fusion protein similar to plant acyl-lipid desaturases[J]. European Journal of Biochemistry, 1995, 232(3):798-805.
[7]	Sayamrat P, Dmitry A L, Norio M. Biochemical characterization of a acyl-lipid desaturase after overexpression of the enzyme in Escherichia coli[J]. Biochimicaet Biophysica Acta, 1998, 1390(3):323-332.
[8]	庄瑞林.中国油茶[M].2版.北京:中国林业出版社, 2008.
[9]	姚小华, 王开良, 任华东, 等.油茶资源与科学利用研究[M].北京:科学出版社, 2012.
[10]	林萍,曹永庆,姚小华, 等. 普通油茶种子4个发育时期的转录组分析[J].分子植物育种, 2011,9(4):498-505.
[11]	Zhou C F, Lin P, Yao X H, et al. Selection of reference genes for quantitative real-time PCR in six oil-tea camellia based on RNA-seq[J]. Molecular Biology, 2013, 47(6):836-851.
[12]	周长富,姚小华,林萍,等. 油茶种子发育过程组分含量动态研究[J].中国油料作物学报, 2013,35(6):680-685.
[13]	Smith M A, Cross A R, Jones O T G, et al. Electron-transport components of the 1-acyl-2-oleoyl-sn-glycero-3-phosphocholine delta 12-desaturase in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons[J]. The Biochemical Journal, 1990, 272(1):23-29.
[14]	戴晓峰,肖玲,武玉花,等. 植物脂肪酸去饱和酶及其编码基因研究进展[J].植物学通报, 2007, 24(1):105-113.
[15]	Liu Q, Singh S P, Brubaker C L, et al. Molecular cloning and expression of a cDNA encoding a microsomal ω-6 fatty acid desaturase from cotton (Gossypium hirsutum)[J]. Austrialian Journal of Plant Physiology,1999, 26:101-106.
[16]	Pirtle I L, Kongcharoensuntorn W, Nampaisansuk M, et al. Molecular cloning and functional expression of the gene for a cotton Δ-12 fatty acid desaturase (FAD2)[J]. Biochimica et Biophysica Acta, 2001,1522(2):122-129.
[17]	Mccartney A W, Dyer J M, Dhanoa P K, et al. Membrane-bound fatty acid desaturases are inserted co-translationally into the ER and contain different ER retrieval motifs at their carboxy termini[J]. The Plant Journal, 2004, 37(2):156-173.
[18]	林萍, 汪阳东, 齐力旺, 等.中间锦鸡儿fad2基因克隆及序列分析[J].分子植物育种, 2008,6(1):148-154.
[19]	Martinez-rivas J M, Sperling P, Luhs W, et al. Spatial and temporal regulation of three different microsomal oleate desaturase genes (FAD2) from normal-type and high-oleic varieties of sunflower (Helianthus annuus L.)[J]. Molecular Breeding, 2001,8:159-168.
[20]	Mikkilineni V, Rocheford T R. Sequence variation and genomic organization of fatty acid desaturase-2(fad2) and fatty acid desaturase-6(fad6) cDNAs in maize[J]. Theoretical and Applied Genetics, 2003,106(7):1326-1332.
[21]	Heppard E P, Kinney A J, Stecca K L,et al. Developmental and growth temperature regulation of two different microsomal ω-6 desaturase genes in soybeans[J]. Plant Physiology, 1996,110(1):311-319.
[22]	Schlueter J A, Vasylenko-sanders I F, Deshpande S, et al. The FAD2 gene family of soybean:Insights into the structural and functional divergence of a paleopolyploid genome[J]. Crop Science, 2007, 47(suppl 1):14-26.
[23]	Pham A T, Shannon G J, Bilyeu K D. Combinations of mutant FAD2 and FAD3 genes to produce high oleic acid and low linolenic acid soybean oil[J]. Theoretical and Applied Genetics, 2012,125(3):503-515.
[24]	Hernandez M L, Mancha M, Martinez-rivas J M. Molecular cloning and characterization of genes encoding two microsomal oleate desaturases(FAD2)from olive[J].Phytochemistry, 2005, 66(12):1417-1426.
[25]	林萍, 汪阳东, 齐力旺, 等. 中间锦鸡儿FAD2基因拷贝数检测及组织表达谱分析[J].西北农林科技大学学报:自然科学版, 2010,38(1):119-124.
[26]	肖钢, 张宏军, 彭琪, 等. 甘蓝型油菜油酸脱氢酶基因(fad2)多个拷贝的发现及分析[J].作物学报, 2008,34(9):1563-1568.
[27]	刘永红, 张丽静, 张洪荣,等.Δ-12脂肪酸脱氢酶及其编码基因研究进展[J].草业学报, 2011,20(3):256-267.
[28]	谭晓风, 陈鸿鹏, 张党权, 等. 油茶FAD2基因全长cDNA的克隆和序列分析[J].林业科学, 2008, 4(3):70-75.
[29]	Scheffler J A, Sharpe A G, Schmidt H, et al. Desaturase multigene families of Brassica napus arose through genome duplication[J]. Theoretical and Applied Genetics,1997, 94(5):583-591.
[30]	胡姣.油茶AACT基因和FAD6基因的全长cDNA克隆及原核表达[D]. 长沙:中南林业科技大学, 2010
[31]	Okuley J, Lightner J, Feldmann K, et al. Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis[J]. Plant Cell, 1996, 6(1):l47-l58.
[32]	Ohlrogge J, Browse J. Lipid biosynthesis[J]. Plant Cell, 1995, 7(7):957-970.
[33]	Ohlrogge J, Jaworski J G. Regulation of fatty acid synthesis[J]. Plant Molecular Biology, 1997, 48(48):109-136.
[34]	肖钢, 张振乾, 邬贤梦.六个甘蓝型油菜油酸脱氢酶(fad2)假基因的克隆与分析[J].作物学报, 2010,36(3):435-441.
[35]	Schierholt A, Becker H C, Ecke W. Mapping a high oleic acid mutation in winter oilseed rape (Brassica napus L.)[J]. Theoretical and Applied Genetics, 2000, 101(5):897-901.

Sequence and Expression Characterization of Two Genes Encoding Δ-12 Fatty Acid Desaturases from Camellia oleifera

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通讯作者: 陈斌, bchen63@163.com

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Sequence and Expression Characterization of Two Genes Encoding Δ-12 Fatty Acid Desaturases from Camellia oleifera

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