芥菜型油菜BjuA09DFR基因及其启动子的克隆与转化和表达
|
摘要
该研究利用实时荧光定量(qRT-PCR)检测了BjuA09 DFR基因的时空表达特异性,并通过克隆BjuA09 DFR基因启动子片段,构建该基因的启动子GUS融合表达载体,利用农杆菌介导法将重组质粒转入野生型拟南芥,最后对拟南芥转基因材料不同发育时期的不同组织部位进行GUS组织化学染色,分析BjuA09 DFR基因启动子的表达模式,为BjuA09 DFR基因启动子功能的进一步研究提供理论依据。结果表明:(1)BjuA09 DFR基因在芥菜型油菜的多个组织部位都有表达,尤其是在叶、花、角果和授粉后15d种子中表达量较高。(2)成功构建了BjuA09 DFR基因启动子和GUS基因融合表达载体(pBjuA09 DFR∷GUS),采用农杆菌介导法将重组质粒转入野生型拟南芥,经卡那霉素筛选和PCR检测抗性苗,获得转基因拟南芥阳性苗。(3)GUS组织化学分析结果显示,转基因拟南芥材料的GUS活性具有明显的时空特异性,在叶、花、角果和种子中的染色较深,具有很强的GUS活性。
In this study,qRT-PCR was applied to detect the temporal and spatial expression specificity of BjuA09 DFRgene.In addition,the BjuA09 DFRgene promoter was cloned,and the promoter GUS fusion expression vector of BjuA09 DFRgene was constructed.We transferred the fusion expression vector into Arabidopsis thaliana by Agrobacterium-mediated floral dip method. The expression pattern of BjuA09 DFRgene promoter was analyzed by GUS histochemical staining in different tissues during the different development periods,which provided theoretical basis for further study of the function of BjuA09 DFRgene promoter.The results showed that:(1)BjuA09 DFR gene could express in multiple tissues of Brassica juncea,especially highly expressed in leaves,flowers,silicles and seeds of 15 days afterpollination.(2)The fusion expression vector of GUS gene drived by BjuA09 DFRgene promoter(pBjuA09 DFR∷GUS)was successfully constructed,and transformed into wild-type A.thalianaby Agrobacterium-mediated method.We successfully obtained positive transgenic plants by screening test of resistance to kanamycin and PCR detection.(3)Histochemical analysis of GUS showed that the GUS activity of transgenic A.thaliana displayed obviously temporal and spatial specificity,with the deeper staining in leaves,flowers,silicles and seeds of 15 days after pollination.
In this study,qRT-PCR was applied to detect the temporal and spatial expression specificity of BjuA09 DFRgene.In addition,the BjuA09 DFRgene promoter was cloned,and the promoter GUS fusion expression vector of BjuA09 DFRgene was constructed.We transferred the fusion expression vector into Arabidopsis thaliana by Agrobacterium-mediated floral dip method. The expression pattern of BjuA09 DFRgene promoter was analyzed by GUS histochemical staining in different tissues during the different development periods,which provided theoretical basis for further study of the function of BjuA09 DFRgene promoter.The results showed that:(1)BjuA09 DFR gene could express in multiple tissues of Brassica juncea,especially highly expressed in leaves,flowers,silicles and seeds of 15 days afterpollination.(2)The fusion expression vector of GUS gene drived by BjuA09 DFRgene promoter(pBjuA09 DFR∷GUS)was successfully constructed,and transformed into wild-type A.thalianaby Agrobacterium-mediated method.We successfully obtained positive transgenic plants by screening test of resistance to kanamycin and PCR detection.(3)Histochemical analysis of GUS showed that the GUS activity of transgenic A.thaliana displayed obviously temporal and spatial specificity,with the deeper staining in leaves,flowers,silicles and seeds of 15 days after pollination.
