紫色马铃薯花青素StAN1基因的克隆及功能分析
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摘要
MYB是植物花青素合成代谢途径中最主要的转录因子。该研究以紫色马铃薯B-5为试验材料,通过同源克隆技术,克隆了马铃薯花青素StAN1基因,并对StAN1基因的表达、遗传转化及其转基因烟草的花青素含量进行分析。结果表明:(1)StAN1基因全长774bp,编码了258个氨基酸;系统进化树分析发现,StAN1与辣椒、茄子、芦竹的亲缘关系最近,与矮牵牛、苹果等的亲缘关系最远。(2)荧光定量PCR分析显示,StAN1基因在马铃薯不同组织均有表达,其表达量从小到大依次为:根<叶<茎<匍匐茎<薯皮<薯肉。(3)成功构建了表达载体pJAM1502-AN1,并经农杆菌(Gv3101)转化获得根、茎、叶片及叶脉均为紫红色的转StAN1基因烟草,PCR鉴定表明目的基因StAN1已成功转入烟草中。(4)花青素含量分析表明,野生型烟草叶片中花青素含量为2mg/g,叶片颜色为绿色,而转StAN1基因烟草叶片花青素含量达20mg/g,叶片颜色变成了紫红色。
MYB is the most important transcription factor in plant anthocyanin biosynthesis pathway.In this study,the anthocyanin StAN1 gene of purple potato was cloned by homologous cloning technique.The results showed that:(1)the full length of the StAN1 gene was 774 bp,encoded 258 amino acids;Phylogenetic tree analysis showed that StAN1 had the closest relationship with pepper,eggplant and asparagus,and had the furthest relationship with Petunia and apple.(2)Fluorescence quantitative PCR analysis showed that StAN1 gene was expressed in different tissues of potato.(3)The expression vector pJAM1502-AN1 was successfully constructed by Agrobacterium(Gv3101)transformation to obtain the root,stem,leaf and vein are purple-red transgenic tobacco,the expression vector StAN1 gene was transformed from small to large as the root< leaf< stolon < tuber skin < tuber.(3)Transgenic tobacco with StAN1 gene was constructed successfully through Agrobacterium(Gv3101)transformation.PCR identification showed that the target gene StAN1 had been successfully transferred into tobacco.(4)Theanthocyanin content of wild type tobacco leaves was 2 mg/g,and the color of leaves was green.However,the anthocyanin content of the transgenic tobacco leaves reached 20 mg/g,and the color of the leaves changed to purplish red.
MYB is the most important transcription factor in plant anthocyanin biosynthesis pathway.In this study,the anthocyanin StAN1 gene of purple potato was cloned by homologous cloning technique.The results showed that:(1)the full length of the StAN1 gene was 774 bp,encoded 258 amino acids;Phylogenetic tree analysis showed that StAN1 had the closest relationship with pepper,eggplant and asparagus,and had the furthest relationship with Petunia and apple.(2)Fluorescence quantitative PCR analysis showed that StAN1 gene was expressed in different tissues of potato.(3)The expression vector pJAM1502-AN1 was successfully constructed by Agrobacterium(Gv3101)transformation to obtain the root,stem,leaf and vein are purple-red transgenic tobacco,the expression vector StAN1 gene was transformed from small to large as the root< leaf< stolon < tuber skin < tuber.(3)Transgenic tobacco with StAN1 gene was constructed successfully through Agrobacterium(Gv3101)transformation.PCR identification showed that the target gene StAN1 had been successfully transferred into tobacco.(4)Theanthocyanin content of wild type tobacco leaves was 2 mg/g,and the color of leaves was green.However,the anthocyanin content of the transgenic tobacco leaves reached 20 mg/g,and the color of the leaves changed to purplish red.
引文
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[11]MATHEWS H,CLENDENNEN S K,CALDWELL C G,et al.Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis,modification,and transport[J].The Plant Cell,2003,15(8):1 689-1 703.
[12]CHU H,JEONG J C,KIM W J,et al.Expression of the sweetpotato R2R3-type IbMYB1agene induces anthocyanin accumulation in Arabidopsis[J].Physiologia Plantarum,2013,148(2):189-199.
[13]ENDT D V,KIJNE J W,MEMELINK J.Transcription factors controlling plant secondary metabolism:what regulates the regulators[J].Phytochemistry,2002,61(2):107-114.
[14]BOMAL C,BEDON F,CARON S,et al.Involvement of Pinus taeda MYB1and MYB8in phenylpropanoid metabolism and secondary cell wall biogenesis:a comparative in planta analysis[J].Journal of Experimental Botany,2008,59(14):3 925-3 939.
[15]TAYLOR L P,GROTEWOLD E.Flavonoids as developmental regulators[J].Current Opinion in Plant Biology,2005,8(3):317-323.
[16]VERDONK J C,HARING M A,VAN A T,et al.ODOR-ANT1regulates fragrance biosynthesis in petunia flowers[J].Plant Cell,2005,17(5):1 612-1 624.
