甘蓝冷胁迫相关基因BobHLH18克隆与表达分析
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摘要
利用同源克隆方法在耐寒,迟抽薹甘蓝自交系Y923中克隆到一个甘蓝响应冷胁迫b HLH转录因子基因Bob HLH18的DNA和c DNA全长,基因组搜索分析结果表明,该基因位于甘蓝1号染色体上,属于LF1亚基因组编码基因;序列分析结果表明,该基因含有4个外显子和3个内含子,编码338个氨基酸,蛋白质分子量为38 380,等电点为6. 80,其编码蛋白质的N端含有一个b HLH结构域;亚细胞定位分析指出该基因编码蛋白质定位在细胞核上,表明该基因编码蛋白质为核定位蛋白质,与其为转录因子特征相符;序列比对结果显示,Bob HLH18蛋白与白菜和拟南芥中的b HLH蛋白具有较高的同源性,相似度分别为90. 5%和89. 0%;聚类分析指出Bob HLH18及其同源蛋白分别聚类成2个进化分支,且来自十字花科植物的b HLH18同源蛋白质都聚在同一进化分支上; qRT-PCR分析结果表明Bob HLH18基因受冷胁迫诱导,能在叶片中被较高的诱导表达,表明该基因可能在甘蓝叶片应答冷胁迫过程中起重要作用。
In this study,a gene full-length sequence of the cold stress-related bH LH transcription factor BobH LH18,was isolated from the cabbage inbred line Y923 with cold and bolting resistance by homologous cloning methods. Genome search analysis results showed that this gene was located on C01 chromosome of cabbage,which belonged to the LF1 subgenome. Sequence analysis results showed that this gene contained four exons and three introns,and its encoded protein contained 338 amino acids with the molecular weight of 38 380 and the isoelectric point of 6.80,its N terminal contained bH LH domain. Subcellular localization predication analysis results indicated that the protein encoded by BobH LH18 was located on the nucleus and had nuclear protein features,which was consistent with transcription factor characteristics. Homology analysis results showed that BobH LH18 protein had high homology with bH LH18 protein in Chinese cabbage and Arabidopsis, and the similarities were 90. 5% and 89. 0%,respectively. Cluster analysis results showed that BobH LH18 and its homologous proteins could be divided into two evolutionary branches,and the bH LH18 homologs from the cruciferous plants could be clustered into the same division. qRT-PCR analysis results indicated that BobH LH18 gene was induced by cold stress and had high expression levels at 12 h after cold treatment,suggesting that BobH LH18 gene may play an important role in the response of cabbage leaves to cold stress.
In this study,a gene full-length sequence of the cold stress-related bH LH transcription factor BobH LH18,was isolated from the cabbage inbred line Y923 with cold and bolting resistance by homologous cloning methods. Genome search analysis results showed that this gene was located on C01 chromosome of cabbage,which belonged to the LF1 subgenome. Sequence analysis results showed that this gene contained four exons and three introns,and its encoded protein contained 338 amino acids with the molecular weight of 38 380 and the isoelectric point of 6.80,its N terminal contained bH LH domain. Subcellular localization predication analysis results indicated that the protein encoded by BobH LH18 was located on the nucleus and had nuclear protein features,which was consistent with transcription factor characteristics. Homology analysis results showed that BobH LH18 protein had high homology with bH LH18 protein in Chinese cabbage and Arabidopsis, and the similarities were 90. 5% and 89. 0%,respectively. Cluster analysis results showed that BobH LH18 and its homologous proteins could be divided into two evolutionary branches,and the bH LH18 homologs from the cruciferous plants could be clustered into the same division. qRT-PCR analysis results indicated that BobH LH18 gene was induced by cold stress and had high expression levels at 12 h after cold treatment,suggesting that BobH LH18 gene may play an important role in the response of cabbage leaves to cold stress.
引文
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[17]靳哲,张扬勇,方智远,等.结球甘蓝BoCBF1与BoCBF2基因的CAPS标记及其对耐寒性的影响[J].中国蔬菜,2012(14):23-30.
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[23] ZHAO H,LI X,MA L. Basic helix-loop-helix transcription factorsand epidermal cell fate determination in Arabidopsis[J]. Plant Sig-nal Behav,2012,7(12):1556-1560.
[24] GROSZMANN M,BYLSTRA Y,LAMPUGNANI E R,et al. Reg-ulation of tissue-specific expression of SPATULA,a b HLH geneinvolved in carpel development,seedling germination,and lateralorgan growth in Arabidopsis[J]. J Exp Bot,2010,61(5):1495-1508.