引文
[1]ANDERSEN M,JORDHEIM M.Anthocyanins[M]//Encyclopedia of Life Sciences(ELS).Chichester:John Wiley and Sons Ltd,2010:1-12.
[2]GOULD K,DAVIES K,WINEFIELD C.Anthocyanins:Biosynthesis,Functions,and Application[M].New York:Springer,2009.
[3]WINKEL-SHIRLEY B.Biosynthesis of flavonoids and effects of stress[J].Current Opinion in Plant Biology,2002,5(3):218-223.
[4]GERATS A G,VLAMINGP D,DOODEMANM,et al.Genetic control of the conversion of dihydroflavonols into flavonols and anthocyanins in flowers of Petunia hybrida[J].Planta,1982,155(4):364-368.
[5]HARBORNE J B,WILLIAMS C A.Advances in flavonoid research since 1992[J].Phytochemistry,2000,55(6):481-504.
[6]WINKEL-SHIRLEY B.Flavonoid biosynthesis.A colorful model for genetics,biochemistry,cell biology,and biotechnology[J].Plant Physiol.,2001,126(2):485-493.
[7]GONZALEZ A,ZHAO M,LEAVITT J M,et al.Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/MYB transcriptional complex in Arabidopsis seedlings[J].Plant J.,2008,53(5):814-827.
[8]PETRONI K,TONELLI C.Recent advances on the regulation of anthocyanin synthesis in reproductive organs[J].Plant Sci.,2011,181(3):219-229.
[9]POURCEL L,BOHORQUEZ-RESTREPO A,IRANI N G,et al.Anthocyanin biosynthesis,regulation,and transport:new insights from model species[J].Recent Advances in Polyphenol Research,2012,3:143-160.
[10]ESPLEY R V,BOVY A,BAVA C,et al.Analysis of genetically modified red-fleshed apples reveals effects on growth and consumer attributes[J].Plant Biotechnol.J.,2013,11(4):408-419.
[11]李亚丽,李欣,肖婕,等.二氢黄酮醇-4-还原酶在花青素合成中的功能及调控研究进展[J].西北植物学报,2018,38(1):187-196.LI Y L,LI X,XIAO J,et al.Function and regulation characterization of dihydroflavonol 4-reductase in anthocyanin biosynthesis[J].Acta Botanica Boreali-Occidentalia Sinica,2018,38(1):187-196.
[12]MIOSIC S,THILL J,MILOSEVIC M,et al.Dihydroflavonol4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in fragaria species[J].PloS One,2014,9(11):e112707.
[13]LOU Q,LIU Y.Transcriptome sequencing and metabolite analysis reveals the role of delphinidin metabolism in flower colour in grape hyacinth[J].Journal of Experimental Botany,2014,65(12):3 157-3 164.
[14]KIM S,BINZEL M L,PARK S,et al.Inactivation of DFR(dihydroflavonol 4-reductase)gene transcription results in blockage of anthocyanin production in yellow onions(Allium cepa)[J].Molecular Breeding,2004,14(3):253-263.
[15]KIM S,PARK J Y,YANG T J.Characterization of three active transposable elements recently inserted in three independent DFR-A alleles and one high-copy DNA transposon isolated from the pink allele of the ANSgene in onion(Allium cepa L.)[J].Mol.Genet Genomics,2015,290(3):1 027-1 037.
[16]WENDELL D L,VAZIRI A,SHERGILL G.The gene encoding dihydroflavonol 4-reductase is a candidate for the anthocyaninless locus of rapid cycling Brassica rapa(Fast Plants Type)[J].PloS One,2016,11(8):e0161394.
[17]严明理,刘显军,刘忠松,等.芥菜型油菜4-二氢黄酮醇还原酶基因的克隆和表达分析[J].作物学报,2008,34(1):1-7.YAN M L,LIU X J,LIU Z S,et al.Cloning and expression analysis of the dihydroflavonol 4-reductase gene in Brassica juncea[J].Acta Agronomica Sinica 2008,34(1):1-7.
