种子有刺毛与无刺毛胡萝卜转录因子DcTTG1的分离及表达分析
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摘要
胡萝卜种子中刺毛严重影响胡萝卜播种质量。在胡萝卜播种前需要将种子上的刺毛用机械或者人工方法去除。转录因子TTG1(TRANSPARENT TESTA GLABRA 1)是调节植物刺毛发育的主要因子之一,而在胡萝卜中未有研究报道。本研究报道一种从武汉洪山区野生胡萝卜(武野)中,筛选到植株、茎叶及种子均无毛的材料(命名为:武野-无毛),并对其毛发育调节转录因子DcTTG1进行研究。结果表明,武野和武野-无毛材料的DcTTG1长度均为1011 bp,均可编码336个氨基酸残基。两个材料的DcTTG1序列上有3个位置不同,导致其推导的氨基酸有3个位点不同。两个材料DcTTG1蛋白序列,均含有4个WD-40基序。以拟南芥为外群,进化分析上胡萝卜的DcTTG1与其他伞形科的TTG1处在同一分枝上,其他科植物的TTG1也处在同一分枝上。表达分析表明,DcTTG1在武野材料中的茎、叶及授粉5 d的花,表达量显著高于武野-无毛,但在未授粉花及形成的种子(授粉20 d)不存在显著性。
Seed hairs in carrot negatively affect the germination of carrot seeds.They were necessary to be removed by mechanical or manual approaches before sowing.The development of seed hairs has been shown to be relevant to the regulation mechanism of transcription factor DcTTG1.In this study,we analyzed the genomic and transcriptional variation of DcTTG1 in a seed-hairless carrot genotype(named:Wuyehairless),which was identified from the wild carrot germplasm Wuye in Hongshan district of Wuhan city.In both genotypes Wuye and Wuye-hairless,DcTTG1 contained an open reading frame with a length of 1011 bp,encoding for 336 amino acid residues.Three amino acid substitutions caused by three nucleotide mutations were observed between two genotypes.By bioinformatic analysis,the deduced DcTTG1 protein sequence contained four WD-40 motifs in both genotypes.The cluster analysis showed that the DcTTG1 of carrot and other TTG1 of Apiaceae plant were clustered in same branch.The transcriptional level of DcTTG1 in Wuye was significantly higher than Wuye-hairless in the stem,leaf and 5 d after pollination of flowers,while no significant difference was observed at the stages of un-pollinated flowers and seeds formation(20 d after pollination).
Seed hairs in carrot negatively affect the germination of carrot seeds.They were necessary to be removed by mechanical or manual approaches before sowing.The development of seed hairs has been shown to be relevant to the regulation mechanism of transcription factor DcTTG1.In this study,we analyzed the genomic and transcriptional variation of DcTTG1 in a seed-hairless carrot genotype(named:Wuyehairless),which was identified from the wild carrot germplasm Wuye in Hongshan district of Wuhan city.In both genotypes Wuye and Wuye-hairless,DcTTG1 contained an open reading frame with a length of 1011 bp,encoding for 336 amino acid residues.Three amino acid substitutions caused by three nucleotide mutations were observed between two genotypes.By bioinformatic analysis,the deduced DcTTG1 protein sequence contained four WD-40 motifs in both genotypes.The cluster analysis showed that the DcTTG1 of carrot and other TTG1 of Apiaceae plant were clustered in same branch.The transcriptional level of DcTTG1 in Wuye was significantly higher than Wuye-hairless in the stem,leaf and 5 d after pollination of flowers,while no significant difference was observed at the stages of un-pollinated flowers and seeds formation(20 d after pollination).
