doi:

DOI: 10.3724/SP.J.1006.2016.01471

Acta Agronomica Sinica (作物学报) 2016/42:10 PP.1471-1478

Functional Analysis of BnFAD2-C5 Promoter and Intron at Expression Level in Brassica napus


Abstract:
High oleic rapeseed breeding and the formation mechanism of oleic acid have become a central issue after finding the important economic value of rapeseed oil with high oleic acid. The fatty acid dehydrogenase gene (FAD2) is a key enzyme gene to control oleic acid content, but the regulation of FAD2 gene is not well understood. According to the homology between rapeseed and oleracea, the BnFAD2-C5 promoter sequence of 1257 bp was cloned. Promoter and intron of BnFAD2-C5 gene were analyzed using β-glucuronidase (GUS) reporter and green fluorescent protein (GFP) reporter system to construct deleted vectors and trans-form Arabidopsis thaliana. Deletion analysis of BnFAD2-C5 promoter through GUS stainning revealed that -319 to -1 bp was the minimum promoter region. And deletion analysis of BnFAD2-C5 promoter and intron through GFP reporter system using western technique showed that -1257 to -1020 bp and -319 to -1 bp regions of BnFAD2-C5 promoter could induce expression of reporter genes effectively in transgenic Arabidopsis seed in the mid stage of seed development, while BnFAD2-C5 intron could confer the enhancement of promoter's function and the intron-mediated enhancement region was mainly located in +631 to +1033 bp.

Key words:Brassica napus,BnFAD2-C5 gene,Intron-mediated enhancement,Arabidopsis thalina

ReleaseDate:2017-01-12 13:30:45



[1] Jung S, Swift D, Sengoku E, Patel M, Teule F, Powell G, Abbott A. The high oleate trait in the cultivated peanut (Arachis hypogaea L.). Isolation and characterization of two genes encoding microsomal oleoyl-PC desaturases. Mol Gen Genet, 2000, 263: 796-805

Guan C Y, Liu C L, Chen S Y, Peng Q, Li X, Guan M. High oleic acid content materials of rapeseed (Brassica napus) produced by radiation breeding. Acta Agron Sin, 2006, 32: 1625-1629

[2] Terés S, Barceló-Coblijn G, Benet M, Alvarez R, Bressani R, Halver J E, EscribáP V. Oleic acid content is responsible for the reduction in blood pressure induced by olive oil. Proc Natl Acad Sci USA, 2008, 105: 13811-13816

He J M, Wang J Q, Chen W, Li G Z, Dong Y S, Cun S X. Studies on rapidly obtaining high oleic acid germplasm of Brassica napus by mutagen EMS and microspore culture. Southwest China J Agric Sci, 2003, 16(2): 34-36 (in Chinese with English abstract)

[3] Wijesundera C, Ceccato C, Fagan P, Shen Z, Burton W, Salisbury P. Canola quality Indian mustard oil (Brassica juncea) is more stable to oxidation than conventional Canola oil (Brassica napus). Am J Clin Nutr, 2008, 85:693-699

Liu F, Liu R Y, Peng Y, Guan C Y. Cloning and expression of BnFAD2-C1 gene involved in Brassica napus and analysis of transcription regulation elements. Acta Agron Sin, 2015, 41: 1663-1670 (in Chinese with English abstract)

[4] Tang G Q, Novitzky W P, Carol Griffin H. Oleate desaturase enzymes of soybean: evidence of regulation through differential stability and phosphorylation. Plant J, 2005, 44: 433-446

[5] Rojas-Barros P, de Haro A, Fernandez-Martinez J M. Inheritance of high oleic/low ricinoleic acid content in the seed oil of castor mutant OLE-1. Cropence, 2005, 45: 157-162

[6] Nabloussi A, Fernandez-Martinez J M, Velasco L. Inheritance of mid and high oleic acid content in Ethiopian mustard. Cropence, 2006, 46: 2361-2367

[7] 官春云, 刘春林, 陈社员, 彭琦, 李栒, 官梅. 辐射育种获得油菜(Brassica napus)高油酸材料. 作物学报, 2006, 32: 1625-1629 (in Chinese with English abstract)

[8] 和江明, 王敬乔, 陈薇, 李根泽, 董云松, 寸守铣. 用EMS诱变和小孢子培养快速获得甘蓝型油菜高油酸种质材料的研究. 西南农业学报, 2003, 16(2): 34-36

[9] Hu X, Sullivan-Gilbert M, Gupta M, Thompson S A. Mapping of the loci controlling oleic and linolenic acid contents and development of fad 2 and fad 3 allele-specific markers in canola (Brassica napus L.). Theor Appl Genet, 2006, 113: 497-507

[10] Stoutjesdijk P A, Hurlestone C, Singh S P, Green A G. High-oleic acid Australian Brassica napus and B. juncea varieties produced by co-suppression of endogenous D12-desaturases. Biochem Soc T, 2000, 28: 938-940

