苎麻转录因子基因BnMYB3的克隆及表达分析
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摘要
该研究采用RACE技术,从苎麻中克隆到1个MYB转录因子基因(BnMYB3)的全长cDNA序列(GenBank登录号为MF741320.1)。生物信息学分析表明,BnMYB3基因cDNA全长为1 216bp,包括900bp编码区序列,编码含有299个氨基酸的蛋白,其分子量约为33.63kD,理论等电点为9.16;该蛋白质含有2个典型的MYB结构域,属于R2R3-MYB。从苎麻基因组中克隆了BnMYB3基因1 681bp启动子序列,该序列包含ABRE、GARE-motif、CGTCA-motif和TGACG-motif等多个逆境相关的顺式作用元件。实时荧光定量PCR分析表明,BnMYB3为组成型表达基因,在茎和叶中的表达量显著高于根;BnMYB3基因能够响应镉胁迫,且表达量随镉胁迫处理时间和处理浓度的增加而显著上升。
In this paper,a MYB gene named BnMYB3 was isolated from ramie(Boehmeria nivea L.)by amplification of cDNA ends(RACE),whose GenBank accession number is MF741320.1.The sequence analysis showed that the full-length cDNA of BnMYB3 contained 1 216 base pairs,with a coding region of900 base pairs.This sequence encoded a protein of 299 amino acids in length with a molecular weight of33.63 kD and a theoretical isoelectric point of 9.16.The protein contains two typical MYB domains which belong to R2 R3-MYB gene family.The 1 681 base pairs promoter sequence of BnMYB3 was cloned from the genome of ramie.By bioinformatics analysis,the promoter sequence contained several stress-related cis-acting elements,such as ABRE,GARE-motif,CGTCA-motif and TGACG-motif.Real-time quantitative PCR analysis showed that BnMYB3 was a constitutive gene,but the expression levels of BnMYB3 in stems and leaves were significantly higher than that in roots.Furthermore,BnMYB3 gene was apparently induced by cadmium,and the relative expression level of BnMYB3 increased dramatically with the prolongation of treatment time and the increasement of Cd~(2+) concentration.
In this paper,a MYB gene named BnMYB3 was isolated from ramie(Boehmeria nivea L.)by amplification of cDNA ends(RACE),whose GenBank accession number is MF741320.1.The sequence analysis showed that the full-length cDNA of BnMYB3 contained 1 216 base pairs,with a coding region of900 base pairs.This sequence encoded a protein of 299 amino acids in length with a molecular weight of33.63 kD and a theoretical isoelectric point of 9.16.The protein contains two typical MYB domains which belong to R2 R3-MYB gene family.The 1 681 base pairs promoter sequence of BnMYB3 was cloned from the genome of ramie.By bioinformatics analysis,the promoter sequence contained several stress-related cis-acting elements,such as ABRE,GARE-motif,CGTCA-motif and TGACG-motif.Real-time quantitative PCR analysis showed that BnMYB3 was a constitutive gene,but the expression levels of BnMYB3 in stems and leaves were significantly higher than that in roots.Furthermore,BnMYB3 gene was apparently induced by cadmium,and the relative expression level of BnMYB3 increased dramatically with the prolongation of treatment time and the increasement of Cd~(2+) concentration.
引文
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[12]CHEN Y,CHEN Z L,KANG J Q,et al.AtMYB14regulates cold tolerance in Arabidopsis[J].Plant Molecular Biology Reporter,2013,31(1):87-97.
[13]RAFFAELE S,RIVAS S,ROBY D.An essential role for salicylic acid in AtMYB30-mediated control of the hypersensitive cell death program in Arabidopsis[J].FEBS Letters,2006,580(14):3 498-3 504.
[14]雷梅,岳庆玲,陈同斌,等.湖南柿竹园矿区土壤重金属含量及植物吸收特征[J].生态学报,2005,25(5):1 146-1 151.LEI M,YUE Q L,CHEN T B,et al.Heavy metal concentrations in soils and plants around Shizhuyuan mining area of Hunan Province[J].Acta Ecologica Sinica,2005,25(5):1 146-1 151.
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[16]佘玮,揭雨成,邢虎成,等.湖南冷水江锑矿区苎麻对重金属的吸收和富集特性[J].农业环境科学学报,2010,29(1):91-96.SHE W,JIE Y C,XING H C,et al.Uptake and accumulation of heavy metal by ramie(Boehmeria nivea)growing on antimony mining area in Lengshuijiang City of Hunan Province[J].Journal of Agro-Environment Science,2010,29(1):91-96.
