GT和GATA转录因子对甘蓝型油菜BnA5.FAD2和BnC5.FAD2启动子功能的调控
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摘要
【目的】脂肪酸去饱和酶2基因(FAD2)是控制油菜中油酸含量的重要基因,通过研究GATA和GT转录因子与BnA5.FAD2和BnC5.FAD2启动子的互作关系及其转录调控机制,为高油酸油菜育种提供分子理论基础。【方法】通过缺失启动子片段及生物信息学分析,预测BnA5.FAD2和BnC5.FAD2启动子区潜在顺式作用元件;从PlantTFDB转录因子数据库中筛选候选转录因子,通过实时荧光定量PCR(qRT-PCR)检测转录因子的表达规律,并与BnA5.FAD2和BnC5.FAD2表达规律对比,进一步筛选候选转录因子;利用酵母单杂交验证转录因子与启动子序列的互作情况;将转录因子与BnA5.FAD2和BnC5.FAD2启动子序列共转入拟南芥,通过Western blot检测报告基因GFP的蛋白表达情况,分析转录因子对于启动子功能的影响。【结果】单独敲除BnA5.FAD2和BnC5.FAD2启动子中的GT或GATA顺式作用元件后,GFP蛋白丰度均下降,表明GT和GATA顺式作用元件在调节基因转录中起重要作用。酵母单杂交结果显示,GATA家族转录因子(Bna010243-1、Bna026124-1和Bna026124-2)和GT家族转录因子(Bna010915和Bna013749)可以与BnA5.FAD2和BnC5.FAD2启动子相互作用。Western blot结果显示,当GATA家族转录因子与含有GATA元件的启动子片段共转化拟南芥时,GFP蛋白含量明显高于无转录因子时,当敲除GATA元件时,GFP蛋白含量无明显变化;当GT家族转录因子与含有GT元件的启动子片段共转化拟南芥时,GFP蛋白含量有明显变化,当敲除GT元件时,GFP蛋白含量无明显变化。【结论】GATA家族的转录因子Bna010243-1、Bna026124-1和Bna026124-2可以与BnA5.FAD2和BnC5.FAD2启动子中GATA元件相互作用,增强基因的表达水平。GT家族的转录因子Bna010915可以与BnA5.FAD2和BnC5.FAD2启动子中的GT元件发生互作,正向调节基因表达;Bna013749通过与BnA5.FAD2和BnC5.FAD2启动子中的GT元件发生互作,负向调节基因表达。
【Objective】 Fatty acid desaturase 2 gene(FAD2) is an important gene for controlling oleic acid content in rapeseed. And in this paper, the interaction between GATA family and BnA5.FAD2 and BnC5.FAD2 gene promoters or GT family transcription factors and BnA5.FAD2 and BnC5.FAD2 gene promoters was studied to make sure the transcription regulation mechanism to provide the theoretical basis for high oleic acid breeding. 【Method】 The potential cis-elements were predicted by deletion of promoter fragments and bioinformatics analysis, and candidate transcription factors were screened from PlantTFDB transcription factor database. Real-time PCR(q RT-PCR) was used to detect the expression pattern of transcription factors to further screen candidate transcription factors by comparison with BnA5.FAD2 and BnC5.FAD2 gene expression patterns. Transcription factors were co-transformed with BnA5.FAD2 and BnC5.FAD2 gene promoters into yeast and verified interaction relationship between promoter and transcription factors by yeast one-hybrid. The transcription factors were transformed into Arabidopsis thaliana with BnA5.FAD2 and BnC5.FAD2 gene promoter sequence, and the reporter gene GFP was detected by western blot to analyze the effects of transcription factors on promoters. 【Result】 After knocking out the GT or GATA cis-elements from BnA5.FAD2 and BnC5.FAD2 gene promoters respectively, the GFP protein abundance decreased, which indicate that GT and GATA cis-elements play important roles in regulating gene transcription. Yeast one-hybrid results showed that the GATA family transcription factors(Bna010243-1, Bna026124-1, Bna026124-2) and the GT family transcription factors(Bna010915 and Bna013749) can interact with BnA5.FAD2 and BnC5.FAD2 gene promoters. Western blot analysis showed that when the GATA family transcription factor and the promoter fragment containing GATA elements were co-transformed into Arabidopsis thaliana, the GFP protein content was significantly higher than that of no transcription factor. When the GATA element was knocked out, the GFP protein content did not change significantly. When the GT family transcription factor and the promoter fragment containing the GT element were co-transformed into Arabidopsis thaliana, the GFP protein content changed significantly. When the GT element was knocked out, the GFP protein content did not change significantly. 【 Conclusion 】 The GATA family transcription factors Bna010243-1, Bna026124-1, Bna026124-2 can interact with the GATA elements in the BnA5.FAD2 and BnC5.FAD2 gene promoters to enhance gene expression levels. The GT family transcription factor Bna010915 interacts with the GT elements in the BnA5.FAD2 and BnC 5.FAD2 gene promoters to positively regulate gene expression; Bna013749 interacts with the GT elements in the BnA5.FAD2 and BnC5.FAD2 gene promoters to negatively regulate gene expression.
