油葵HaFAD2-2基因的克隆及表达分析
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摘要
脂肪酸脱氢酶2(fatty acid desaturase,FAD2)催化油酸生成亚油酸,是植物体内生成多不饱和脂肪酸的关键酶。根据已报道的向日葵(Helianthus annuus L.)FAD2基因序列,设计引物进行RT-PCR,克隆得到油葵FAD2-2基因全长cDNA,命名为HaFAD2-2。该基因开放阅读框为1 152bp,编码383个氨基酸,相对分子质量43.96kD,等电点为8.56。对基因组进行内含子调查发现,该基因在编码区内没有内含子。多序列比对和系统进化分析发现,FAD2-2基因编码蛋白与金盏菊(Calendula officinalis)、斑鸠菊(Vernonia galamensis)等菊科植物具有较近的亲缘关系。qRT-PCR分析表明,HaFAD2-2基因在根、茎、叶、花、子叶和未成熟种子中均有表达,且以叶中的表达量最高,未成熟种子中的表达量最低;低温(5℃、15℃)胁迫处理能显著促进该基因在根中的表达,抑制其在叶中的表达;盐胁迫(300 mmol/L NaCl)处理对其表达也具有抑制作用。该研究结果可为进一步探讨HaFAD2-2基因的功能奠定基础。
Linoleic acid is synthesized by fatty acid desaturase 2(FAD2),which play an important role for produce polyunsaturated fatty acid in plant.The key enzyme for the conversion of oleic acid into linoleic acid was isolated from sunflower(Helianthus annuus L.),and designated as HaFAD2-2.The open reading frame(ORF)of HaFAD2-2was 1 152 bp in length,which encoded 383 amino acids,with the isoelectric point of 8.56 and molecular mass of 43.96 kD.The genome intron survey showed that there is no introns in open reading frame region.Phylogenetic analysis showed that HaFAD2-2protein shares high homologies with calendula(Calendula officinalis)and dove(Vernonia galamensis).qRT-PCR analysis indicated that HaFAD2-2was expressed in roots,stems,leaves,cotyledon,flowers and developing seeds,and the highest in leaves,the lowest in developing seeds.Furthermore,HaFAD2-2in roots could be induced significantly by low temperature(5 ℃,15 ℃),but repressed in leaves by low temperature(5 ℃,15 ℃)and NaCl stress(300mmol/L).Results in this experiment provide reference for further functional study on HaFAD2-2.
Linoleic acid is synthesized by fatty acid desaturase 2(FAD2),which play an important role for produce polyunsaturated fatty acid in plant.The key enzyme for the conversion of oleic acid into linoleic acid was isolated from sunflower(Helianthus annuus L.),and designated as HaFAD2-2.The open reading frame(ORF)of HaFAD2-2was 1 152 bp in length,which encoded 383 amino acids,with the isoelectric point of 8.56 and molecular mass of 43.96 kD.The genome intron survey showed that there is no introns in open reading frame region.Phylogenetic analysis showed that HaFAD2-2protein shares high homologies with calendula(Calendula officinalis)and dove(Vernonia galamensis).qRT-PCR analysis indicated that HaFAD2-2was expressed in roots,stems,leaves,cotyledon,flowers and developing seeds,and the highest in leaves,the lowest in developing seeds.Furthermore,HaFAD2-2in roots could be induced significantly by low temperature(5 ℃,15 ℃),but repressed in leaves by low temperature(5 ℃,15 ℃)and NaCl stress(300mmol/L).Results in this experiment provide reference for further functional study on HaFAD2-2.
引文
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[15]WANG Y,ZHANG X,ZHAO Y,et al.Insights into the novel members of the FAD2gene family involved in high-oleate fluxes in peanut[J].Genome,2015,58(8):375-383.
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[17]KARGIOTIDOU A,DELI D,GALANOPOULOU D,et al.Low temperature and light regulate delta 12fatty acid desaturases(FAD2)at a transcriptional level in cotton(Gossypium hirsutum)[J].Journal of Experimental Botany,2008,59:2 043-2 056.
