Molecular cloning and characterisation of an acyl carrier protein thioesterase gene (CocoFatB1) expressed in the endosperm of coconut (Cocos nucifera) and its heterologous expression in Nicotiana tabacum to engineer the accumulation of different fatty acids
Yijun Yuan A B , Yinhua Chen B , Shan Yan A , Yuanxue Liang A , Yusheng Zheng A C and Li Dongdong A C
+ Author Affiliations
- Author Affiliations
A Department of Biotechnology, Hainan University, Haikou, Hainan 570228, China.
B Hainan Key Laboratory for Sustainable Utilisation of Tropical Bioresource, Hainan University, Haikou, Hainan 570228, China.
C Corresponding authors. Emails: liddfym@hotmail.com; hainanzyh@yahoo.com.cn
Functional Plant Biology 41(1) 80-86 https://doi.org/10.1071/FP13050
Submitted: 6 March 2013 Accepted: 31 May 2013 Published: 8 July 2013
Abstract
Coconut (Cocos nucifera L.) contains large amounts of medium chain fatty acids, which mostly recognise acyl-acyl carrier protein (ACP) thioesterases that hydrolyse acyl-ACP into free fatty acids to terminate acyl chain elongation during fatty acid biosynthesis. A full-length cDNA of an acyl-ACP thioesterase, designated CocoFatB1, was isolated from cDNA libraries prepared from coconut endosperm during fruit development. The gene contained an open reading frame of 1254 bp, encoding a 417-amino acid protein. The amino acid sequence of the CocoFatB1 protein showed 100% and 95% sequence similarity to CnFatB1 and oil palm (Elaeis guineensis Jacq.) acyl-ACP thioesterases, respectively. Real-time fluorescent quantitative PCR analysis indicated that the CocoFatB1 transcript was most abundant in the endosperm from 8-month-old coconuts; the leaves and endosperm from 15-month-old coconuts had ~80% and ~10% of this level. The CocoFatB1 coding region was overexpressed in tobacco (Nicotiana tabacum L.) under the control of the seed-specific napin promoter following Agrobacterium tumefaciens-mediated transformation. CocoFatB1 transcript expression varied 20-fold between different transgenic plants, with 21 plants exhibiting detectable levels of CocoFatB1 expression. Analysis of the fatty acid composition of transgenic tobacco seeds showed that the levels of myristic acid (14 : 0), palmitic acid (16 : 0) and stearic acid (18 : 0) were increased by 25%, 34% and 17%, respectively, compared with untransformed plants. These results indicated that CocoFatB1 acts specifically on 14 : 0-ACP, 16 : 0-ACP and 18 : 0-ACP, and can increase medium chain saturated fatty acids. The gene may valuable for engineering fatty acid metabolism in crop improvement programmes.
Additional keywords: medium chain fatty acids, myristic acid, palmitic acid, stearic acid, transgenic tobacco.
References
Beermann C, Winterling N, Green A, Mobius M, Schmitt JJ, Boehm G (2007) Comparison of the structures of triacylglycerols from native and transgenic medium-chain fatty acid-enriched rape seed oil by liquid chromatography-atmospheric pressure chemical ionization ion-trap mass spectrometry (LC-APCI-ITMS). Lipids 42, 383–394.| Comparison of the structures of triacylglycerols from native and transgenic medium-chain fatty acid-enriched rape seed oil by liquid chromatography-atmospheric pressure chemical ionization ion-trap mass spectrometry (LC-APCI-ITMS).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXkvFCmtLk%3D&md5=bb41d57eb4da05e2e50352990471f88dCAS | 17406932PubMed |
Bhatnagar AS, Prasanth Kumar PK, Hemavathy J, Gopala Krishna AG (2009) Fatty acid composition, oxidative stability, and radical scavenging activity of vegetable oil blends with coconut oil. Journal of the American Oil Chemists’ Society 86, 991–999.
