花生脂肪酸延长酶基因AhFAE1及其启动子的克隆与功能分析
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摘要
为探讨花生脂肪酸中低芥酸含量的原因,从花生中克隆了脂肪酸延长酶FAE1及其启动子序列,并进行了功能分析。结果表明,AhFAE1全长cDNA为2 202bp,含有441bp的5′非翻译区及201bp的3′非翻译区,编码蛋白含519aa,分子量58.17Da,理论等电点9.15,AhFAE1与麻疯树(Jatropha curcas ALB76796.1)、黄麻(Corchorus capsularis OMO87584.1)、拟南芥AtKCS4亲缘关系较近。亚细胞定位结果显示AhFAE1定位于内质网。qRT-PCR结果显示,AhFAE1在各个组织中均有表达,在种子中随着种子的发育成"钟"型变化,花后60d表达量最高。构建了植物表达载体,通过农杆菌介导法转化拟南芥研究启动子功能,利用GUS组织化学染色研究其表达特征,在任何组织中未发现GUS活性。推测AhFAE1可能参与了花生长链脂肪酸的合成,但是该基因启动子转录活性弱可能是造成花生中低芥酸含量的主要原因。
In order to explore the reasons for low erucic acid content in peanut seeds, FAE1 cDNA and its promoter sequence were isolated and their functionality were analyzed. Results showed that a full-length cDNA sequence and its promoter were isolated. Sequence analysis showed that cDNA fragment length was 2 202 bp containing 5′-untranslated region of 441 bp, and 3′-untranslated region of 201 bp, encoding a protein of 517 amino acids with a predicted molecular mass of 58.17 Da and theoretical pI of 9.15. Phylogenetic tree analysis revealed that AhFAE1 shared closer genetic relationship with homologues from Jatropha curcas(ALB76796.1),Corchorus capsularis(OMO87584.1) and Arabidopsis(AtKCS4). Subcellular localization assays showed that AhFAE1 protein was located in endoplasmic reticulum. qRT-PCR revealed that AhFAE1 transcripts were constitutively expressed in all examined tissues and displayed a bell-shaped pattern throughout seed development with expression peaking at 60 DAF. To study the function of this promoter, we constructed a binary plant vector pBI-PAhFAE1, which was introduced into Arabidopsis by Agrobacterium-mediated transformation. Histochemical staining analysis indicated that no GUS activity was detected in any tissue. AhFAE1 was potentially involved in very long chain fatty acid synthesization. Low transcriptional activity of AhFAE1 promoter could have caused the low content of erucic acid in peanut.
In order to explore the reasons for low erucic acid content in peanut seeds, FAE1 cDNA and its promoter sequence were isolated and their functionality were analyzed. Results showed that a full-length cDNA sequence and its promoter were isolated. Sequence analysis showed that cDNA fragment length was 2 202 bp containing 5′-untranslated region of 441 bp, and 3′-untranslated region of 201 bp, encoding a protein of 517 amino acids with a predicted molecular mass of 58.17 Da and theoretical pI of 9.15. Phylogenetic tree analysis revealed that AhFAE1 shared closer genetic relationship with homologues from Jatropha curcas(ALB76796.1),Corchorus capsularis(OMO87584.1) and Arabidopsis(AtKCS4). Subcellular localization assays showed that AhFAE1 protein was located in endoplasmic reticulum. qRT-PCR revealed that AhFAE1 transcripts were constitutively expressed in all examined tissues and displayed a bell-shaped pattern throughout seed development with expression peaking at 60 DAF. To study the function of this promoter, we constructed a binary plant vector pBI-PAhFAE1, which was introduced into Arabidopsis by Agrobacterium-mediated transformation. Histochemical staining analysis indicated that no GUS activity was detected in any tissue. AhFAE1 was potentially involved in very long chain fatty acid synthesization. Low transcriptional activity of AhFAE1 promoter could have caused the low content of erucic acid in peanut.
引文
[1] 石东乔,周弈华,陈正华.植物脂肪酸调控基因工程研究[J].生命科学,2002,14(5):291-244.
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[3] Ghanevati M,Jaworski J G.Engineering and mechanistic studies of the Arabidopsis FAE1β-ketoacyl-CoA synthase,FAE1KCS [J].Europ J Biochem,2002,269(14):3 531-3 539.
[4] Puyaubert J,Garbay B,Costaglioli P,et al.Acyl-CoA elongase expression during seed development in Brassica napus[J].Biochimica et Biophysica Acta,2001,1533(2):141-152.
