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Acta Agron Sin ›› 2016, Vol. 42 ›› Issue (07): 1000-1008.doi: 10.3724/SP.J.1006.2016.01000


Cloning and Analyses of Expression and for BnFAD2 Genes in Brassica napus

LIU Rui-Yang,LIU Fang,GUAN Chun-Yun   

  1. College of Agronomy, Hunan Agricultural University / National Oilseed Crops Improvement Center in Hunan, Changsha 410128, China
  • Received:2016-01-11 Revised:2016-05-09 Online:2016-07-12 Published:2016-05-11
  • Contact: 官春云, E-mail: guancy2011@yahoo.com.cn E-mail:ruiyang_liu2007@126.com
  • Supported by:

    This study was supported by Graduate Innovation Foundation of Hunan (CX2013A012) and the Major State Basic Research Development Program of China (2015CB150200).


The oil containing high oleic acid is high nutritional. In Brassica napus, the fatty acid desaturase gene (FAD2) is the key gene controlling oleic acid content. In this study, the full-length cDNA sequences of three genes located on chromosome A5, C5 and A1 in Brassica napus were cloned and named BnFAD2-A5, BnFAD2-C5 and BnFAD2-A1. The three genes encode proteins with 384, 384 and 136 amino acid residues, respectively. TMHMM was used to predict transmembrane domain and Clust X software was used to analyze the activity center of FAD2 genes. Both of the results showed that BnFAD2-A1 did not have the function of dehydrogenase. The yeast complementary experiment on four genes (including published BnFAD2-C1 gene) showed that the desaturation capability of BnFAD2-A5 gene was next to that of BnFAD2-C5 gene, and both of them were greater than that of BnFAD2-C1 gene. The expression patterns of the four genes were analyzed by using qRT-PCR technique in different tissues and the protein stability of BnFAD2-C1, BnFAD2-A5 and BnFAD2-C5 was analyzed by using Hemagglutinin labeling method. Both of the results revealed that BnFAD2-A5 and BnFAD2-C5 aremajor genes affecting the accumulation of oleic acid in rape seed.

Key words: Brassica napus, BnFAD2, Bioinformation, Protein activity, Protein stability