| Fatty acid composition, oxidative stability, and radical scavenging activity of vegetable oil blends with coconut oil.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtFaqt77P&md5=9dd08726bf45c15354562ffab7396686CAS |
Brown AP, Slabas AR, Rafferty JB (2010) ‘Lipids in photosynthesis: essential and regulatory functions.’ (Springer:Dordrecht, The Netherlands)
Ceniza MS, Ueda S, Sugimura Y (1991) In vitro culture of coconut endosperm: callus induction and its fatty acids. Plant Cell Reports 11, 546–549.
| In vitro culture of coconut endosperm: callus induction and its fatty acids.Crossref | GoogleScholarGoogle Scholar |
Chi X, Yang Q, Pan L, Chen M, He Y, Yang Z, Yu S (2011) Isolation and characterization of fatty acid desaturase genes from peanut (Arachis hypogaea L.). Plant Cell Reports 30, 1393–1404.
| Isolation and characterization of fatty acid desaturase genes from peanut (Arachis hypogaea L.).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXovVWgtr4%3D&md5=3c8ba0b90d82357f316117a0fd5cc6f3CAS | 21409552PubMed |
Davies HM, Anderson L, Fan C, Hawkins DJ (1991) Developmental induction, purification, and further characterization of 12 : 0-ACP thioesterase from immature cotyledons of Umbellularia californica. Archives of Biochemistry and Biophysics 290, 37–45.
| Developmental induction, purification, and further characterization of 12 : 0-ACP thioesterase from immature cotyledons of Umbellularia californica.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK38Xis1Sh&md5=01f526da3737650c1bafb8dce2571ecdCAS | 1898097PubMed |
Davies HM, Hawkins DJ, Nelsen JS (1995) Lysophosphatidic acid acyltransferase from immature coconut endosperm having medium chain length substrate specificity. Phytochemistry 39, 989–996.
| Lysophosphatidic acid acyltransferase from immature coconut endosperm having medium chain length substrate specificity.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXnsFSnsro%3D&md5=0aaddf4c5d01023299eb34cde042d8e3CAS |
Dehesh K, Jones A, Knutzon DS, Voelker TA (1996) Production of high levels of 8 : 0 and 10 : 0 fatty acids in transgenic canola by overexpression of ChFatB2, a thioesterase cDNA from Cuphea hookeriana. The Plant Journal 9, 167–172.
| Production of high levels of 8 : 0 and 10 : 0 fatty acids in transgenic canola by overexpression of ChFatB2, a thioesterase cDNA from Cuphea hookeriana.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK28XitVyjur8%3D&md5=d2ca325a8e2996e7db538fb00abb1236CAS | 8820604PubMed |
Dormann P, Voelker TA, Ohlrogge JB (1995) Cloning and expression in Escherichia coli of a novel thioesterase from Arabidopsis thaliana specific for long-chain acyl-acyl carrier proteins. Archives of Biochemistry and Biophysics 316, 612–618.
| Cloning and expression in Escherichia coli of a novel thioesterase from Arabidopsis thaliana specific for long-chain acyl-acyl carrier proteins.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DyaK2M7kt1KrtA%3D%3D&md5=f1f7f4a871309304102b79080f9dc484CAS | 7840673PubMed |
Enig MG (1998) Lauric oils as antimicrobial agents: theory of effect, scientific rationale, and dietary applications as adjunct nutritional support for HIV-infected individuals. In ‘Nutrients and foods in AIDS’. (Ed RR Watson) pp. 81–87. (CRC Press, Boca Raton)
Hitr WD, Yadav NS (1992) ‘Nucleotide sequences of soybean acyl-ACP thioesterase genes. International Patent WO 92/11373.’ (Wilmington Del)
Hoekema A, Hirsch P, Hooykaas PJ, Schilperoort R (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303, 179–180.
| A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL3sXktVWhsrY%3D&md5=353167e3076e43eef8a5aeb1994d330bCAS |
Jeukendrup AE, Sarah A (2004) Fat supplementation, health, and endurance performance. Nutrition 20, 678–688.