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[6] James D W J R,Lim E,Keller J,et al.Directedtagging of the Arabidopsis FATTYACIDELONGATION 1 (FAE1) genewith the maizetransposon Activator[J].Plant Cell,1995,7(3):309-319.
[7] Millar A A,Kunst L.Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme[J].Plant J,1997,12(1):121-131.
[8] Lassner M W,Lardizabal K,Metz J G.A jojoba beta-ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants[J].Plant Cell,1996,8(2):281-292.
[9] Mietkiewska E,Giblin E M,Wang S,et al.Seed-specific heterologous expression of a nasturtium FAE gene in Arabidopsis results in a dramatic increase in the proportion of erucic acid[J].Plant Physiol,2004,136(1):2 665-2 675.
[10] Mietkiewska E,Brost J M,Giblin E M,et al.Cloning and functional characterization of the fatty acid elongase 1 (FAE1) gene from high erucic Crambeabyssinica cv.Prophet[J].Plant Biotechnol J,2007,5:636-645.
[11] Rossak M,Smith M,Kunst L.Expressionof the FAE1 gene and FAE1 promoter activity in developing seeds of Arabidopsis thaliana[J].Plant Mol Biol,2001,46(6):717-725.
[12] Han J,Lühs W,Sonntag K,et al.Functional characterization of β-ketoacyl-CoA synthase genes from Brassica napus L.[J].Plant Mol Biol,2001,46(2):229-239.
[13] Roscoe T J,Lessire R,Puyaubert J,et al.Mutations in the fatty acid elongation 1 geneare associated with a loss of beta-ketoacyl-CoA synthase activity in low erucic acid rapeseed[J].FEBS Lett,2001,492:107-111.
[14] Katavic V,Friesen W,Barton D L,et al.Utility of the Arabidopsis FAE1 and yeast SLC1-1 genes for improvements in erucic acid and oil content in rapeseed[J].Biochem Soc Transact,2000,28(6):935-937.
[15] Kanrar S,Venkateswari J,Dureja P,et al.Modification of erucic acid content in Indian mustard (Brassica juncea) by up-regulation and down-regulation of the Brassica juncea FATTY ACID ELONGATION1 (BjFAE1) gene[J].Plant Cell Rep,2006,25(2):148-155.
[16] Qi Q,Rose P A,Abrams G D,et al.(+)-Abscisic acid metabolism,3-ketoacyl-coenzyme A synthase gene expression,and very-long-chain monounsaturated fatty acid biosynthesis in Brassica napus embryos[J].Plant Physiol,1998,117(3):979-987.
[17] Peng Q,Hu Y,Wei R,et al.Simultaneous silencing of FAD2and FAE1 genes affects both oleic acid and erucic acid contents in Brassica napus seeds[J].Plant Cell Rep,2010,29(4):317-325.
[18] Shi J,Lang C,Wu X,et al.RNAi knockdown of fatty acid elongase 1 alters fatty acid composition in Brassica napus[J].Biochem Biophys Res Commun,2015,466(3):518-522.
[19] Li X,Mei D,Liu Q,et al.Down-regulation of crambe fatty acid desaturase and elongase in Arabidopsis and crambe resulted in significantly increased oleic acid content in seed oil[J].Plant Biotechnol J,2016,14(1):323-331.
[20] Brand Y,Hovav R.Identification of suitable internal control genes for quantitative real-time PCR expression analyses in peanut (Arachis hypogaea)[J].Peanut Sci,2010,37:12-19.
[21] 石磊,苗利娟,齐飞艳,等.花生Δ9-硬脂酰-ACP脱氢酶基因启动子的克隆及功能分析[J].作物学报,2016,42:1 629-1 637.
[22] Sparkes I A,Runions J,Kearns A,et al.Rapid,transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants[J].Nat Prot,2006,1(4):2 019-2 025.
[23] Lemieux B,Miquel M,Somerville C,et al.Mutants of Arabidopsis with alterations in seed lipid fatty acid composition[J].Theor Appl Genet,1990,80(2):234-240.
[24] Bertioli D J,Cannon S B,Froenicke L,et al.The genome sequences of Arachis duranensis and Arachis ipaensis,the diploid ancestors of cultivated peanut[J].Nat Genet,2016,48(4):438-446.