[17]DAMELIA V,AVERSANO R,BATELLI G,et al.High AN1variability and interaction with basic helix-loop-helix cofactors related to anthocyanin biosynthesis in potato leaves[J].The Plant,2014,80(3):527-540.
[18]黄艳岚.马铃薯花青素转录激活基因的克隆与序列分析[D].广州:华南热带农业大学,2006.
[19]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408.
[20]YANG J H,PARK H Y,KIM Y S,et al.Quality characteristics of vacuum-fried snacks prepared from various sweet potato cultivars[J].Food Science and Biotechnology,2012,21(2):525-530.
[21]ZHANG Y,CHENG S,JONG D D,et al.The potato R locus codes for dihydroflavonol 4-reductase[J].Theoretical&Applied Genetics,2009,119(5):931-937.
[22]邢文,金晓玲.调控植物类黄酮生物合成的MYB转录因子研究进展[J].分子植物育种,2015,13(3):689-696.XING W,JIN X L.Advances in the study of MYB transcription factors regulating biosynthesis of flavonoids in plants[J].Molecular Plant Breeding Mol Plant Breed,2015,13(3):689-696.
[23]李永杰,谢让金,邓烈.R2R3MYB转录因子在果树花青素合成中的调控作用[J].中国南方果树,2014,43(6):18-23.LI Y J,XIE R J,DENG L.Regulation of R2R3MYB transcription factors in anthocyanin synthesis of fruit trees[J].Fruit Trees in Southern China,2014,43(6):18-23.
[24]段岩娇,张鲁刚,何琼,等.紫心大白菜花青素积累特性及相关基因表达分析[J].园艺学报,2012,39(11):2 159-2 167.DUAN Y J,ZHANG L G,HE Q,et al.Expression of transcriptional factors and structural genes of anthocyanin biosynthesis in purple-heading Chinese cabbage[J].Acta Horticulturae Sinica,2012,39(11):2 159-2 167.
[25]JUNG C S,GRIFFITHS H M,JONG D M D,et al.The potatodeveloper(D)locus encodes an R2R3 MYB transcription factor that regulates expression of multiple anthocyanin structural genes in tuber skin[J].Theoretical and Applied Genetics,2009,120(1):45-57.
[26]LI W,WANG B,WANG M,et al.Cloning and characterization of a potato\r,StAN11\r,gene involved in anthocyanin biosynthesis regulation[J].Journal of Integrative Plant Biology,2014,56(4):364-372.
[27]LIU Y,LINWANG K,ESPLEY R V,et al.Functional diversification of the potato R2R3MYB anthocyanin activators AN1,MYBA1,and MYB113and their interaction with basic helix-loop-helix cofactors[J].Journal of Experimental Botany,2016,67(8):2 159-2 176.
[28]谈欢,刘玉汇,李丽霞.马铃薯块茎花色素苷合成相关R2R3MYB蛋白基因的克隆和功能分析[J].作物学报,2018,44(7):1 021-1 031.TAN H,LIU Y H,LI L X.Cloning and functional analysis of R2R3MYB genes involved in anthocyanin biosynthesis in potato tuber[J].Acta Agronomica Sinica,2018,44(7):1 021-1 031.
[2]STUSHNOFF C,HOLM D,THOMPSON M D,et al.Antioxidant properties of cultivars and selections from the colorado potato breeding program[J].American Journal of Potato Research,2008,85(4):267-276.
[3]戴妙妙,王婷婷,马壮,等.紫娟茶中花青素的抗氧化性研究[J].中国食品添加剂,2016,37(7):117-122.DAI M M,WANG T T,MA Z,et al.The study on the antioxidant activity of anthocyanins from Zi Juan Tea[J].China Food Additives,2016,37(7):117-122.
[4]王华,李茂福,杨媛.果实花青素生物合成分子机制研究进展[J].植物生理学报,2015,51(1):29-43.WANG H,LI M F,YANG Y.Recent advances on the molecular mechanisms of anthocyanin synthesis in fruits[J].Plant Physiology Journal,2015,51(1):29-43.
[5]罗春丽,王林,李杏,等.紫薯花青素体外抗氧化及对H2O2诱导HepG2细胞氧化损伤的保护作用[J].食品科学,2015,36(17):225-230.LUO C L,WANG L,LI X et al.Antioxidant activities and protective effect of anthocyanins from purple sweet potato on HepG2cell injury induced by H2O2[J].Sitiology,2015,36(17):225-230.
[6]樊锦涛,蒋琛茜,邢继红.拟南芥R2R3-MYB家族22亚族的结构与功能遗传[J].遗传,2014,36(10):985-994.FAN J T,JIANG C Q,XING J H.Structure and function of the 22nd subfamily in Arabidopsis R2R3-MYB family[J].Heredetas,2014,36(10):985-994.
[7]BALDONI E,GENGA A,COMINELLI E.Plant MYB transcription factors:fheir role in drought response mechanisms[J].International Journal of Molecular Sciences,2015,16(7):15 811-15 851.
