菜心BclUBE2基因的克隆与表达分析
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摘要
采用同源克隆法从菜心中获得cDNA全长459 bp的E2基因,命名为BclUBE2。该基因编码153个氨基酸,分子量为17.24ku,理论等电点为5.37,为亲水性非分泌蛋白。结构分析显示,该蛋白含有一个泛素结合酶E2活性位点、一个WD重复序列和一个高度保守的半胱氨酸。进化分析发现,菜心BclUBE2蛋白与芸薹属芜菁和油菜的亲缘关系较近。qRT-PCR分析表明,菜心BclUBE2基因在根、茎、叶、叶柄中均有表达,且表达丰度和变化趋势不同。在低温胁迫下,根中的表达量呈现先降低后升高的变化趋势,且在处理1 h时表达量最低;在茎、叶、叶柄中均呈现先升高后降低的表达趋势,茎和叶柄处理6 h时的表达量最高,叶片处理1 h时的表达量最高。说明菜心BclUBE2基因在响应低温胁迫中发挥作用。
The ubiquitin-conjugating enzyme E2 gene was cloned from Brassica campestris L. ssp. chinensis var. utilisTsen et Lee and designated as BclUBE2. The full cDNA length of BclUBE2 was 459 bp, encoding 153 amino acid residues with an estimated molecular mass of 17.24 ku and a calculated pI of 5.37. This protein was hydrophilic non-secretory. Structural analysis revealed that the sequence contained an E2 ubiqitin-conjugating enzyme active site, a WD-repeated sequence and a highly conserved cysteine. Phylogenetic analysis showed that BclUBE2 protein had the closest relationship with Brassica napus and Brassica rapa. qRT-PCR results showed that the existence of BclUBE2 was detectable in the root, stem, leaf and petiole with different expressive abundance and change trend. Under low temperature stress, the expression level in the root showed the trend of decreasing first and then increasing, and reached the lowest level at 1 h. However, the expression level in the stem, leaf and petiole increased first and then decreased with the highest level at 6 h in stem and petiole and at 1 h in leaf. These results indicated that BclUBE2 gene was involved in plant response to low temperature stress.
The ubiquitin-conjugating enzyme E2 gene was cloned from Brassica campestris L. ssp. chinensis var. utilisTsen et Lee and designated as BclUBE2. The full cDNA length of BclUBE2 was 459 bp, encoding 153 amino acid residues with an estimated molecular mass of 17.24 ku and a calculated pI of 5.37. This protein was hydrophilic non-secretory. Structural analysis revealed that the sequence contained an E2 ubiqitin-conjugating enzyme active site, a WD-repeated sequence and a highly conserved cysteine. Phylogenetic analysis showed that BclUBE2 protein had the closest relationship with Brassica napus and Brassica rapa. qRT-PCR results showed that the existence of BclUBE2 was detectable in the root, stem, leaf and petiole with different expressive abundance and change trend. Under low temperature stress, the expression level in the root showed the trend of decreasing first and then increasing, and reached the lowest level at 1 h. However, the expression level in the stem, leaf and petiole increased first and then decreased with the highest level at 6 h in stem and petiole and at 1 h in leaf. These results indicated that BclUBE2 gene was involved in plant response to low temperature stress.
引文
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[15]蔡佳文,金晓霞,于丽杰,等.龙葵E2泛素结合酶基因SorUBC克隆及表达特性分析[J].东北农业大学学报,2016, 47(11):26-36.
[16]安红强,范静,梁易,等.铁皮石斛泛素结合酶基因DoUBC24的克隆及表达分析[J].生物技术通讯,2016,27(5):643-648.
[17]决登伟,桑雪莲,舒波,等.玉米泛素结合酶基因ZmUBC-76的功能分析[J].热带作物学报,2017,38(8):1 507-1 511.
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[21]GlickmanMH,CiechanoverA.Theubiquitin-proteasome proteolyticpathway:destructionforthesakeofconstruction[J]. Physiological Reviews, 2002, 82(2):373-428.
[22]WanX,MoA,LiuS,etal.Constitutiveexpressionofa peanutubiquitin-conjugatingenzymegeneinArabidopsis confersimprovedwater-stresstolerancethroughregulation ofstress-responsivegeneexpression[J].JournalofBioscience&Bioengineering, 2011, 111(4):478-484.
[23]王安邦,金志强,刘菊华,等.香蕉泛素结合酶基因MaUCE2在非生物胁迫下的表达分析[J].生物技术通报,2013(5):77-80.