[25] Chen Z,Wang M,Barkley N A,et al.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[J].Plant Mol Biol Rep,2010,28:542-548.
[26] 郭安强,万勇善,刘风珍.花生2-甲基-6-植基-1,4-苯醌甲基转移酶基因VTE3的克隆及多态性分析[J].中国农业科学,2012,45(9):1 685-1 695.
[27] 东金玉,万勇善,刘风珍.花生Δ9-硬脂酰-ACP脱氢酶基因(SAD)的序列分析[J].作物学报,2012,38(7):1 167-1 177.
[28] Nath U K,Wilmer J A,Wallington E J,et al.Increasing erucic acid content through combination of endogenous low polyunsaturated fatty acids alleles with Ld-LPAAT+Bn-fae1 transgenes in rapeseed (Brassica napus L.)[J].Theor Appl Genet,2009,118(4):765-773.
[29] Wang N,Shi L,Tian F,et al.Assessment of FAE1 polymorphisms in three Brassica species using Eco TILLING and their association with differences in seed erucic acid contents[J].BMC Plant Biol,2010,10:137.
[30] Wu G,Wu Y,Xiao L,et al.Zero erucic acid trait of rapeseed (Brassica napus L.) results from a deletion of four base pairs in the fatty acid elongase 1 gene[J].Theor Appl Genet,2008,116(4):491-499.
[31] 武玉花,吴刚,肖玲,等.十字花科植物中低芥酸野生种的发掘和FAE1基因的功能验证[J].中国农业科学,2009,42(11):3 819-3 827.
[32] Ghanevati M,Jaworski J G.Active-site residues of a plant membrane-bound fatty acid elongase beta-ketoacyl-CoA synthase,FAE1 KCS[J].Biochimica et Biophysica Acta,2001,1530(1):77-85.
[2] Barret P,Delourme R,Renard M ,et al.A rapeseed FAE1 gene is linked to the E1 locus associated with variation in the content of erucic acid[J].Theoret Appl Genet,1998,96:177-186.
[3] Ghanevati M,Jaworski J G.Engineering and mechanistic studies of the Arabidopsis FAE1β-ketoacyl-CoA synthase,FAE1KCS [J].Europ J Biochem,2002,269(14):3 531-3 539.
[4] Puyaubert J,Garbay B,Costaglioli P,et al.Acyl-CoA elongase expression during seed development in Brassica napus[J].Biochimica et Biophysica Acta,2001,1533(2):141-152.
[5] Blacklock B J,Jaworski J G.Substrate specificity of Arabidopsis 3-ketoacyl-CoA synthases[J].Biochem Biophys Res Commun,2006,346(2):583-590.
[6] James D W J R,Lim E,Keller J,et al.Directedtagging of the Arabidopsis FATTYACIDELONGATION 1 (FAE1) genewith the maizetransposon Activator[J].Plant Cell,1995,7(3):309-319.
[7] Millar A A,Kunst L.Very-long-chain fatty acid biosynthesis is controlled through the expression and specificity of the condensing enzyme[J].Plant J,1997,12(1):121-131.
[8] Lassner M W,Lardizabal K,Metz J G.A jojoba beta-ketoacyl-CoA synthase cDNA complements the canola fatty acid elongation mutation in transgenic plants[J].Plant Cell,1996,8(2):281-292.
[9] Mietkiewska E,Giblin E M,Wang S,et al.Seed-specific heterologous expression of a nasturtium FAE gene in Arabidopsis results in a dramatic increase in the proportion of erucic acid[J].Plant Physiol,2004,136(1):2 665-2 675.
[10] Mietkiewska E,Brost J M,Giblin E M,et al.Cloning and functional characterization of the fatty acid elongase 1 (FAE1) gene from high erucic Crambeabyssinica cv.Prophet[J].Plant Biotechnol J,2007,5:636-645.
[11] Rossak M,Smith M,Kunst L.Expressionof the FAE1 gene and FAE1 promoter activity in developing seeds of Arabidopsis thaliana[J].Plant Mol Biol,2001,46(6):717-725.
[12] Han J,Lühs W,Sonntag K,et al.Functional characterization of β-ketoacyl-CoA synthase genes from Brassica napus L.[J].Plant Mol Biol,2001,46(2):229-239.
[13] Roscoe T J,Lessire R,Puyaubert J,et al.Mutations in the fatty acid elongation 1 geneare associated with a loss of beta-ketoacyl-CoA synthase activity in low erucic acid rapeseed[J].FEBS Lett,2001,492:107-111.