【Objective】 Fatty acid desaturase 2 gene(FAD2) is an important gene for controlling oleic acid content in rapeseed. And in this paper, the interaction between GATA family and BnA5.FAD2 and BnC5.FAD2 gene promoters or GT family transcription factors and BnA5.FAD2 and BnC5.FAD2 gene promoters was studied to make sure the transcription regulation mechanism to provide the theoretical basis for high oleic acid breeding. 【Method】 The potential cis-elements were predicted by deletion of promoter fragments and bioinformatics analysis, and candidate transcription factors were screened from PlantTFDB transcription factor database. Real-time PCR(q RT-PCR) was used to detect the expression pattern of transcription factors to further screen candidate transcription factors by comparison with BnA5.FAD2 and BnC5.FAD2 gene expression patterns. Transcription factors were co-transformed with BnA5.FAD2 and BnC5.FAD2 gene promoters into yeast and verified interaction relationship between promoter and transcription factors by yeast one-hybrid. The transcription factors were transformed into Arabidopsis thaliana with BnA5.FAD2 and BnC5.FAD2 gene promoter sequence, and the reporter gene GFP was detected by western blot to analyze the effects of transcription factors on promoters. 【Result】 After knocking out the GT or GATA cis-elements from BnA5.FAD2 and BnC5.FAD2 gene promoters respectively, the GFP protein abundance decreased, which indicate that GT and GATA cis-elements play important roles in regulating gene transcription. Yeast one-hybrid results showed that the GATA family transcription factors(Bna010243-1, Bna026124-1, Bna026124-2) and the GT family transcription factors(Bna010915 and Bna013749) can interact with BnA5.FAD2 and BnC5.FAD2 gene promoters. Western blot analysis showed that when the GATA family transcription factor and the promoter fragment containing GATA elements were co-transformed into Arabidopsis thaliana, the GFP protein content was significantly higher than that of no transcription factor. When the GATA element was knocked out, the GFP protein content did not change significantly. When the GT family transcription factor and the promoter fragment containing the GT element were co-transformed into Arabidopsis thaliana, the GFP protein content changed significantly. When the GT element was knocked out, the GFP protein content did not change significantly. 【 Conclusion 】 The GATA family transcription factors Bna010243-1, Bna026124-1, Bna026124-2 can interact with the GATA elements in the BnA5.FAD2 and BnC5.FAD2 gene promoters to enhance gene expression levels. The GT family transcription factor Bna010915 interacts with the GT elements in the BnA5.FAD2 and BnC 5.FAD2 gene promoters to positively regulate gene expression; Bna013749 interacts with the GT elements in the BnA5.FAD2 and BnC5.FAD2 gene promoters to negatively regulate gene expression.
引文
[1]YANG Y Y,YANG S Q,CHEN Z H,GUAN C Y,CHEN S Y,LIU ZS.QTL analysis of 18-C unsaturated fatty acid contents in zero-erucic rapeseed(Brassica napus L.).Acta Agronomica Sinica,2011,37(8):1342-1350.
[2]NABLOUSSI A,FERNáNDEZMARTíNEZ J M,VELASCO L.Inheritance of mid and high oleic acid content in Ethiopian mustard.Crop Science,2006,46(6):2361-2367.
[3]CHAPMAN K D,DYER J M,MULLEN R T.Biogenesis and functions of lipid droplets in plants:Thematic Review Series:Lipid Droplet Synthesis and Metabolism:from Yeast to Man.Journal of Lipid Research,2012,53(2):215-226.
[4]KARGIOTIDOU,A,DELI D,GALANOPOULOU D,TSAFTARIS A,FARMAKI T.Low temperature and light regulate delta 12 fatty acid desaturase(FAD2)at a transcriptional level in cotton(Gossypium hirsutum).Journal of Experimental Botany,2008,59(8):2043-2056.
[5]MARTIN C E,OH C,JIANG Y.Regulation of long chain unsaturated fatty acid synthesis in yeast.Biochimica et Biophysica Acta,2007,1771(3):271-285.