引文
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[12]刘凯歌,齐双慧,段绍伟,李东,金倡宇,高晨浩,刘绚霞,陈明训.甘蓝型油菜BnTTG1-1基因的功能分析.植物学报,2017,52(6):713-722Liu K G,Qi S H,Duan S W,Li D,Jin C Y,Gao C H,Liu XX,Chen M X.Functional analysis of Brassica napus BnTTG1-1gene.Chinese Bulletin of Botany,2017,52(6):713-722
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[14]Alahakoon U.Effect of TRANSPARENT TESTA GLABRA1on trichome development,growth,and insect resistance in a Brassica napus AtGLABRA3+background.Canada Saskatoon:University of Saskatchewan Saskatoon,2014
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[16]Koornneef M.The complex syndrome of ttg mutants.Arabidopsis Information Service,1981,18:45-51
[17]Lloyd A M.The TTG gene is required to specify epidermal cell fate and cell patterning in the Arabidopsis root.Developmental Biology,1994,166(2):740-754
[18]Jia X L,Wang G L,Xiong F,Yu X R,Xu Z S,Wang F,Xiong A S.De novo assembly,transcriptome characterization,lignin accumulation,and anatomic characteristics:novel insights into lignin biosynthesis during celery leaf development.Scientific Reports,2015,5:e8259
[19]Li M Y,Tan H W,Wang F,Jiang Q,Xiong A S.De novo transcriptome sequence assembly and identification of AP2/ERF transcription factor related to abiotic stress in parsley(Petroselinum crispum).PLoS One,2014,9(9):e108977
[20]Tan G F,Wang F,Li M Y,Wang G L,Jiang Q,Xiong AS.De novo assembly and transcriptome characterization:novel insights into the temperature stress in Cryptotaenia japonica Hassk.Acta Physiologiae Plantarum,2015,37(1):1-12
[21]Jiang Q,Wang F,Tan H W,Li M Y,Xu Z S,Tan G F,Xiong A S.De novo transcriptome assembly,gene annotation,marker development,and miRNA potential target genes validation under abiotic stresses in Oenanthe javanica.Molecular Genetics&Genomics,2015,290(2):671-683
[22]Kumar S,Nei M,Dudley J,Tamura K.MEGA:a biologistcentric software for evolutionary analysis of DNA and protein sequences.Briefings in Bioinformatics,2008,9(4):299-306
[23]Tian C,Jiang Q,Wang F,Xu Z S,Xiong A S.Selection of suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves.PLoS One,2015,10(2):e0117569
[24]Pfaffl M W.A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Research,2001,29:e45
[25]Wagner G J,Wang E,Shepherd R W.New approaches for studying and exploiting an old protuberance,the plant trichome.Annals of Botany,2004,93(1):3-11
[26]Werker E.Trichome diversity and development.Advances in Botanical Research,2000,31:1-35
[27]Cardoso M Z.Herbivore handling of a plants trichome:the case of Heliconius charithonia(L.)(Lepidoptera:Nymphalidae)and Passiflora lobata(Killip)Hutch.(Passifloraceae).Neotropical Entomology,2008,37(3):247-252
[28]庄飞云,朱德蔚.胡萝卜种质资源描述规范和数据标准.北京:中国农业出版社,2007Zhuang F Y,Zhu D W.Descriptors and Data Standard for Carrot(Daucus carota L.).Beijing:China Agriculture Press,2007
[29]Simon P W,Freeman R E,Vieira J V,Boiteux L S,Briard M,Nothnagel T.Carrot.Handbook Plant Breeding,2017,16(S1):165-178
[30]Tan G F,Wang F,Zhang X Y,Xiong A S.Different lengths,copies and expression levels of the mitochondrial atp6 gene in male sterile and fertile lines of carrot(Daucus carota L.).Mitochondrial DNA Part A,2017,29(3):446-454
[2]Schmaelzle S,Tanumihardjo S A.Carrots of various colors.Totowa:Humana Press,2013:21-28
[3]谭国飞,王枫,马静,田畅,陈逸云,熊爱生.室温贮藏过程中胡萝卜口感及部分营养成分含量变化.植物资源与环境学报,2014,23(1):107-109Tan G F,Wang F,Ma J,Tian C,Chen Y Y,Xiong A S.Changes of taste and content of some nutritional components of carrot during storage period under room temperature.Journal of Plant Resources and Environment,2014,23(1):107-109
[4]谭国飞,王枫,马静,张馨月,熊爱生.野生瓣化型雄性不育胡萝卜‘武野-不育’的特征鉴定与分析.植物遗传资源学报,2017,18(6):1216-1220Tan G F,Wang F,Ma J,Zhang X Y,Xiong A S.Identification and analysis of the characteristics of a wild petaloid male-sterile carrot‘Wuye-BY’.Journal of Plant Genetic Resources,2017,18(6):1216-1220
[5]单人骅,佘孟兰.中国植物志:第55(3)卷.北京:科学出版社,1992:225-226Shan R H,She M L.Flora of China:Volume 55(3).Beijing:Science Press,1992:225-226
[6]Gyudong O,Eunjo S,Sangjin J,Youngdoo P.Development of SNP molecular markers related to seed-hair characteristic based on EST sequences in carrot.Korean Journal of Horticultural Science&Technology,2013,31(1):80-88
[7]Gyudong O,Eunjo S,Sangjin J,Youngdoo P.Construction of cDNA library and EST analysis related to seed-hair characteristics in carrot.Korean Journal of Horticultural Science&Technology,2013,31(6):782-789