[18]文添龙,刘雪梅,冀亚萍,等.高等植物胁迫诱导型启动子的研究进展[J].西北植物学报,2014,34(1):206-214.WEN T L,LIU X M,JI Y P,et al.Resarech progress of stress-induced promoter in higher plant[J].Acta Botanica Boreali-Occidentalia Sinica,2014,34(1):206-214.
[19]CHANDNA R,AUGUSTINE R,BISHT N C.Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR[J].PLoS One,2012,7(5):e36918.
[20]LIU X J,LU Y,YAN M L,et al.Genome-wide identification,localization,and expression analysis of proanthocyanidin-associated genes in Brassica[J].Frontiers in Plant Science,2016,7:1831.
[21]CHEN M,LIU H,KONG J,et al.RopGEF7regulates PLETHORA-dependent maintenance of the root stem cell niche in Arabidopsis[J].Plant Cell,2011,23(8):2 880-2 894.
[22]TIAN J,HAN Z,ZHANG J,et al.The balance of expression of dihydroflavonol 4-reductase and flavonol synthase regulates flavonoid biosynthesis and red foliage coloration in crabapples[J].Scientific Reports,2015,5:12 228.
[23]GOLLOP R,EVEN S,COLOVATSOLOVA V,et al.Expression of the grape dihydroflavonol reductase gene and analysis of its promoter region[J].Journal of Experimental Botany,2002,53(373):1 397-1 409.
[24]MORI K,SUGAYA S,GEMMA H.Decreased anthocyanin biosynthesis in grape berries grown under elevated night temperature condition[J].Sci.Hortic.,2005,105(3):319-330.
[25]AHMED N U,PARK J I,JUNG H J,et al.Characterization of dihydroflavonol 4-reductase(DFR)genes and their association with cold and freezing stress in Brassica rapa[J].Gene,2014,550(1):46-55.
[2]GOULD K,DAVIES K,WINEFIELD C.Anthocyanins:Biosynthesis,Functions,and Application[M].New York:Springer,2009.
[3]WINKEL-SHIRLEY B.Biosynthesis of flavonoids and effects of stress[J].Current Opinion in Plant Biology,2002,5(3):218-223.
[4]GERATS A G,VLAMINGP D,DOODEMANM,et al.Genetic control of the conversion of dihydroflavonols into flavonols and anthocyanins in flowers of Petunia hybrida[J].Planta,1982,155(4):364-368.
[5]HARBORNE J B,WILLIAMS C A.Advances in flavonoid research since 1992[J].Phytochemistry,2000,55(6):481-504.
[6]WINKEL-SHIRLEY B.Flavonoid biosynthesis.A colorful model for genetics,biochemistry,cell biology,and biotechnology[J].Plant Physiol.,2001,126(2):485-493.
[7]GONZALEZ A,ZHAO M,LEAVITT J M,et al.Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/MYB transcriptional complex in Arabidopsis seedlings[J].Plant J.,2008,53(5):814-827.
[8]PETRONI K,TONELLI C.Recent advances on the regulation of anthocyanin synthesis in reproductive organs[J].Plant Sci.,2011,181(3):219-229.
[9]POURCEL L,BOHORQUEZ-RESTREPO A,IRANI N G,et al.Anthocyanin biosynthesis,regulation,and transport:new insights from model species[J].Recent Advances in Polyphenol Research,2012,3:143-160.
[10]ESPLEY R V,BOVY A,BAVA C,et al.Analysis of genetically modified red-fleshed apples reveals effects on growth and consumer attributes[J].Plant Biotechnol.J.,2013,11(4):408-419.
[11]李亚丽,李欣,肖婕,等.二氢黄酮醇-4-还原酶在花青素合成中的功能及调控研究进展[J].西北植物学报,2018,38(1):187-196.LI Y L,LI X,XIAO J,et al.Function and regulation characterization of dihydroflavonol 4-reductase in anthocyanin biosynthesis[J].Acta Botanica Boreali-Occidentalia Sinica,2018,38(1):187-196.