[14] Katavic V,Friesen W,Barton D L,et al.Utility of the Arabidopsis FAE1 and yeast SLC1-1 genes for improvements in erucic acid and oil content in rapeseed[J].Biochem Soc Transact,2000,28(6):935-937.
[15] Kanrar S,Venkateswari J,Dureja P,et al.Modification of erucic acid content in Indian mustard (Brassica juncea) by up-regulation and down-regulation of the Brassica juncea FATTY ACID ELONGATION1 (BjFAE1) gene[J].Plant Cell Rep,2006,25(2):148-155.
[16] Qi Q,Rose P A,Abrams G D,et al.(+)-Abscisic acid metabolism,3-ketoacyl-coenzyme A synthase gene expression,and very-long-chain monounsaturated fatty acid biosynthesis in Brassica napus embryos[J].Plant Physiol,1998,117(3):979-987.
[17] Peng Q,Hu Y,Wei R,et al.Simultaneous silencing of FAD2and FAE1 genes affects both oleic acid and erucic acid contents in Brassica napus seeds[J].Plant Cell Rep,2010,29(4):317-325.
[18] Shi J,Lang C,Wu X,et al.RNAi knockdown of fatty acid elongase 1 alters fatty acid composition in Brassica napus[J].Biochem Biophys Res Commun,2015,466(3):518-522.
[19] Li X,Mei D,Liu Q,et al.Down-regulation of crambe fatty acid desaturase and elongase in Arabidopsis and crambe resulted in significantly increased oleic acid content in seed oil[J].Plant Biotechnol J,2016,14(1):323-331.
[20] Brand Y,Hovav R.Identification of suitable internal control genes for quantitative real-time PCR expression analyses in peanut (Arachis hypogaea)[J].Peanut Sci,2010,37:12-19.
[21] 石磊,苗利娟,齐飞艳,等.花生Δ9-硬脂酰-ACP脱氢酶基因启动子的克隆及功能分析[J].作物学报,2016,42:1 629-1 637.
[22] Sparkes I A,Runions J,Kearns A,et al.Rapid,transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants[J].Nat Prot,2006,1(4):2 019-2 025.
[23] Lemieux B,Miquel M,Somerville C,et al.Mutants of Arabidopsis with alterations in seed lipid fatty acid composition[J].Theor Appl Genet,1990,80(2):234-240.
[24] Bertioli D J,Cannon S B,Froenicke L,et al.The genome sequences of Arachis duranensis and Arachis ipaensis,the diploid ancestors of cultivated peanut[J].Nat Genet,2016,48(4):438-446.
[25] Chen Z,Wang M,Barkley N A,et al.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[J].Plant Mol Biol Rep,2010,28:542-548.
[26] 郭安强,万勇善,刘风珍.花生2-甲基-6-植基-1,4-苯醌甲基转移酶基因VTE3的克隆及多态性分析[J].中国农业科学,2012,45(9):1 685-1 695.
[27] 东金玉,万勇善,刘风珍.花生Δ9-硬脂酰-ACP脱氢酶基因(SAD)的序列分析[J].作物学报,2012,38(7):1 167-1 177.
[28] Nath U K,Wilmer J A,Wallington E J,et al.Increasing erucic acid content through combination of endogenous low polyunsaturated fatty acids alleles with Ld-LPAAT+Bn-fae1 transgenes in rapeseed (Brassica napus L.)[J].Theor Appl Genet,2009,118(4):765-773.
[29] Wang N,Shi L,Tian F,et al.Assessment of FAE1 polymorphisms in three Brassica species using Eco TILLING and their association with differences in seed erucic acid contents[J].BMC Plant Biol,2010,10:137.
[30] Wu G,Wu Y,Xiao L,et al.Zero erucic acid trait of rapeseed (Brassica napus L.) results from a deletion of four base pairs in the fatty acid elongase 1 gene[J].Theor Appl Genet,2008,116(4):491-499.
[31] 武玉花,吴刚,肖玲,等.十字花科植物中低芥酸野生种的发掘和FAE1基因的功能验证[J].中国农业科学,2009,42(11):3 819-3 827.
[32] Ghanevati M,Jaworski J G.Active-site residues of a plant membrane-bound fatty acid elongase beta-ketoacyl-CoA synthase,FAE1 KCS[J].Biochimica et Biophysica Acta,2001,1530(1):77-85.