[8]孙美玲,吕爽,邱李梅.浅析我国胡萝卜育种概况.赤子,2012(1):133Sun M L,Lv S,Qiu L M.Brief analysis of carrot breeding in china.Chizi,2012(1):133
[9]Gyudong O,Eunmi H,Eunjo S,Sangjin J,Youngdoo P.ESTprofiling for seed-hair characteristic and development of EST-SSR and SNP markers in carrot.Korean Journal of Horticultural Science&Technology,2010,28(6):1025-1038
[10]Eunjo S,Sungkwan P,Gyudong O,Sangjin J,Youngdoo P.Development of RAPD-SCAR molecular marker related to seed-hair characteristic in carrot.Korean Journal of Horticultural Science&Technology,2013,31(6):756-763
[11]Hwang E M,Oh G D,Shim E J,Jeon S J.Analysis of seed hair formation related genes by EST profiling in carrot(Daucus carota var.sativa).Korean J Hortic Sci,2010,28(6):1039-1050
[12]刘凯歌,齐双慧,段绍伟,李东,金倡宇,高晨浩,刘绚霞,陈明训.甘蓝型油菜BnTTG1-1基因的功能分析.植物学报,2017,52(6):713-722Liu K G,Qi S H,Duan S W,Li D,Jin C Y,Gao C H,Liu XX,Chen M X.Functional analysis of Brassica napus BnTTG1-1gene.Chinese Bulletin of Botany,2017,52(6):713-722
[13]Walker A R,Davison P A,Bolognesi-Winfield A C,James C M,Srinivasan N,Esch J J,Marks M D,Gray J C.The TRANSPARENT TESTA GLABRA1 locus,which regulates trichome differentiation and anthocyanin biosynthesis in Arabidopsis,encodes a WD40 repeat protein.Plant Cell,1999,11(7):1337-1350
[14]Alahakoon U.Effect of TRANSPARENT TESTA GLABRA1on trichome development,growth,and insect resistance in a Brassica napus AtGLABRA3+background.Canada Saskatoon:University of Saskatchewan Saskatoon,2014
[15]Larkin J C,Oppenheimer D G,Lloyd A M,Paparozzi E T,Marks M.Roles of the GLABROUS1 and TRANSPARENTTESTA GLABRA genes in Arabidopsis trichome development.Plant Cell,1994,6(8):1065-1076
[16]Koornneef M.The complex syndrome of ttg mutants.Arabidopsis Information Service,1981,18:45-51
[17]Lloyd A M.The TTG gene is required to specify epidermal cell fate and cell patterning in the Arabidopsis root.Developmental Biology,1994,166(2):740-754
[18]Jia X L,Wang G L,Xiong F,Yu X R,Xu Z S,Wang F,Xiong A S.De novo assembly,transcriptome characterization,lignin accumulation,and anatomic characteristics:novel insights into lignin biosynthesis during celery leaf development.Scientific Reports,2015,5:e8259
[19]Li M Y,Tan H W,Wang F,Jiang Q,Xiong A S.De novo transcriptome sequence assembly and identification of AP2/ERF transcription factor related to abiotic stress in parsley(Petroselinum crispum).PLoS One,2014,9(9):e108977
[20]Tan G F,Wang F,Li M Y,Wang G L,Jiang Q,Xiong AS.De novo assembly and transcriptome characterization:novel insights into the temperature stress in Cryptotaenia japonica Hassk.Acta Physiologiae Plantarum,2015,37(1):1-12
[21]Jiang Q,Wang F,Tan H W,Li M Y,Xu Z S,Tan G F,Xiong A S.De novo transcriptome assembly,gene annotation,marker development,and miRNA potential target genes validation under abiotic stresses in Oenanthe javanica.Molecular Genetics&Genomics,2015,290(2):671-683
[22]Kumar S,Nei M,Dudley J,Tamura K.MEGA:a biologistcentric software for evolutionary analysis of DNA and protein sequences.Briefings in Bioinformatics,2008,9(4):299-306
[23]Tian C,Jiang Q,Wang F,Xu Z S,Xiong A S.Selection of suitable reference genes for qPCR normalization under abiotic stresses and hormone stimuli in carrot leaves.PLoS One,2015,10(2):e0117569
[24]Pfaffl M W.A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Research,2001,29:e45
[25]Wagner G J,Wang E,Shepherd R W.New approaches for studying and exploiting an old protuberance,the plant trichome.Annals of Botany,2004,93(1):3-11
[26]Werker E.Trichome diversity and development.Advances in Botanical Research,2000,31:1-35
[27]Cardoso M Z.Herbivore handling of a plants trichome:the case of Heliconius charithonia(L.)(Lepidoptera:Nymphalidae)and Passiflora lobata(Killip)Hutch.(Passifloraceae).Neotropical Entomology,2008,37(3):247-252
[28]庄飞云,朱德蔚.胡萝卜种质资源描述规范和数据标准.北京:中国农业出版社,2007Zhuang F Y,Zhu D W.Descriptors and Data Standard for Carrot(Daucus carota L.).Beijing:China Agriculture Press,2007
[29]Simon P W,Freeman R E,Vieira J V,Boiteux L S,Briard M,Nothnagel T.Carrot.Handbook Plant Breeding,2017,16(S1):165-178
[30]Tan G F,Wang F,Zhang X Y,Xiong A S.Different lengths,copies and expression levels of the mitochondrial atp6 gene in male sterile and fertile lines of carrot(Daucus carota L.).Mitochondrial DNA Part A,2017,29(3):446-454