[25] FARQUHARSON K L. A domain in the b HLH transcription factorDYT1 Is critical for anther development[J]. Plant Cell,2016,28(5):997-998.
[26] RAJANI S,SUNDARESAN V. The Arabidopsis myc/b HLH geneALCATRAZ enables cell separation in fruit dehiscence[J]. CurrBiol,2001,11(24):1914-1922.
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[28] LEIVAR P,MONTE E,OKA Y,et al. Multiple phytochrome-in-teracting b HLH transcription factors repress premature seedlingphotomorphogenesis in darkness[J]. Curr Biol,2008,18(23):1815-1823.
[29] NAKATA M,MITSUDA N,HERDE M,et al. A bHLH-type tran-scription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIP-TION FACTOR/JA-ASSOCIATED MYC2-LIKE1,acts as a re-pressor to negatively regulate jasmonate signaling in Arabidopsis[J]. Plant Cell,2013,25(5):1641-1656.
[30] KAVAS M,BALOGLU M C,ATABAY E S,et al. Genome-widecharacterization and expression analysis of common bean bHLHtranscription factors in response to excess salt concentration[J].Mol Genet Genomics,2016,291(1):129-143.
[31] LONG T A,TSUKAGOSHI H,BUSCH W,et al. The bHLH tran-scription factor POPEYE regulates response to iron deficiency inArabidopsis roots[J].Plant Cell,2010,22(7):2219-2236.
[32]王翠,兰海燕.植物bHLH转录因子在非生物胁迫中的功能研究进展[J].生命科学研究,2016,20(4):358-364.
[33] TOLEDO-ORTIZ G,HUQ E,QUAIL P H. The Arabidopsis basic/helix-loop-helix transcription factor family[J]. Plant Cell,2003,15(8):1749-1770.
[34]曹宁,张启翔,郝瑞杰,等.梅花PmICE1基因的克隆及低温条件下的表达[J].东北林业大学学报,2014,42(4):21-25.
[35]向殿军,殷奎德,满丽莉,等.大白菜低温胁迫转录因子BcICE1的克隆及表达分析[J].分子植物育种,2011,9(3):364-369.
[36]刘海霞,康俊根,颉建明,等.甘蓝Ogu CMS相关的花药优势表达转录因子BoBHLH1的克隆与表达分析[J].园艺学报,2010,37(12):1953-1960.
[37]豆玉娟,曹飞,马跃,等.栽培草莓果实中特异表达的b HLH78基因的克隆及过量表达载体构建[J].分子植物育种,2014,12(3):456-465.
[38]杨娜.独行菜种子低温萌发耐受性特性及bHLH类转录因子分析[D].乌鲁木齐:新疆师范大学,2017.
[39] FOWLER S,THOMASHOW M F. Arabidopsis transcriptome profi-ling indicates that multiple regulatory pathways are activated dur-ing cold acclimation in addition to the CBF cold response pathway[J]. Plant Cell,2002,14(8):1675-1690.
[40]姚攀锋,赵学荣,李茂菲,等.苦荞转录因子基因FtbHLH3的克隆及其非生物胁迫下的表达分析[J].基因组学与应用生物学,2016,35(2):429-435.
[41]李晓刚,李慧,杨青松,等.杜梨b HLH转录因子家族两成员的序列特征及对非生物胁迫的转录响应[J].江苏农业科学,2017,45(22):40-45.
[42]李宇伟,连瑞丽,王新民.蒺藜苜蓿低温胁迫响应基因Mt-b HLH-1的克隆及功能分析[J].中国农业科学,2012,45(16):3430-3436.
[43]袁琳琳,王亚茹,曾卫军,等.独行菜种子bHLH类转录因子基因家族及幼苗laICE1表达对冷胁迫的响应[J].西北植物学报,2018,38(1):26-34.
[44]何洁,顾秀容,魏春华,等.西瓜b HLH转录因子家族基因的鉴定及其在非生物胁迫下的表达分析[J].园艺学报,2016,43(2):281-294.
[2] PABO C O,SAUER R T. Transcription factors:structural familiesand principles of DNA recognition[J]. Annu Rev Biochem,1992,61:1053-1095.
[3] WANAPU C,SHINMYO A. Cis-regulatory elements of the peroxi-dase gene in Arabidopsis thaliana involved in root-specific expres-sion and responsiveness to high-salt stress[J]. Ann N Y Acad Sci,1996,782:107-114.