| Fat supplementation, health, and endurance performance.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXltVGitbk%3D&md5=71ed9ba15c7aa3d31723873bc626f579CAS | 15212751PubMed |
Jha JK, Maiti MK, Bhattacharjee A, Basu A, Sen PC, Sen SK (2006) Cloning and functional expression of an acyl-ACP thioesterase FatB type from Diploknema (Madhuca) butyracea seeds in Escherichia coli. Plant Physiology and Biochemistry 44, 645–655.
| Cloning and functional expression of an acyl-ACP thioesterase FatB type from Diploknema (Madhuca) butyracea seeds in Escherichia coli.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhsl2jtQ%3D%3D&md5=4ec6e2b5b30caa24b730c86c833108a6CAS | 17092734PubMed |
Jha SS, Jha JK, Chattopadhyaya B, Basu A, Sen SK, Maiti MK (2010) Cloning and characterization of cDNAs encoding for long-chain saturated acyl-ACP thioesterases from the developing seeds of Brassica juncea. Plant Physiology and Biochemistry 48, 476–480.
| Cloning and characterization of cDNAs encoding for long-chain saturated acyl-ACP thioesterases from the developing seeds of Brassica juncea.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXlvVeisrc%3D&md5=cdde5d262c7ae3be68236a3ab69a7c1dCAS | 20356753PubMed |
Jing F, Cantu DC, Tvaruzkova J, Chipman JP, Nikolau BJ, Yandeau-Nelson MD, Reilly PJ (2011) Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity. BMC Biochemistry 12, 44–59.
| Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3MXhtFKms7rJ&md5=31103d41c4112054c3c7ef0116a15d89CAS | 21831316PubMed |
Jones A, Davies HM, Voelker TA (1995) Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases. The Plant Cell 7, 359–371.
| Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2MXks1Chsb0%3D&md5=5d0633b302fc912cc7fa158f4bb9beeaCAS | 7734968PubMed |
Knutzon DS, Hayes TR, Wyrick A, Xiong H, Maelor Davies H, Voelker TA (1999) Lysophosphatidic acid acyltransferase from coconut endosperm mediates the insertion of laurate at the sn-2 position of triacylglycerols in lauric rapeseed oil and can increase total laurate levels. Plant Physiology 120, 739–746.
| Lysophosphatidic acid acyltransferase from coconut endosperm mediates the insertion of laurate at the sn-2 position of triacylglycerols in lauric rapeseed oil and can increase total laurate levels.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXks1amtLg%3D&md5=433848a6085b5f1da6de9af65e0192cdCAS | 10398708PubMed |
Kridl JC, McCarter DW, Rose RE, Scherer DE, Knutzon DS, Radke SE, Knauf VC (1991) Isolation and characterization of an expressed napin gene from Brassica rapa. Seed Science Research 1, 202–219.
| Isolation and characterization of an expressed napin gene from Brassica rapa.Crossref | GoogleScholarGoogle Scholar |
Leonard JM, Slabaugh MB, Knapp SJ (1997) Cuphea wrightii thioesterases have unexpected broad specificities on saturated fatty acids. Plant Molecular Biology 34, 669–679.
| Cuphea wrightii thioesterases have unexpected broad specificities on saturated fatty acids.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXltFertLw%3D&md5=c27e7e09ecbf03b49d28ef092ffec478CAS | 9247548PubMed |
Leonard JM, Knapp SJ, Slabaugh MB (1998) A Cuphea beta-ketoacyl-ACP synthase shifts the synthesis of fatty acids towards shorter chains in Arabidopsis seeds expressing Cuphea FatB thioesterases. The Plant Journal 13, 621–628.
| A Cuphea beta-ketoacyl-ACP synthase shifts the synthesis of fatty acids towards shorter chains in Arabidopsis seeds expressing Cuphea FatB thioesterases.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXisVKgtLg%3D&md5=9f271d0f99e27942a25692857a4b4405CAS | 9681004PubMed |
Li DD, Fan YM (2007) Extraction and quality analysis of total RNA from pulp of coconut (Cocos nucifera L.). Molecular Plant Breeding 5, 883–886. [In Chinese]
Li DD, Fan YM (2009) Cloning, characterization, and expression analysis of an oleosin gene in coconut (Cocos nucifera L.) pulp. The Journal of Horticultural Science & Biotechnology 84, 483–488.