[2]吴焱秋,柴家科.泛素-蛋白酶体途径的组成及其生物学作用[J].生理科学进展, 2001, 32(4):331-333.
[3]YeY,RapeM.Buildingubiquitinchains:E2enzymesat work[J].NatureReviewsMolecularCellBiology,2009,10(11):755-764.
[4]PictonS,GrayJE,LoweA,etal.Sequenceofacloned tomatoubiquitinconjugatingenzyme[J].PlantPhysiology,1993, 103(4):1 471-1 472.
[5]ZhangXD,JenkinsJN,CallahanFE,etal.Molecular cloning,differentialexpression,andfunctionalcharacterization of a family of class I ubiquitin-conjugating enzyme(E2)genesincotton(Gossypium)[J].BBA-GeneStructureand Expression, 2003, 1 625(3):269-279.
[6]Xu L, Ménard R, Berr A, et al. The E2 ubiquitin-conjugating enzymes,At UBC1andAt UBC2,playredundantrolesand are involved in activation of FLC expression and repression offloweringinArabidopsisthaliana[J].ThePlantJournal,2009, 57(2):279-288.
[7]UnverT,TurktasM,BudakH.Inplantaevidenceforthe involvementofaubiquitinconjugatingenzyme(UBCE2clade)innegativeregulationofdiseaseresistance[J].Plant Molecular Biology Reporter, 2013, 31(2):323-334.
[8]覃碧.巴西橡胶树HbUBC22基因克隆与表达分析[J].中国农学通报, 2013, 29(19):1-8.
[9]徐晨曦,姜静,刘甜甜,等.柽柳泛素结合酶基因(E2s)的序列分析及功能验证[J].东北林业大学学报,2007,35(11):1-4.
[10]赵瑞丽,钟凤林,林义章,等.小白菜BcUBCE2基因的克隆及表达分析[J].西北植物学报, 2014, 34(1):60-65.
[11]刘炎霖,陈钰辉,刘富中,等.水茄泛素结合酶E2基因St UBCc的克隆及黄萎病菌诱导表达分析[J].园艺学报,2015, 42(6):1 185-1 194.
[12]尹丽娟,陈阳,刘沛,等.小麦泛素结合酶Ta E2的表达分析及蛋白互作[J].植物遗传资源学报,2014,15(1):144-152.
[13]朱家红,徐靖,畅文军,等.巴西橡胶树泛素结合酶基因Hb UBC5的克隆和表达分析[J].热带作物学报,2014,35(9):1 710-1 714.
[14]蒋忠荣,申飞,刘志,等.津田芜菁泛素结合酶BrUBC11基因的克隆及表达分析[J].华北农学报,2015,30(1):97-102.
[15]蔡佳文,金晓霞,于丽杰,等.龙葵E2泛素结合酶基因SorUBC克隆及表达特性分析[J].东北农业大学学报,2016, 47(11):26-36.
[16]安红强,范静,梁易,等.铁皮石斛泛素结合酶基因DoUBC24的克隆及表达分析[J].生物技术通讯,2016,27(5):643-648.
[17]决登伟,桑雪莲,舒波,等.玉米泛素结合酶基因ZmUBC-76的功能分析[J].热带作物学报,2017,38(8):1 507-1 511.
[18]Dreher K,CallisJ.Ubiquitin,hormonesandbioticstressin plants[J]. Annals of Botany, 2007, 99(5):787-822.
[19]Cui F, Liu L, Zhao Q, et al. Arabidopsis ubiquitin conjugase UBC32 is an ERAD componentthat functions in brassinosteroid-mediatedsaltstresstolerance[J].PlantCell,2012,24(1):233-244.
[20]Shi S Q, Shi Z, Jiang Z P, et al. Effects of exogenous GABA ongeneexpressionofCaraganaintermediarootsunder NaClstress:regulatoryrolesfor H2O2and ethyleneproduction[J]. Plant, Cell&Environment, 2010, 33(2):149-162.
[21]GlickmanMH,CiechanoverA.Theubiquitin-proteasome proteolyticpathway:destructionforthesakeofconstruction[J]. Physiological Reviews, 2002, 82(2):373-428.
[22]WanX,MoA,LiuS,etal.Constitutiveexpressionofa peanutubiquitin-conjugatingenzymegeneinArabidopsis confersimprovedwater-stresstolerancethroughregulation ofstress-responsivegeneexpression[J].JournalofBioscience&Bioengineering, 2011, 111(4):478-484.
[23]王安邦,金志强,刘菊华,等.香蕉泛素结合酶基因MaUCE2在非生物胁迫下的表达分析[J].生物技术通报,2013(5):77-80.