[6]吴秀丽,李扬秋.造血转录因子GATA-3的研究进展.国外医学输血及血液学分册.2001,24(5):387-390.WU X L,LI Y Q.Research progress of hematopoietic transcription factor GATA-3.Foreign Medical Blood Transfusion and Hematology,2001,24(5):387-390.(in Chinese)
[7]袁岐,张春利,赵婷婷,许向阳.植物中GATA转录因子的研究进展.分子植物育种,2017(5):1702-1707.YUAN Q,ZHANG C L,ZHAO T T,XU X Y.Research of advances GATA transcription factor in plant.Molecular Plant Breeding,2017(5):1702-1707.(in Chinese)
[8]RASTOGI R,BATE N J,SIVASANKAR S,ROTHSTEIN S J.Footprinting of the spinach nitrite reductase gene promoter reveals the preservation of nitrate regulatory elements between fungi and higher plants.Plant Molecular Biology,1997,34(3):465-476.
[9]GREEN P J,KAY S A,CHUA N H.Sequence-specific interactions of a pea nuclear factor with light-responsive elements upstream of the rbcS-3A gene.The EMBO Journal,1987,6(9):2543-2549.
[10]MCCARTY D R,CHORY J.Conservation and innovation in plant signaling pathways.Cell,2000,103(2):201-209.
[11]VILLAIN P,CLABAULT G,MACHE R,ZHOU D X.S1F binding site is related to but different from the light-responsive GT-1 binding site and differentially represses the spinach rps1 promoter in transgenic tobacco.The Journal of Biological Chemistry,1994,269(24):16626-16630.
[12]DEHESH K,SMITH L G,TEPPERMAN J M,QUAIL P H.Twin autonomous bipartite nuclear localization signals direct nuclear import of GT-2.The Plant Journal,1995,8(1):25-36.
[13]DEHESH K,HUNG H,TEPPERMAN J M,QUAIL P H.GT-2:Atranscription factor with twin autonomous DNA-binding domains of closely related but different target sequence specificity.The EMBOJournal,1992,11(11):4131-4144.
[14]SUPRUNOVA T,KRUGMAN T,DISTELFELD A,FAHIMA T,NEVO E,KOROL A.Identification of a novel gene(Hsdr4)involved in water-stress tolerance in wild barley.Plant Molecular Biology,2007,64(1/2):17-34.
[15]MEHROTRA R,KIRAN K,CHATURVEDI C P,ANSARI S A,LODHI N,SAWANT S,TULI R.Effect of copy number and spacing of the ACGT and GT cis elements on transient expression of minimal promoter in plants.Journal of Genetics,2005,84(2):183-187.
[16]GUAN Q L,CHEN H X,ZHANG Y,LI Q L.Progresses on GTelements and GT factors in plants.Hereditas,2009,31(2):123-130.
[17]LE GOURRIEREC J,LI Y F,ZHOU D X.Transcriptional activation by Arabidopsis GT-1 may be through interaction with TFIIA-TBP-TATA complex.The Plant Journal,1999,18(6):663-668.
[18]AN G.Binary ti vectors for plant transformation and promoter analysis.Methods in Enzymology,1987,153(153C):292-305.
[19]CLOUGH S J,BENT A F.Floral dip:A simplified method for Agrobaacterium-mediated transformation of Arabidopsis thaliana.The Plant Journal,1998,16:735-743.
[20]刘睿洋,刘芳,张振乾,官春云.甘蓝型油菜BnFAD2-C5基因启动子及内含子在表达水平的功能分析.作物学报,2016,42(10):1471-1478.LIU R Y,LIU F,ZHANG Z Q,GUAN C Y.Functional analysis of BnFAD2-C5 promoter and intron at expression level in Brassica napus.Acta Agronomica Sinica,2016,42(10):1471-1478.(in Chinese)
[21]XIAO G,ZHANG Z Q,YIN C F,LIU R Y,WU X M,TAN T L,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.
[22]LIU F,WANG G L,LIU R Y,GUAN C Y.The promoter of fatty acid desaturase on chromosome C5 in Brassica napus,drives high-level expression in seeds.Plant Biotechnology Reports,2016,10(6):369-381.
[23]KAPLAN-LEVY R N,BREWER P B,QUON T,DAVID R S.The trihelix family of transcription factors--light,stress and development.Trends in Plant Science,2012,17(3):163-171.
[24]FAN S D,WANG Y.Cloning and activity analysis of promoter of GRAS transcription factor gene HcSCL13 from Halostachys caspica.Biotechnology Bulletin,2017,33:131-138.