Livak K, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2–
Ct) method. Methods 25, 402–408.| Analysis of relative gene expression data using real-time quantitative PCR and the 2–
Ct) method.Crossref | Ct) method.&journal=Methods&volume=25&pages=402-408&publication_year=2001&author=K%20Livak&hl=en&doi=10.1006/meth.2001.1262" target="_blank" rel="nofollow noopener noreferrer" class="reftools">GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XhtFelt7s%3D&md5=cda22da73d6034ad2e4c74827f633baaCAS | 11846609PubMed | Marone M, Mozzetti S, De Ritis D, Pierelli L, Scambia G (2001) Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample. Biological Procedures Online 3, 19–25.
| Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XjsFWrtQ%3D%3D&md5=93637584cbaf884926cfacd7601e919cCAS | 12734582PubMed |
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiology 115, 493–497.
| A revised medium for rapid growth and bioassays with tobacco tissue cultures.Crossref | GoogleScholarGoogle Scholar |
Ohlrogge JB, Kuhn DN, Stumpf PK (1979) Subcelluar localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea. Proceedings of the National Academy of Sciences of the United States of America 76, 1194–1198.
| Subcelluar localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE1MXhvVeitbo%3D&md5=7c27e1a35b7e056642aabf6d12143728CAS | 286305PubMed |
Oo KC, Stumpf PK (1979) Fatty acid biosynthesis in the developing endosperm of Cocos nucifera. Lipids 14, 132–143.
| Fatty acid biosynthesis in the developing endosperm of Cocos nucifera.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE1MXhsVymsb8%3D&md5=eb4f088bbde96245a746ded67ee217a7CAS |
Othman A, Lazarus C, Fraser T, Stobart K (2000) Cloning of a palmitoyl–acyl carrier protein thioesterase from oil palm. Biochemical Society Transactions 28, 619–622.
| Cloning of a palmitoyl–acyl carrier protein thioesterase from oil palm.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXhsVGrtbo%3D&md5=70df1e1d1308f8e29565160bffff6ecdCAS | 11171146PubMed |
Pollard MR, Anderson L, Fan C, Hawkins DJ, Davies HM (1991) A specific acyl-ACP thioesterase implicated in medium-chain fatty acid production in immature cotyledons of Umbellularia californica. Archives of Biochemistry and Biophysics 284, 306–312.
| A specific acyl-ACP thioesterase implicated in medium-chain fatty acid production in immature cotyledons of Umbellularia californica.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3MXnvVOhuw%3D%3D&md5=ce4bd110fd586a5df161067dcca6c344CAS | 1989513PubMed |
Ramírez M, Amate L, Gil A (2001) Absorption and distribution of dietary fatty acids from different sources. Early Human Development 65, S95–S101.
| Absorption and distribution of dietary fatty acids from different sources.Crossref | GoogleScholarGoogle Scholar | 11755040PubMed |
Rivera R, Edvards KJ, Barker JHA, Aranold GM, Ayad G, Hodgkin T (1999) Isolation and characterization of polymorphic microsatellites in Cocos nucifera L. Genome 42, 668–675.
Samsudeen K, Jacob PM, Niral V, Kumaran PM, Salooja R, Moosa H (2006) Exploration and collection of coconut germplasm in Kadmat and Amini Islands of Lakshadweep, India. Genetic Resources and Crop Evolution 53, 1721–1728.
| Exploration and collection of coconut germplasm in Kadmat and Amini Islands of Lakshadweep, India.Crossref | GoogleScholarGoogle Scholar |
Sánchez-García A, Moreno-Pérez AJ, Muro-Pastor AM, Salas JJ, Garcés R, Martínez-Force E (2010) Acyl-ACP thioesterases from castor (Ricinus communis L.): an enzymatic system appropriate for high rates of oil synthesis and accumulation. Phytochemistry 71, 860–869.