[17]朱守晶,史文娟,揭雨成,等.苎麻镉响应转录因子BnMYB1的克隆和表达分析[J].农业生物技术学报,2018,26(5):774-783.ZHU S J,SHI W J,JIE Y C,et al.Cloning and expression analysis of cadmium-responsive transcription factor gene BnMYB1from ramie(Boehmeria nivea)[J].Journal of Agricultural Biotechnology,2018,26(5):774-783.
[18]SHE W,ZHU S J,JIE Y C,et al.Expression profiling of cadmium response genes in ramie(Boehmeria nivea L.)root[J].Bulletin of Environmental Contamination and Toxicology,2015,94(4):453-459.
[19]AMBAWAT S,SHARMA P,YADAV N R,et al.MYBtranscription factor genes as regulators for plant responses:an overview[J].Physiology and Molecular Biology of Plants,2013,19(3):307-321.
[20]DUBOS C,STRACKE R,GROTEWOLD E,et al.MYBtranscription factors in Arabidopsis[J].Trends in Plant Science,2010,15(10):573-581.
[21]BALDONI E,GENGA A,COMINELLI E.Plant MYBtranscription factors:their role in drought response mechanisms[J].International Journal of Molecular Sciences,2015,16(7):15 811-15 851.
[22]KASUGA M,LIU Q,MIURA S,et al.Improving plant drought,salt,and freezing tolerance by gene transfer of a single stress-inducible transcription factor[J].Nature Biotechnology,1999,17(3):287-291.
[23]樊锦涛,蒋琛茜,邢继红,等.拟南芥R2R3-MYB家族第22亚族的结构与功能分析[J].遗传,2014,36(10):985-994.FAN J T,JIANG C X,XING J H,et al.Structure and function of the 22nd subfamily in Arabidopsis R2R3-MYBfamily[J].Hereditas,2014,36(10):985-994.
[24]SKRIVER K,OLSEN F L,ROGERS J C,et al.Cis-acting DNA elements responsive to gibberellin and its antagonist abscisic acid[J].Proceedings of the National Academy of Sciences of the United States of America,1991,88(16):7 266-7 270.
[25]ISHIBASHI Y,TAWARATSUMIDA T,KONDO K,et al.Reactive oxygen species are involved in gibberellin/abscisic acid signaling in barley aleurone cells[J].Plant Physiology,2012,158(4):1 705-1 714.
[26]VETTAKKORUMAKANKAV N N,FALK D,SAXENAP,et al.A crucial role for gibberellins in stress protection of plants[J].Plant and Cell Physiology,1999,40(5):542-548.
[27]SARKAR S,PERRAS M R,FALK D E,et al.Relationship between gibberellins,height,and stress tolerance in barley(Hordeum vulgare L.)seedlings[J].Plant Growth Regulation,2004,42(2):125-135.
[28]MAEDA K,KIMURA S,DEMURA T,et al.DcMYB1acts as a transcriptional activator of the carrot phenylalanine ammonia-lyase gene(DcPAL1)in response to elicitor treatment,UV-B irradiation and the dilution effect[J].Plant Molecular Biology,2005,59(5):739-752.
[29]JIN H,COMINELLI E,BAILEY P,et al.Transcriptional repression by ATMYB4controls production of UV-protecting sunscreens in Arabidopsis[J].The EMBO Journal,2000,19(22):6 150-6 161.
[30]KRANZ H D,DENEKAMP M,GRECO R,et al.Towards functional characterisation of the members of the R2R3-MYBgene family fromArabidopsis thaliana[J].The Plant Journal:for Cell and Molecular Biology,1998,16(2):263-276.
[31]JEONG S T,GOTO-YAMAMOTO N,et al.Effects of plant hormones and shading on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins[J].Plant Science,2004,167(2):247-252.
[32]FUSCO N,MICHELETTO L,DAL CORSO G,et al.Identification of cadmium-regulated genes by cDNA-AFLP in the heavy metal accumulator Brassica juncea L.[J].Journal of Experimental Botany,2005,56(421):3 017-3 027.
[33]CHEN Y H,YANG X Y,HE K,et al.The MYB transcription factor superfamily of Arabidopsis:expression analysis and phylogenetic comparison with the rice MYB family[J].Plant Molecular Biology,2006,60(1):107-124.