[8]MARTIN C,PAZ_ARES J.MYB transcription factors in plants[J].Trends in Genetics,1997,13(2):67-73.
[9]BOREVITZ J O,XIA Y,BLOUNT J,et al.Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis[J].The Plant Cell,2000,12(12):2 383-2 393.
[10]QUATTROCCHIO F,WING J,WOUDE K V D,et al.Molecular analysis of the anthocyanin2gene of Petunia and its role in the evolution of flower color[J].Plant Cell,1999,11(8):1 433-1 444.
[11]MATHEWS H,CLENDENNEN S K,CALDWELL C G,et al.Activation tagging in tomato identifies a transcriptional regulator of anthocyanin biosynthesis,modification,and transport[J].The Plant Cell,2003,15(8):1 689-1 703.
[12]CHU H,JEONG J C,KIM W J,et al.Expression of the sweetpotato R2R3-type IbMYB1agene induces anthocyanin accumulation in Arabidopsis[J].Physiologia Plantarum,2013,148(2):189-199.
[13]ENDT D V,KIJNE J W,MEMELINK J.Transcription factors controlling plant secondary metabolism:what regulates the regulators[J].Phytochemistry,2002,61(2):107-114.
[14]BOMAL C,BEDON F,CARON S,et al.Involvement of Pinus taeda MYB1and MYB8in phenylpropanoid metabolism and secondary cell wall biogenesis:a comparative in planta analysis[J].Journal of Experimental Botany,2008,59(14):3 925-3 939.
[15]TAYLOR L P,GROTEWOLD E.Flavonoids as developmental regulators[J].Current Opinion in Plant Biology,2005,8(3):317-323.
[16]VERDONK J C,HARING M A,VAN A T,et al.ODOR-ANT1regulates fragrance biosynthesis in petunia flowers[J].Plant Cell,2005,17(5):1 612-1 624.
[17]DAMELIA V,AVERSANO R,BATELLI G,et al.High AN1variability and interaction with basic helix-loop-helix cofactors related to anthocyanin biosynthesis in potato leaves[J].The Plant,2014,80(3):527-540.
[18]黄艳岚.马铃薯花青素转录激活基因的克隆与序列分析[D].广州:华南热带农业大学,2006.
[19]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method[J].Methods,2001,25(4):402-408.
[20]YANG J H,PARK H Y,KIM Y S,et al.Quality characteristics of vacuum-fried snacks prepared from various sweet potato cultivars[J].Food Science and Biotechnology,2012,21(2):525-530.
[21]ZHANG Y,CHENG S,JONG D D,et al.The potato R locus codes for dihydroflavonol 4-reductase[J].Theoretical&Applied Genetics,2009,119(5):931-937.
[22]邢文,金晓玲.调控植物类黄酮生物合成的MYB转录因子研究进展[J].分子植物育种,2015,13(3):689-696.XING W,JIN X L.Advances in the study of MYB transcription factors regulating biosynthesis of flavonoids in plants[J].Molecular Plant Breeding Mol Plant Breed,2015,13(3):689-696.
[23]李永杰,谢让金,邓烈.R2R3MYB转录因子在果树花青素合成中的调控作用[J].中国南方果树,2014,43(6):18-23.LI Y J,XIE R J,DENG L.Regulation of R2R3MYB transcription factors in anthocyanin synthesis of fruit trees[J].Fruit Trees in Southern China,2014,43(6):18-23.
[24]段岩娇,张鲁刚,何琼,等.紫心大白菜花青素积累特性及相关基因表达分析[J].园艺学报,2012,39(11):2 159-2 167.DUAN Y J,ZHANG L G,HE Q,et al.Expression of transcriptional factors and structural genes of anthocyanin biosynthesis in purple-heading Chinese cabbage[J].Acta Horticulturae Sinica,2012,39(11):2 159-2 167.
[25]JUNG C S,GRIFFITHS H M,JONG D M D,et al.The potatodeveloper(D)locus encodes an R2R3 MYB transcription factor that regulates expression of multiple anthocyanin structural genes in tuber skin[J].Theoretical and Applied Genetics,2009,120(1):45-57.
[26]LI W,WANG B,WANG M,et al.Cloning and characterization of a potato\r,StAN11\r,gene involved in anthocyanin biosynthesis regulation[J].Journal of Integrative Plant Biology,2014,56(4):364-372.
[27]LIU Y,LINWANG K,ESPLEY R V,et al.Functional diversification of the potato R2R3MYB anthocyanin activators AN1,MYBA1,and MYB113and their interaction with basic helix-loop-helix cofactors[J].Journal of Experimental Botany,2016,67(8):2 159-2 176.
[28]谈欢,刘玉汇,李丽霞.马铃薯块茎花色素苷合成相关R2R3MYB蛋白基因的克隆和功能分析[J].作物学报,2018,44(7):1 021-1 031.TAN H,LIU Y H,LI L X.Cloning and functional analysis of R2R3MYB genes involved in anthocyanin biosynthesis in potato tuber[J].Acta Agronomica Sinica,2018,44(7):1 021-1 031.