[11] Peng Q, Hu Y, Wei R, Zhang Y, Guan C, Ruan Y, Liu C. Simultaneous silencing of FAD2 and FAE1 genes affects both oleic acid and erucic acid contents in Brassica napus seeds. Plant Cell Rep, 2010, 29: 317-325

[12] Kim M J, Kim H, Shin J S, Chung C H, Ohlrogge J B, Suh M C. Seed-specific expression of sesame microsomal oleic acid desaturase is controlled by combinatorial properties between negative cis-regulatory elements in the SeFAD2 promoter and enhancers in the 5′-UTR intron. Mol Genet Genom, 2006, 276: 351-368

[13] Xiao G, Zhang Z Q, Yin C F, Liu R Y, Wu X M, Tan T L, Chen S Y, Lu C M, Guan C Y. Characterization of the promoter and 5′-UTR intron of oleic acid desaturase (FAD2) gene in Brassica napus. Gene, 2014, 545: 45-55

[14] 刘芳, 刘睿洋, 彭烨, 官春云. 甘蓝型油菜BnFAD2-C1 基因全长序列的克隆, 表达及转录调控元件分析. 作物学报, 2015, 41: 1663-1670

[15] Jefferson R A, Kavanagh T A, Bevan M W. GUS fusion: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J, 1987, 6: 3901-3907

[16] Heppard E P, Kinney A J, Stecca K L, Miao G H. Developmental and growth temperature regulation of two different microsomal [omega]-6 desaturase genes in soybeans. Plant Physiol, 1996, 110: 311-319

[17] Pirtle I L, Kongcharoensuntorn W, Nampaisansuk M, Knesek J E, Chapman K D, Pirtle R M. Molecular cloning and functional expression of the gene for a cotton Δ-12 fatty acid desaturase (FAD2). BBA-Gene Struc Exp, 2001, 1522: 122-129

[18] Mascarenhas D, Mettler I J, Pierce D A, Lowe H W. Intron-mediated enhancement of heterologous gene expression in maize. Plant Mol Biol, 1990, 15: 913-920

[19] Chung B Y, Simons C, Firth A E, Brown C M, Hellens R P. Effect of 5'UTR introns on gene expression in Arabidopsis thaliana. BMC Genom, 2006, 7: 120

[20] Parra G, Bradnam K, Rose A B, Korf I. Comparative and functional analysis of intron-mediated enhancement signals reveals conserved features among plants. Nucl Acids Res, 2011, 39: 5328-5337

[21] Carola M, Finer J J. The intron and 5′ distal region of the soybean Gmubi promoter contribute to very high levels of gene expression in transiently and stably transformed tissues. Plant Cell Rep, 2015, 34: 111-120

[22] Rose A B. Requirements for intron-mediated enhancement of gene expression in Arabidopsis. RNA, 2002, 8: 1444-1453

[23] Stålberg K, Ellerstöm M, Ezcurra I, Ablov S, Rask L. Disruption of an overlapping E-box/ABRE motif abolished high transcription of the napA storage-protein promoter in transgenic Brassica napus seeds. Planta, 1996, 199: 515-519

[24] Eulgem T, Rushton P J, Robatzek S, Somssich I E. The WRKY superfamily of plant transcription factors. Trends Plant Sci, 2000, 5: 199-206

[25] Park H C, Kim M L, Kang Y H, Jeon J M, Yoo J H, Kim M C, Yoon H W. Pathogen-and NaCl-induced expression of the SCaM-4 promoter is mediated in part by a GT-1 box that interacts with a GT-1-like transcription factor. Plant Physiol, 2004, 135: 2150-2161

[26] Andreasson E, Taipalensuu J, Rask L, Meijer J. Age-dependent wound induction of a myrosinase-associated protein from oilseed rape (Brassica napus). Plant Mol Biol, 1999, 41: 171-180

[27] Yanagisawa S. Dof1 and Dof2 transcription factors are associated with expression of multiple genes involved in carbon metabolism in maize. Plant J, 2000, 21: 281-288

[28] Rouster J, Leah R, Mundy J, Cameron-Mills V. Identification of a methyl jasmonate-responsive region in the promoter of a lipoxygenase 1 gene expressed in barley grain. Plant J, 1997, 11: 513-523

[29] Lam E, Chua N H. ASF-2: a factor that binds to the cauliflower mosaic virus 35S promoter and a conserved GATA motif in Cab promoters. Plant Cell, 1989, 1: 1147-1156

[30] Kusnetsov V, Landsberger M, Meurer J. The assembly of the CAAT-box binding complex at a photosynthesis gene promoter is regulated by light, cytokinin, and the stage of the plastids. J Biol Chem, 1999, 274: 36009-36011