[34]LIU T M,ZHU S Y,TANG Q M,et al.Genome-wide transcriptomic profiling of ramie(Boehmeria nivea L.Gaud)in response to cadmium stress[J].Gene,2015,558(1):131-137.
[2]CHANEY R L,REEVES P G,RYAN J A,et al.An improved understanding of soil Cd risk to humans and low cost methods to phytoextract Cd from contaminated soils to prevent soil Cd risks[J].BioMetals,2004,17(5):549-553.
[3]GRANT C A,CLARKE J M,DUGUID S,et al.Selection and breeding of plant cultivars to minimize cadmium accumulation[J].The Science of the Total Environment,2008,390(2):301-310.
[4]VERBRUGGEN N,HERMANS C,SCHAT H.Mechanisms to cope with arsenic or cadmium excess in plants[J].Current Opinion in Plant Biology,2009,12(3):364-372.
[5]LIPPOLD F,SANCHEZ D H,MUSIALAK M,et al.AtMyb41regulates transcriptional and metabolic responses to osmotic stress in Arabidopsis[J].Plant Physiology,2009,149(4):1 761-1 772.
[6]MARTIN C,PAZ-ARES J.MYB transcription factors in plants[J].Trends in Genetics,1997,13(2):67-73.
[7]ROMERO I,FUERTES A,BENITO M J,et al.More than80R2R3-MYB regulatory genes in the genome of Arabidopsis thaliana[J].Plant Journal,1998,14(3):273-284.
[8]COMINELLI E,GALBIATI M,VAVASSEUR A,et al.Aguard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance[J].Current Biology,2005,15(13):1 196-1 200.
[9]JUNG C,SEO J S,HAN S W,et al.Overexpression of AtMYB44enhances stomatal closure to confer abiotic stress tolerance in transgenic Arabidopsis[J].Plant Physiology,2008,146(2):623-635.
[10]CUI M H,YOO K S,HYOUNG S,et al.An Arabidopsis R2R3-MYB transcription factor,AtMYB20,negatively regulates type 2Cserine/threonine protein phosphatases to enhance salt tolerance[J].FEBS Letters,2013,587(12):1 773-1 778.
[11]AGARWAL M,HAO Y J,KAPOOR A,et al.A R2R3type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance[J].The Journal of Biological Chemistry,2006,281(49):37 636-37 645.
[12]CHEN Y,CHEN Z L,KANG J Q,et al.AtMYB14regulates cold tolerance in Arabidopsis[J].Plant Molecular Biology Reporter,2013,31(1):87-97.
[13]RAFFAELE S,RIVAS S,ROBY D.An essential role for salicylic acid in AtMYB30-mediated control of the hypersensitive cell death program in Arabidopsis[J].FEBS Letters,2006,580(14):3 498-3 504.
[14]雷梅,岳庆玲,陈同斌,等.湖南柿竹园矿区土壤重金属含量及植物吸收特征[J].生态学报,2005,25(5):1 146-1 151.LEI M,YUE Q L,CHEN T B,et al.Heavy metal concentrations in soils and plants around Shizhuyuan mining area of Hunan Province[J].Acta Ecologica Sinica,2005,25(5):1 146-1 151.
[15]朱守晶,史文娟,揭雨成.不同苎麻品种对土壤中镉、铅富集的差异[J].江苏农业学报,2018,34(2):320-326.ZHU S J,SHI W J,JIE Y C.Variety difference in cadmium and lead accumulation by ramie(Boehmeria nivea)from soil[J].Jiangsu Journal of Agricultural Sciences,2018,34(2):320-326.
[16]佘玮,揭雨成,邢虎成,等.湖南冷水江锑矿区苎麻对重金属的吸收和富集特性[J].农业环境科学学报,2010,29(1):91-96.SHE W,JIE Y C,XING H C,et al.Uptake and accumulation of heavy metal by ramie(Boehmeria nivea)growing on antimony mining area in Lengshuijiang City of Hunan Province[J].Journal of Agro-Environment Science,2010,29(1):91-96.
[17]朱守晶,史文娟,揭雨成,等.苎麻镉响应转录因子BnMYB1的克隆和表达分析[J].农业生物技术学报,2018,26(5):774-783.ZHU S J,SHI W J,JIE Y C,et al.Cloning and expression analysis of cadmium-responsive transcription factor gene BnMYB1from ramie(Boehmeria nivea)[J].Journal of Agricultural Biotechnology,2018,26(5):774-783.