[4] YAMAGUCHI-SHINOZAKI K,SHINOZAKI K. A novel cis-actingelement in an Arabidopsis gene is involved in responsiveness todrought,low-temperature,or high-salt stress[J]. Plant Cell,1994,6(2):251-264.
[5] MATYS V,KEL-MARGOULIS O V,FRICKE E,et al. TRANS-FAC and its module TRANS Compel:transcriptional gene regula-tion in eukaryotes[J]. Nucleic Acids Res,2006,34(Database is-sue):D108-D110.
[6] ATCHLEY W R,TERHALLE W,DRESS A. Positional depend-ence,cliques,and predictive motifs in the bHLH protein domain[J]. J Mol Evol,1999,48(5):501-516.
[7] HEIM M A,JAKOBY M,WERBER M,et al. The basic helix-loop-helix transcription factor family in plants:a genome-widestudy of protein structure and functional diversity[J]. Mol BiolEvol,2003,20(5):735-747.
[8] PIRES N,DOLAN L. Origin and diversification of basic-helix-loop-helix proteins in plants[J]. Mol Biol Evol,2010,27(4):862-874.
[9] CARRETERO-PAULET L,GALSTYAN A,ROIG-VILLANOVAI,et al. Genome-wide classification and evolutionary analysis ofthe b HLH family of transcription factors in Arabidopsis,poplar,rice,moss,and algae[J]. Plant Physiol,2010,153(3):1398-1412.
[10] ATCHLEY W R,FITCH W M. A natural classification of the basichelix-loop-helix class of transcription factors[J]. Proc Natl AcadSci USA,1997,94(10):5172-5176.
[11] LI X,DUAN X,JIANG H,et al. Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis[J]. Plant Physiol,2006,141(4):1167-1184.
[12] SONG X M,HUANG Z N,DUAN W K,et al. Genome-wide a-nalysis of the b HLH transcription factor family in Chinese cabbage(Brassica rapa ssp. pekinensis)[J]. Mol Genet Genomics,2014,289(1):77-91.
[13] RUSHTON P J,BOKOWIEC M T,HAN S,et al. Tobacco tran-scription factors:novel insights into transcriptional regulation in theSolanaceae[J]. Plant Physiol,2008,147(1):280-295.
[14] NIU X,GUAN Y,CHEN S,et al. Genome-wide analysis of basichelix-loop-helix(b HLH)transcription factors in Brachypodiumdistachyon[J]. BMC Genomics,2017,18(1):619.
[15] SUN H,FAN H J,LING H Q. Genome-wide identification andcharacterization of the bHLH gene family in tomato[J]. BMC Ge-nomics,2015,16:9.
[16]杨丽梅,方智远,刘玉梅,等.“十一五”我国甘蓝遗传育种研究进展[J].中国蔬菜,2011(2):1-10.
[17]靳哲,张扬勇,方智远,等.结球甘蓝BoCBF1与BoCBF2基因的CAPS标记及其对耐寒性的影响[J].中国蔬菜,2012(14):23-30.
[18]夏瑞祥,肖宁,洪义欢,等.东乡野生稻苗期耐冷性的QTL定位[J].中国农业科学,2010,43(3):443-451.
[19] HUANG G T,MA S L,BAI L P,et al. Signal transduction duringcold,salt,and drought stresses in plants[J]. Mol Biol Rep,2012,39(2):969-987.
[20] CHINNUSAMY V,OHTA M,KANRAR S,et al. ICE1:a regula-tor of cold-induced transcriptome and freezing tolerance in Arabi-dopsis[J]. Genes Dev,2003,17(8):1043-1054.
[21] LIU C,WU Y,WANG X. bZIP transcription factor Osb ZIP52/RISBZ5:a potential negative regulator of cold and drought stressresponse in rice[J]. Planta,2012,235(6):1157-1169.
[22] OH E,KIM J,PARK E,et al. PIL5,a phytochrome-interactingbasic helix-loop-helix protein,is a key negative regulator of seedgermination in Arabidopsis thaliana[J]. Plant Cell,2004,16(11):3045-3058.
[23] ZHAO H,LI X,MA L. Basic helix-loop-helix transcription factorsand epidermal cell fate determination in Arabidopsis[J]. Plant Sig-nal Behav,2012,7(12):1556-1560.