| Acyl-ACP thioesterases from castor (Ricinus communis L.): an enzymatic system appropriate for high rates of oil synthesis and accumulation.Crossref | GoogleScholarGoogle Scholar | 20382402PubMed |
Shimakata T, Stumpf PK (1983) Purification and characterization of beta-ketoacyl-ACP synthetase I from Spinacia oleracea leaves. Archives of Biochemistry and Biophysics 220, 39–45.
| Purification and characterization of beta-ketoacyl-ACP synthetase I from Spinacia oleracea leaves.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL3sXosFamtg%3D%3D&md5=a8040c027be8eb306178127a8a35e361CAS | 6830245PubMed |
Stumpf PK (1987) The biosynthesis of saturated fatty acids. The Biochemistry of Plants 9, 121–136.
Voelker T (1996) Plant acyl-ACP thioesterases: chain-length determining enzymes in plant fatty acid biosynthesis. Genetic Engineering (NY) 18, 111–133.
Voelker TA, Davies HM (1994) Alteration of the specificity and regulation of fatty acid synthesis of Escherichia coli by expression of a plant medium-chain acyl-acyl carrier protein thioesterase. Journal of Bacteriology 176, 7320–7327.
Voelker TA, Worrell AC, Anderson L, Bleibaum J, Fan C, Hawkins DJ, Radke SE, Davies HM (1992) Fatty acid biosynthesis redirected to medium chain in transgenic oilseed plants. Science 257, 72–74.
| Fatty acid biosynthesis redirected to medium chain in transgenic oilseed plants.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3sXksVGhtbs%3D&md5=7d0a4a17a770f684a67c2d7fcc93ef80CAS | 1621095PubMed |
Voelker TA, Jones A, Cranmer AM, Davies HM, Knutzon DS (1997) Broad-range and binary-range acyl-acyl-carrier-protein thioesterases suggest an alternative mechanism for medium-chain production in seeds. Plant Physiology 114, 669–677.
| Broad-range and binary-range acyl-acyl-carrier-protein thioesterases suggest an alternative mechanism for medium-chain production in seeds.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXktVWjsrY%3D&md5=35fcb64ba9ea428dd4de1ce3383f9a8dCAS | 9193098PubMed |
Weaire PJ, Kekwick RG (1975) The synthesis of fatty acids in avocado mesocarp and cauliflower bud tissue. Biochemical Journal 146, 425–437.
Wiberg E, Banas A, Stymne S (1997) Fatty acid distribution and lipid metabolism in developing seeds of laurate-producing rape (Brassica napus L.). Planta 203, 341–348.
| Fatty acid distribution and lipid metabolism in developing seeds of laurate-producing rape (Brassica napus L.).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK2sXnt1OitLs%3D&md5=4fbd3747a38bc9051b367197040ce2acCAS | 9431681PubMed |
Wiberg E, Edwards P, Byrne J, Stymne S, Dehesh K (2000) The distribution of caprylate, caprate and laurate in lipids from developing and mature seeds of transgenic Brassica napus L. Planta 212, 33–40.
| The distribution of caprylate, caprate and laurate in lipids from developing and mature seeds of transgenic Brassica napus L.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXosVers70%3D&md5=6160d0dae7cc47064570e44bdf24359aCAS | 11219581PubMed |
Wu PZ, Lin J, Wei Q, Zeng L, Chen YP, Li MR, Jiang HW, Wu GJ (2009) Cloning and functional characterization of an acyl-acyl carrier protein thioesterase (JcFATB1) from Jatropha curcas. Tree Physiology 29, 1299–1305.
| Cloning and functional characterization of an acyl-acyl carrier protein thioesterase (JcFATB1) from Jatropha curcas.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtlKqtLjK&md5=ee5715cb49a53e1b7b962e1b50d39498CAS | 19671567PubMed |
Zheng X, Deng W, Luo K, Duan H, Chen Y, McAvoy R, Song S, Pei Y, Li Y (2007) The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters. Plant Cell Reports 26, 1195–1203.
| The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXotFymtLg%3D&md5=a1971e74fd0a36e13265c15b15abcf47CAS | 17340093PubMed |