[18]SHE W,ZHU S J,JIE Y C,et al.Expression profiling of cadmium response genes in ramie(Boehmeria nivea L.)root[J].Bulletin of Environmental Contamination and Toxicology,2015,94(4):453-459.
[19]AMBAWAT S,SHARMA P,YADAV N R,et al.MYBtranscription factor genes as regulators for plant responses:an overview[J].Physiology and Molecular Biology of Plants,2013,19(3):307-321.
[20]DUBOS C,STRACKE R,GROTEWOLD E,et al.MYBtranscription factors in Arabidopsis[J].Trends in Plant Science,2010,15(10):573-581.
[21]BALDONI E,GENGA A,COMINELLI E.Plant MYBtranscription factors:their role in drought response mechanisms[J].International Journal of Molecular Sciences,2015,16(7):15 811-15 851.
[22]KASUGA M,LIU Q,MIURA S,et al.Improving plant drought,salt,and freezing tolerance by gene transfer of a single stress-inducible transcription factor[J].Nature Biotechnology,1999,17(3):287-291.
[23]樊锦涛,蒋琛茜,邢继红,等.拟南芥R2R3-MYB家族第22亚族的结构与功能分析[J].遗传,2014,36(10):985-994.FAN J T,JIANG C X,XING J H,et al.Structure and function of the 22nd subfamily in Arabidopsis R2R3-MYBfamily[J].Hereditas,2014,36(10):985-994.
[24]SKRIVER K,OLSEN F L,ROGERS J C,et al.Cis-acting DNA elements responsive to gibberellin and its antagonist abscisic acid[J].Proceedings of the National Academy of Sciences of the United States of America,1991,88(16):7 266-7 270.
[25]ISHIBASHI Y,TAWARATSUMIDA T,KONDO K,et al.Reactive oxygen species are involved in gibberellin/abscisic acid signaling in barley aleurone cells[J].Plant Physiology,2012,158(4):1 705-1 714.
[26]VETTAKKORUMAKANKAV N N,FALK D,SAXENAP,et al.A crucial role for gibberellins in stress protection of plants[J].Plant and Cell Physiology,1999,40(5):542-548.
[27]SARKAR S,PERRAS M R,FALK D E,et al.Relationship between gibberellins,height,and stress tolerance in barley(Hordeum vulgare L.)seedlings[J].Plant Growth Regulation,2004,42(2):125-135.
[28]MAEDA K,KIMURA S,DEMURA T,et al.DcMYB1acts as a transcriptional activator of the carrot phenylalanine ammonia-lyase gene(DcPAL1)in response to elicitor treatment,UV-B irradiation and the dilution effect[J].Plant Molecular Biology,2005,59(5):739-752.
[29]JIN H,COMINELLI E,BAILEY P,et al.Transcriptional repression by ATMYB4controls production of UV-protecting sunscreens in Arabidopsis[J].The EMBO Journal,2000,19(22):6 150-6 161.
[30]KRANZ H D,DENEKAMP M,GRECO R,et al.Towards functional characterisation of the members of the R2R3-MYBgene family fromArabidopsis thaliana[J].The Plant Journal:for Cell and Molecular Biology,1998,16(2):263-276.
[31]JEONG S T,GOTO-YAMAMOTO N,et al.Effects of plant hormones and shading on the accumulation of anthocyanins and the expression of anthocyanin biosynthetic genes in grape berry skins[J].Plant Science,2004,167(2):247-252.
[32]FUSCO N,MICHELETTO L,DAL CORSO G,et al.Identification of cadmium-regulated genes by cDNA-AFLP in the heavy metal accumulator Brassica juncea L.[J].Journal of Experimental Botany,2005,56(421):3 017-3 027.
[33]CHEN Y H,YANG X Y,HE K,et al.The MYB transcription factor superfamily of Arabidopsis:expression analysis and phylogenetic comparison with the rice MYB family[J].Plant Molecular Biology,2006,60(1):107-124.
[34]LIU T M,ZHU S Y,TANG Q M,et al.Genome-wide transcriptomic profiling of ramie(Boehmeria nivea L.Gaud)in response to cadmium stress[J].Gene,2015,558(1):131-137.