[24] GROSZMANN M,BYLSTRA Y,LAMPUGNANI E R,et al. Reg-ulation of tissue-specific expression of SPATULA,a b HLH geneinvolved in carpel development,seedling germination,and lateralorgan growth in Arabidopsis[J]. J Exp Bot,2010,61(5):1495-1508.
[25] FARQUHARSON K L. A domain in the b HLH transcription factorDYT1 Is critical for anther development[J]. Plant Cell,2016,28(5):997-998.
[26] RAJANI S,SUNDARESAN V. The Arabidopsis myc/b HLH geneALCATRAZ enables cell separation in fruit dehiscence[J]. CurrBiol,2001,11(24):1914-1922.
[27] MATUS J T,POUPIN M J,CANON P,et al. Isolation of WDRand bHLH genes related to flavonoid synthesis in grapevine(Vitisvinifera L.)[J]. Plant Mol Biol,2010,72(6):607-620.
[28] LEIVAR P,MONTE E,OKA Y,et al. Multiple phytochrome-in-teracting b HLH transcription factors repress premature seedlingphotomorphogenesis in darkness[J]. Curr Biol,2008,18(23):1815-1823.
[29] NAKATA M,MITSUDA N,HERDE M,et al. A bHLH-type tran-scription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIP-TION FACTOR/JA-ASSOCIATED MYC2-LIKE1,acts as a re-pressor to negatively regulate jasmonate signaling in Arabidopsis[J]. Plant Cell,2013,25(5):1641-1656.
[30] KAVAS M,BALOGLU M C,ATABAY E S,et al. Genome-widecharacterization and expression analysis of common bean bHLHtranscription factors in response to excess salt concentration[J].Mol Genet Genomics,2016,291(1):129-143.
[31] LONG T A,TSUKAGOSHI H,BUSCH W,et al. The bHLH tran-scription factor POPEYE regulates response to iron deficiency inArabidopsis roots[J].Plant Cell,2010,22(7):2219-2236.
[32]王翠,兰海燕.植物bHLH转录因子在非生物胁迫中的功能研究进展[J].生命科学研究,2016,20(4):358-364.
[33] TOLEDO-ORTIZ G,HUQ E,QUAIL P H. The Arabidopsis basic/helix-loop-helix transcription factor family[J]. Plant Cell,2003,15(8):1749-1770.
[34]曹宁,张启翔,郝瑞杰,等.梅花PmICE1基因的克隆及低温条件下的表达[J].东北林业大学学报,2014,42(4):21-25.
[35]向殿军,殷奎德,满丽莉,等.大白菜低温胁迫转录因子BcICE1的克隆及表达分析[J].分子植物育种,2011,9(3):364-369.
[36]刘海霞,康俊根,颉建明,等.甘蓝Ogu CMS相关的花药优势表达转录因子BoBHLH1的克隆与表达分析[J].园艺学报,2010,37(12):1953-1960.
[37]豆玉娟,曹飞,马跃,等.栽培草莓果实中特异表达的b HLH78基因的克隆及过量表达载体构建[J].分子植物育种,2014,12(3):456-465.
[38]杨娜.独行菜种子低温萌发耐受性特性及bHLH类转录因子分析[D].乌鲁木齐:新疆师范大学,2017.
[39] FOWLER S,THOMASHOW M F. Arabidopsis transcriptome profi-ling indicates that multiple regulatory pathways are activated dur-ing cold acclimation in addition to the CBF cold response pathway[J]. Plant Cell,2002,14(8):1675-1690.
[40]姚攀锋,赵学荣,李茂菲,等.苦荞转录因子基因FtbHLH3的克隆及其非生物胁迫下的表达分析[J].基因组学与应用生物学,2016,35(2):429-435.
[41]李晓刚,李慧,杨青松,等.杜梨b HLH转录因子家族两成员的序列特征及对非生物胁迫的转录响应[J].江苏农业科学,2017,45(22):40-45.
[42]李宇伟,连瑞丽,王新民.蒺藜苜蓿低温胁迫响应基因Mt-b HLH-1的克隆及功能分析[J].中国农业科学,2012,45(16):3430-3436.
[43]袁琳琳,王亚茹,曾卫军,等.独行菜种子bHLH类转录因子基因家族及幼苗laICE1表达对冷胁迫的响应[J].西北植物学报,2018,38(1):26-34.
[44]何洁,顾秀容,魏春华,等.西瓜b HLH转录因子家族基因的鉴定及其在非生物胁迫下的表达分析[J].园艺学报,2016,43(2):281-294.