[12]MIOSIC S,THILL J,MILOSEVIC M,et al.Dihydroflavonol4-reductase genes encode enzymes with contrasting substrate specificity and show divergent gene expression profiles in fragaria species[J].PloS One,2014,9(11):e112707.
[13]LOU Q,LIU Y.Transcriptome sequencing and metabolite analysis reveals the role of delphinidin metabolism in flower colour in grape hyacinth[J].Journal of Experimental Botany,2014,65(12):3 157-3 164.
[14]KIM S,BINZEL M L,PARK S,et al.Inactivation of DFR(dihydroflavonol 4-reductase)gene transcription results in blockage of anthocyanin production in yellow onions(Allium cepa)[J].Molecular Breeding,2004,14(3):253-263.
[15]KIM S,PARK J Y,YANG T J.Characterization of three active transposable elements recently inserted in three independent DFR-A alleles and one high-copy DNA transposon isolated from the pink allele of the ANSgene in onion(Allium cepa L.)[J].Mol.Genet Genomics,2015,290(3):1 027-1 037.
[16]WENDELL D L,VAZIRI A,SHERGILL G.The gene encoding dihydroflavonol 4-reductase is a candidate for the anthocyaninless locus of rapid cycling Brassica rapa(Fast Plants Type)[J].PloS One,2016,11(8):e0161394.
[17]严明理,刘显军,刘忠松,等.芥菜型油菜4-二氢黄酮醇还原酶基因的克隆和表达分析[J].作物学报,2008,34(1):1-7.YAN M L,LIU X J,LIU Z S,et al.Cloning and expression analysis of the dihydroflavonol 4-reductase gene in Brassica juncea[J].Acta Agronomica Sinica 2008,34(1):1-7.
[18]文添龙,刘雪梅,冀亚萍,等.高等植物胁迫诱导型启动子的研究进展[J].西北植物学报,2014,34(1):206-214.WEN T L,LIU X M,JI Y P,et al.Resarech progress of stress-induced promoter in higher plant[J].Acta Botanica Boreali-Occidentalia Sinica,2014,34(1):206-214.
[19]CHANDNA R,AUGUSTINE R,BISHT N C.Evaluation of candidate reference genes for gene expression normalization in Brassica juncea using real time quantitative RT-PCR[J].PLoS One,2012,7(5):e36918.
[20]LIU X J,LU Y,YAN M L,et al.Genome-wide identification,localization,and expression analysis of proanthocyanidin-associated genes in Brassica[J].Frontiers in Plant Science,2016,7:1831.
[21]CHEN M,LIU H,KONG J,et al.RopGEF7regulates PLETHORA-dependent maintenance of the root stem cell niche in Arabidopsis[J].Plant Cell,2011,23(8):2 880-2 894.
[22]TIAN J,HAN Z,ZHANG J,et al.The balance of expression of dihydroflavonol 4-reductase and flavonol synthase regulates flavonoid biosynthesis and red foliage coloration in crabapples[J].Scientific Reports,2015,5:12 228.
[23]GOLLOP R,EVEN S,COLOVATSOLOVA V,et al.Expression of the grape dihydroflavonol reductase gene and analysis of its promoter region[J].Journal of Experimental Botany,2002,53(373):1 397-1 409.
[24]MORI K,SUGAYA S,GEMMA H.Decreased anthocyanin biosynthesis in grape berries grown under elevated night temperature condition[J].Sci.Hortic.,2005,105(3):319-330.
[25]AHMED N U,PARK J I,JUNG H J,et al.Characterization of dihydroflavonol 4-reductase(DFR)genes and their association with cold and freezing stress in Brassica rapa[J].Gene,2014,550(1):46-55.