[18]LIU W,LI W,HE Q,et al.Characterization of 19genes encoding membrane-bound fatty acid desaturases and their expression profiles in Gossypium raimondiiunder low temperature[J].PLoS One,2015,10(4):e0123281.
[2]FALCONE D L,GIBSON S,LEMIEUX B,et al.Identification of a gene that complements an Arabidopsis mutant deficient in chloroplastω6desaturase activity[J].Plant Physiology,1994,106:1 453-1 459.
[3]JIN U H,LEE J W,CHUNG Y S,et al.Characterization and temporal expression of a x-6fatty acid desaturasec DNA from sesame(Sesamum indicum L.)seeds[J].Plant Science,2001,161:935-941.
[4]CHAPMAN K D,AUSTIN-BROWN S,SPARACE S A,et al.Transgenic cotton plants with increased seed oleic acid content[J].Journal of the American Oil ChemistsSociety,2001,78(9):941-947.
[5]TANG G Q,NOVITZKY W P,CAROL GRIFFIN H,et al.Oleate desaturase enzymes of soybean:evidence of regulation through differential stability and phosphorylation[J].The Plant Journal,2005,44:433-446.
[6]XIAOYUAN C,QINGLI Y,LIJUAN P,et al.Isolation and characterization of fatty acid desaturase genes from peanut(Arachis hypogaea L.)[J].Plant Cell Reports,2011,30:1 393-1 404.
[7]黄冰艳,张新友,苗利娟,等.花生FAD2基因RNAi载体转化及转基因籽粒脂肪酸分析[J].中国油料作物学报,2008,30(3):290-293.HUANG B Y,ZHANG X Y,MIAO L J,et al.RNAi trandformation of AhFAD2gene and fatty acid analysis of transgenic seeds[J].Chinese Journal of Oil Crop Sciences,2008,30(3):290-293.
[8]WATANABE K,OURA T,SAKAI H,et al.Yeast△12fatty acid desaturase:gene cloning,expression,and function[J].Bioscience,Biotechnology,and Biochemistry,2004,68(3):721-727.
[9]RODRGUEZV S,SNCHEZ G A,MARTNEZ R J,et al.Fluidization of membrane lipids enhances the tolerance of Saccharomyces cerevisiae to freezing and salt stress[J].Applied and Environmental Microbiology,2007,73(1):110.
[10]刘公社,ALAIN B,彭克敬.向日葵研究与开发[M].北京:中国科学技术出版社,1994:85-87.
[11]TAMURA K,DUDLEY J,NEI M,et al.MEGA4:molecular evolutionary genetics analysis(MEGA)software version4.0[J].Molecular Biology and Evolution,2007,24(8):1 596-1 599.
[12]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the2-ΔΔCTmethod[J].Methods,2001,25:402-408.
[13]LOS D A,MURATA N.Structure and expression of fatty acid desaturases[J].Biochimica Biophysica Acta,1998,1394(1):3-15.
[14]DONG C J,CAO N,ZHANG Z G,et al.Characterization of the fatty acid desaturase genes in cucumber:structure,phylogeny,and expression patterns[J].PLoS One,2016,11(3):e0149917.
[15]WANG Y,ZHANG X,ZHAO Y,et al.Insights into the novel members of the FAD2gene family involved in high-oleate fluxes in peanut[J].Genome,2015,58(8):375-383.
[16]CAO S J,ZHOU X R,CRAIG C W,et al.A large and functionally diverse family of FAD2genes in safflower(Carthamus tinctorius L.)[J].BMC Plant Biology,2013,13(1):5.
[17]KARGIOTIDOU A,DELI D,GALANOPOULOU D,et al.Low temperature and light regulate delta 12fatty acid desaturases(FAD2)at a transcriptional level in cotton(Gossypium hirsutum)[J].Journal of Experimental Botany,2008,59:2 043-2 056.
[18]LIU W,LI W,HE Q,et al.Characterization of 19genes encoding membrane-bound fatty acid desaturases and their expression profiles in Gossypium raimondiiunder low temperature[J].PLoS One,2015,10(4):e0123281.