[25]NI M,DEHESH K,TEPPERMAN J M,QUAIL P H.GT-2:In vivo transcriptional activation activity and definition of novel twin DNAbinding domains with reciprocal target sequence selectivity.The Plant Cell,1996,8(6):1041-1059.
[26]MCCARTY D R,CHORY J.Conservation and innovation in plant signaling pathways.Cell,2000,103(2):201-209.
[27]丁刘军,普明宇,卫波,王献平,范仁春,张相岐.转录因子基因TuGTY-3参与乌拉尔图小麦对条锈病的抗性.遗传,2016,38(12):1090-1101.DING L J,PU M Y,WEI B,WANG X P,FAN R C,ZHANG X Q.Transcription factor gene TuGTY-3 is involved in the stripe rust resistance in Triticum urartu.Hereditas,2016,38(12):1090-1101.(in Chinese)
[28]KAPLANLEVY R N,BREWER P B,QUON T.The trihelix family of transcription factors-light,stress and development.Trends in Plant Science,2012,17:163-171.
[29]GAO M J,LI X,LUI H,GORDON M G,DEREK D L,SHU W,DWAYNE D H.ASIL1 is required for proper timing of seed filling in Arabidopsis.Plant Signaling&Behavior,2011,6(12):1886-1888.
[30]LOWRY J A,ATCHLEY W R.Molecular evolution of the GATAfamily of transcription factors:Conservation within the DNA-binding domain.Journal of Molecular Evolution,2000,50(2):103-115.
[31]剧建芳.南极丝瓜藓GATA转录因子PnGATA1的功能研究[D].济南:山东大学,2014.JU J F.Functional study of GATA transcription factor PnGATA1 in Antarctic[D].Jinan:Shandong University,2014.(in Chinese)
[2]NABLOUSSI A,FERNáNDEZMARTíNEZ J M,VELASCO L.Inheritance of mid and high oleic acid content in Ethiopian mustard.Crop Science,2006,46(6):2361-2367.
[3]CHAPMAN K D,DYER J M,MULLEN R T.Biogenesis and functions of lipid droplets in plants:Thematic Review Series:Lipid Droplet Synthesis and Metabolism:from Yeast to Man.Journal of Lipid Research,2012,53(2):215-226.
[4]KARGIOTIDOU,A,DELI D,GALANOPOULOU D,TSAFTARIS A,FARMAKI T.Low temperature and light regulate delta 12 fatty acid desaturase(FAD2)at a transcriptional level in cotton(Gossypium hirsutum).Journal of Experimental Botany,2008,59(8):2043-2056.
[5]MARTIN C E,OH C,JIANG Y.Regulation of long chain unsaturated fatty acid synthesis in yeast.Biochimica et Biophysica Acta,2007,1771(3):271-285.
[6]吴秀丽,李扬秋.造血转录因子GATA-3的研究进展.国外医学输血及血液学分册.2001,24(5):387-390.WU X L,LI Y Q.Research progress of hematopoietic transcription factor GATA-3.Foreign Medical Blood Transfusion and Hematology,2001,24(5):387-390.(in Chinese)
[7]袁岐,张春利,赵婷婷,许向阳.植物中GATA转录因子的研究进展.分子植物育种,2017(5):1702-1707.YUAN Q,ZHANG C L,ZHAO T T,XU X Y.Research of advances GATA transcription factor in plant.Molecular Plant Breeding,2017(5):1702-1707.(in Chinese)
[8]RASTOGI R,BATE N J,SIVASANKAR S,ROTHSTEIN S J.Footprinting of the spinach nitrite reductase gene promoter reveals the preservation of nitrate regulatory elements between fungi and higher plants.Plant Molecular Biology,1997,34(3):465-476.
[9]GREEN P J,KAY S A,CHUA N H.Sequence-specific interactions of a pea nuclear factor with light-responsive elements upstream of the rbcS-3A gene.The EMBO Journal,1987,6(9):2543-2549.
[10]MCCARTY D R,CHORY J.Conservation and innovation in plant signaling pathways.Cell,2000,103(2):201-209.
[11]VILLAIN P,CLABAULT G,MACHE R,ZHOU D X.S1F binding site is related to but different from the light-responsive GT-1 binding site and differentially represses the spinach rps1 promoter in transgenic tobacco.The Journal of Biological Chemistry,1994,269(24):16626-16630.
[12]DEHESH K,SMITH L G,TEPPERMAN J M,QUAIL P H.Twin autonomous bipartite nuclear localization signals direct nuclear import of GT-2.The Plant Journal,1995,8(1):25-36.