[1]Okuley J, Lightner J, Feldmann K, Yadav N, Lark E, Browseai J. Arabidopsis FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. Plant Cell, 1994, 6: 147–158
[2]Tanhuanpää P, Vilkki J, Vihinen M. Mapping and cloning of FAD2 gene to develop allele-specific PCR for oleic acid in spring turnip rape (Brassica rapa ssp. oleifera). Mol Breed, 1998, 4: 543–550
[3]Hobbs D H, Flintham J E, Hills M J. Genetic control of storage oil synthesis in seeds of Arabidopsis. Plant Physiol, 2004, 136: 3341–3349
[4]Schierholt A, Becker H C, Ecke W. Mapping a high oleic acid mutation in winter oilseed rape (Brassica napus L.). Theor Appl Genet, 2000, 101: 897–901
[5]Hu X, Sullivan-Gilbert M, Gupta M. Mapping of the loci controlling oleic and linolenic acid contents and development of fad2 and fad3 allele-specific markers in canola (Brassica napus L.). Theor Appl Genet, 2006, 113: 497–507
[6]杨燕宇, 杨盛强, 陈哲红, 官春云, 陈社员, 刘忠松. 无芥酸甘蓝型油菜十八碳不饱和脂肪酸含量的QTL定位. 作物学报, 2011, 37: 1342–1350
Yang Y Y, Yang S Q, Chen Z H, Guan C Y, Chen S Y, Liu Z S. QTL analysis of 18-C unsaturated fatty acid contents in zero-erucic rapeseed (Brassica napus L.). Acta Agron Sin, 2011, 37: 1342-1350
[7]Scheffler J A, Sharpe A G, Schmidt H, Sperling P, Parkin I A P, Lühs W, Lydiate D J, Heinz E. Desaturase multigene families of Brassica napus arose through genome duplication. Theor Appl Genet, 1997, 94: 583-591
[8]Smooker A M, Wells R, Morgan C, Beaudoin F, Cho K, Fraser F, Bancroft I. The identification and mapping of candidate genes and QTL involved in the fatty acid desaturation pathway in Brassica napus. Theor Appl Genet, 2011, 122: 1075–1090
[9]肖钢, 张宏军, 彭琪, 官春云. 甘蓝型油菜油酸脱氢酶基因(FAD2)多个拷贝的发现及分析. 作物学报, 2008, 34: 1563–1568
Xiao G, Zhang H J, Peng Q, Guan C Y. Screening and analysis of mutiple copy of oleate desaturase gene (fad2) in Brassica napus. Acta Agron Sin, 2008, 34: 1563–1568
[10]肖钢, 张振乾, 邬贤梦, 谭太龙, 官春云. 六个甘蓝型油菜油酸脱氢酶(FAD2)假基因的克隆和分析. 作物学报, 2010, 36: 435–441
Xiao G, Zhang Z Q, Wu X M, Tan T L, Guan C Y. Cloning and characterization of six oleic acid desaturase pseudogenes of Brassica napus. Acta Agron Sin, 2010, 36: 435−441
[11]Yang Q Y, Fan C C, Guo Z H, Qin J, Wu J Z, Li Q Y, Fu T D, Zhou Y G. Identification of FAD2 and FAD3 genes in Brassica napus genome and development of allele-specific markers for high oleic and low linolenic acid contents. Theor Appl Genet, 2012, 125: 715–729
[12]Kiefer E, Heller W, Ernst D. A simple and efficient protocol for isolation of functional RNA from plant tissues rich in secondary metabolites. Plant Mol Biol Rep, 2000, 18: 33–39
[13]Li L, Wang X, Gai J, Yu D. Isolation and characterization of a seed-specific isoform of microsomal omega-6 fatty acid desaturase gene (FAD2-1B) from soybean. Mito DNA, 2008, 19:28–36
[14]Chen Z, Wang M, Barkley N, Pittman R. 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. Plant Mol Biol Rep, 2010, 28: 542–548
[15]Zavallo D, Lopez Bilbao M, Hopp H, Heinz R. Isolation and functional characterization of two novel seed-specific promoters from sunflower (Helianthus annuus L.). Plant Cell Rep, 2010, 29: 239–248
[16]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. Theor Appl Genet, 2003, 106: 1326–1332
[17]Zhang D, Pirtle I, Park S, Nampaisansuk M, Neogi P, Wanjie S, Pirtle R, Chapman K. Identification and expression of a new delta-12 fatty acid desaturase (FAD2-4) gene in upland cotton and its functional expression in yeast and Arabidopsis thaliana plants. Plant Physiol Biochem, 2009, 47: 462–471
[18]Cao S J, Zhou X R, Wood C C, Green A G, Singh S P, Liu L X, Liu Q. A large and functionally diverse family of fad2 genes in safflower. BMC Plant Biol, 2013, 13: 5
[19]徐荣华, 邱丽俊, 阳天泉, 王如玲, 田波, 刘爱忠. 小桐子磷脂二酰甘油酰基转移酶(JcPDAT1) cDNA的克隆与功能鉴定. 中国油料作物学报, 2013, 35: 123–130
Xu R H, Qiu L J, Yang T Q, Wang R L, Tian B, Li A Z. Cloning and functioning of phospholipids: diacylglycerol acyltransferase (JcPDAT1) cDNA from Jatropha curcas. Chin J Oil Crop Sci, 2013, 35: 123–130
[20]O’Quin J B, Bourassa L, Zhang D, Shockey J M, Gidda S K, Fosnot S, Chapman K D, Mullen R T, Dyer J M. Temperature-sensitive post-translational regulation of plant omega-3 fatty-acid desaturases is mediated by the endoplasmic reticulum-associated degradation pathway. J Biol Chem, 2010, 285: 21781–21796
[21]鲁少平. 甘蓝型油菜转磷脂酶D (PLD)基因在干旱及氮响应中的作用及应用. 华中农业大学博士学位论文, 湖北武汉, 2013. pp 27–29
Lu S P. Roles and Potential Applications of Phospholipase Ds in Brassica napus Response to Drought and Nitrogen. PhD Dissertation of Huazhong Agricultural University, Wuhan, China, 2013. pp 27–29
[22]Sakai H, Kajiwara S. Cloning and functional characterization of a Δ12 fatty acid desaturase gene from the basidiomycete Lentinula edodes. Mol Genet Genom, 2005, 273: 336–341
[23]Mahmood T, Ekuere U, Yeh F, Good A G, Stringam G R. RFLP linkage analysis and mapping genes controlling the fatty acid profile of Brassica juncea using reciprocal DH populations. Theor Appl Genet, 2003, 107: 283–290
[24]Burns M J, Barnes S R, Bowman J G, Clarke M H E, Werner C P, Kearsey M J. QTL analysis of an intervarietal set of substitution lines in Brassica napus: (i) Seed oil content and fatty acid composition. Heredity, 2003, 90: 39–48
[25]Zhao J, Dimov Z, Becker H, Ecke W, Möllers C. Mapping QTL controlling fatty acid composition in a doubled haploid rapeseed population segregating for oil content. Mol Breed, 2008, 21: 115–125
[26]张洁夫, 戚存扣, 浦惠明, 陈松, 陈锋, 高建芹, 陈新军, 顾慧, 傅寿仲. 甘蓝型油菜主要脂肪酸组成的QTL定位. 2008, 34: 54–60
Zhang J F, Qi C K, Pu H M, Chen S, Chen F, Gao J Q, Chen X J, Gu H, Fu S Z. QTL identification for fatty acid content in rapeseed (Brassica napus L.). Acta Agron Sin, 2008, 34: 54–60
[27]McCartney A W, Dyer J M, Dhanoa P K, Kim P K, Andrews D W, McNew J A, Mullen R T. Membrane-bound fatty acid desaturases are inserted co-translationally into the ER and contain different ER retrieval motifs at their carboxy termini. Plant J, 2004, 37: 156–173
[28]Cheung F, Trick M, Drou N, Lim Y P, Park J Y, Kwon S J, Kim J A, Scott R, Pires J C, Paterson A H, Town C, Bancroft I. Comparative analysis between homoeologous genome segments of Brassica napus and its progenitor species reveals extensive sequence-level divergence. Plant Cell, 2009, 21: 1912–1928
[29]Jung J H, Kim H, Go Y S. Identification of functional BrFAD2-1 gene encoding microsomal delta-12 fatty acid desaturase from Brassica rapa and development of Brassica napus containing high oleic acid contents. Plant Cell Rep, 2011, 30: 1881–1892
[30]崔峰, 夏家辉. 关于假基因的研究进展. 生命科学研究, 1999, 3: 203–209
Cui F, Xia J H. On Research Progress of Pseudogene. Life Sci Res, 1999, 3: 203–209
[31]O’Quin J B, Mullen R T, Dyer J M. Addition of an N-terminal epitope tag significantly increases the activity of plant fatty acid desaturases expressed in yeast cells. Appl Microbiol Biot, 2009, 83: 117–125
[32]Laga B, Seurinck J, Verhoye T, Lambert B. Molecular breeding for high oleic and low linolenic fatty acid composition in Brassica napus. Pflanzenschutz-Nachrichten Bayer, 2004, 54: 87–92
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