[13]DEHESH K,HUNG H,TEPPERMAN J M,QUAIL P H.GT-2:Atranscription factor with twin autonomous DNA-binding domains of closely related but different target sequence specificity.The EMBOJournal,1992,11(11):4131-4144.
[14]SUPRUNOVA T,KRUGMAN T,DISTELFELD A,FAHIMA T,NEVO E,KOROL A.Identification of a novel gene(Hsdr4)involved in water-stress tolerance in wild barley.Plant Molecular Biology,2007,64(1/2):17-34.
[15]MEHROTRA R,KIRAN K,CHATURVEDI C P,ANSARI S A,LODHI N,SAWANT S,TULI R.Effect of copy number and spacing of the ACGT and GT cis elements on transient expression of minimal promoter in plants.Journal of Genetics,2005,84(2):183-187.
[16]GUAN Q L,CHEN H X,ZHANG Y,LI Q L.Progresses on GTelements and GT factors in plants.Hereditas,2009,31(2):123-130.
[17]LE GOURRIEREC J,LI Y F,ZHOU D X.Transcriptional activation by Arabidopsis GT-1 may be through interaction with TFIIA-TBP-TATA complex.The Plant Journal,1999,18(6):663-668.
[18]AN G.Binary ti vectors for plant transformation and promoter analysis.Methods in Enzymology,1987,153(153C):292-305.
[19]CLOUGH S J,BENT A F.Floral dip:A simplified method for Agrobaacterium-mediated transformation of Arabidopsis thaliana.The Plant Journal,1998,16:735-743.
[20]刘睿洋,刘芳,张振乾,官春云.甘蓝型油菜BnFAD2-C5基因启动子及内含子在表达水平的功能分析.作物学报,2016,42(10):1471-1478.LIU R Y,LIU F,ZHANG Z Q,GUAN C Y.Functional analysis of BnFAD2-C5 promoter and intron at expression level in Brassica napus.Acta Agronomica Sinica,2016,42(10):1471-1478.(in Chinese)
[21]XIAO G,ZHANG Z Q,YIN C F,LIU R Y,WU X M,TAN T L,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.
[22]LIU F,WANG G L,LIU R Y,GUAN C Y.The promoter of fatty acid desaturase on chromosome C5 in Brassica napus,drives high-level expression in seeds.Plant Biotechnology Reports,2016,10(6):369-381.
[23]KAPLAN-LEVY R N,BREWER P B,QUON T,DAVID R S.The trihelix family of transcription factors--light,stress and development.Trends in Plant Science,2012,17(3):163-171.
[24]FAN S D,WANG Y.Cloning and activity analysis of promoter of GRAS transcription factor gene HcSCL13 from Halostachys caspica.Biotechnology Bulletin,2017,33:131-138.
[25]NI M,DEHESH K,TEPPERMAN J M,QUAIL P H.GT-2:In vivo transcriptional activation activity and definition of novel twin DNAbinding domains with reciprocal target sequence selectivity.The Plant Cell,1996,8(6):1041-1059.
[26]MCCARTY D R,CHORY J.Conservation and innovation in plant signaling pathways.Cell,2000,103(2):201-209.
[27]丁刘军,普明宇,卫波,王献平,范仁春,张相岐.转录因子基因TuGTY-3参与乌拉尔图小麦对条锈病的抗性.遗传,2016,38(12):1090-1101.DING L J,PU M Y,WEI B,WANG X P,FAN R C,ZHANG X Q.Transcription factor gene TuGTY-3 is involved in the stripe rust resistance in Triticum urartu.Hereditas,2016,38(12):1090-1101.(in Chinese)
[28]KAPLANLEVY R N,BREWER P B,QUON T.The trihelix family of transcription factors-light,stress and development.Trends in Plant Science,2012,17:163-171.
[29]GAO M J,LI X,LUI H,GORDON M G,DEREK D L,SHU W,DWAYNE D H.ASIL1 is required for proper timing of seed filling in Arabidopsis.Plant Signaling&Behavior,2011,6(12):1886-1888.
[30]LOWRY J A,ATCHLEY W R.Molecular evolution of the GATAfamily of transcription factors:Conservation within the DNA-binding domain.Journal of Molecular Evolution,2000,50(2):103-115.
[31]剧建芳.南极丝瓜藓GATA转录因子PnGATA1的功能研究[D].济南:山东大学,2014.JU J F.Functional study of GATA transcription factor PnGATA1 in Antarctic[D].Jinan:Shandong University,2014.(in Chinese)