‘凤丹白’牡丹中参与受精过程的PobHLH基因的克隆与表达
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摘要
以油用牡丹‘凤丹白’为试材,基于已构建的雌蕊转录组数据库,筛选出与双受精相关的bHLH基因同源性高的Unigene序列,命名为PobHLH。其开放阅读框(ORF) 1 338 bp,推测编码一个含有445个氨基酸的蛋白。序列分析表明, PobHLH编码的氨基酸序列中包含已确定的保守基序,包括HLH结构域、APB基序和核定位信号NLS。氨基酸序列比对发现PobHLH与拟南芥bHLH家族VII(a+b)亚组的AtUNE10相似度最高,其次是同亚组的AtSPT和AtALC,可能参与双受精中雌配子体的发育。荧光定量PCR分析表明PobHLH在授粉后24 h表达增量最高,在授粉后48 h表达丰度最大,与‘凤丹白’双受精时间进程相吻合,暗示PobHLH可能在双受精过程中起关键作用。
An unigene sequence shared high homology with bHLH putatively involved in plant double fertilization were obtained from previous-constructed oil tree peony(Paeonia ostii ‘Fengdanbai’) gynoecium transcriptome database and named as PobHLH. PobHLH contains a 1 338 bp-long ORF, putatively encoding 445 amino acid residues with typical bHLH domains. The putative amino acid sequence encoded by PobHLH contained conserved motifs, including HLH domain, APB motifs and NLS. By amino acid sequence comparison, it showed that PobHLH shared the highest similarity with that of AtUNE10 belonging to bHLH subgroup of VII(a+b) in Arabidopsis thaliana, followed by AtSPT and AtALC, which were confirmed to regulate the development of female gametophyte during double fertilization. qRT-PCR analysis indicated that PobHLH showed the highest increment appeared after fertilization for 24 hours, while, the highest abundance was detected after fertilization for 48 hours, which was consistent with process of the double fertilization, which implied its putative function in double fertilization.
An unigene sequence shared high homology with bHLH putatively involved in plant double fertilization were obtained from previous-constructed oil tree peony(Paeonia ostii ‘Fengdanbai’) gynoecium transcriptome database and named as PobHLH. PobHLH contains a 1 338 bp-long ORF, putatively encoding 445 amino acid residues with typical bHLH domains. The putative amino acid sequence encoded by PobHLH contained conserved motifs, including HLH domain, APB motifs and NLS. By amino acid sequence comparison, it showed that PobHLH shared the highest similarity with that of AtUNE10 belonging to bHLH subgroup of VII(a+b) in Arabidopsis thaliana, followed by AtSPT and AtALC, which were confirmed to regulate the development of female gametophyte during double fertilization. qRT-PCR analysis indicated that PobHLH showed the highest increment appeared after fertilization for 24 hours, while, the highest abundance was detected after fertilization for 48 hours, which was consistent with process of the double fertilization, which implied its putative function in double fertilization.
引文
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Nakata M,Mitsuda N,Herde M,et al(2013).A bHLH-type transcription factor,ABA-INDUCIBLE BHLH-TYPETRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1,acts as a repressor to negatively regulate jasmonate signaling in Arabidopsis.Plant Cell,25:1641-1656
Pagnussat GC,Yu HJ,Ngo QA,et al(2005).Genetic and molecular identification of genes required for female gametophyte development and function in Arabidopsis.Development,132:603-614
Penfield S,Josse EM,Kannangara R,et al(2005).Cold and light control seed germination through the bHLH transcription factor SPATULA.Curr Biol,15:1998-2006
Pires N,Dolan L(2010).Origin and diversification of basic-helix-loop-helix proteins in plants.Mol Biol Evol,27:862-874
Rajani S,Sundaresan V(2001).The Arabidopsis myc/bHLHgene ALCATRAZ enables cell separation in fruit dehiscence.Curr Biol,11:1914-1922
Wang T,Liang L,Xue Y,et al(2016).A receptor heteromer mediates the male perception of female attractants in plants.Nature,531:241-244
Zhao Q,Ren YR,Wang QJ,et al(2016).Ubiquitination-related MdBT scaffold proteins target a bHLH transcription factor for iron homeostasis.Plant Physiol,172:1973-1988
Zhu E,You C,Wang S,et al(2015).The DYT1-interacting proteins bHLH010,bHLH089 and bHLH091 are redundantly required for Arabidopsis anther development and transcriptome.Plant J,83:976-990
Bailey PC,Martin C,Toledo-Ortiz G,et al(2003).Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana.Plant Cell,15:2497-2502
Castillon A,Shen H,Huq E(2007).Phytochrome Interacting Factors:central players in phytochrome-mediated light signaling networks.Trends Plant Sci,12:514-521
Chinnusamy V,Ohta M,Kanrar S,et al(2003).ICE1:a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.Genes Dev,17:1043-1054
de Lucas M,Daviere J-M,Rodriguez-Falcon M,et al(2008).A molecular framework for light and gibberellin control of cell elongation.Nature,451:480-484
Dong ZL(2010).Studies on the reproductive biology of Paeonia ostii‘Feng Dan’(dissertation).Beijing:Beijing Forest University(in Chinese with English abstract)[董兆磊(2010).‘凤丹’(Paeonia ostii‘Feng Dan’)生殖生物学的初步研究(学位论文).北京:北京林业大学]
Duek PD,Fankhauser C(2003).HFR1,a putative bHLH transcription factor,mediates both phytochrome A and cryptochrome signaling.Plant J,34:827-836
Groszmann M,Paicu T,Alvarez JP,et al(2011).SPATULAand ALCATRAZ,are partially redundant,functionally diverging bHLH genes required for Arabidopsis gynoecium and fruit development.Plant J,68:816-829
Heisler MG,Atkinson A,Bylstra YH,et al(2001).SPATULA,a gene that controls development of carpel margin tissues in Arabidopsis,encodes a bHLH protein.Development,128:1089-1098
Higashiyama T(2010).Peptide signaling in pollen-pistil interactions.Plant Cell Physiol,51:177-189
Jiang Y,Deyholos MK(2006).Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes.BMC Plant Biol,6:25-44
Ko SS,Li MJ,Lin YJ,et al(2017).Tightly controlled expression of bHLH142 is essential for timely tapetal programmed cell death and pollen development in rice.Front Plant Sci,8:1258-1273
Koini MA,Alvey L,Allen T,et al(2009).High temperature-mediated adaptations in plant architecture require the bHLH transcription factor PIF4.Curr Biol,19:408-413
Leivar P,Monte E,Al-Sady B,et al(2008).The Arabidopsis phytochrome-interacting factor PIF7,together with PIF3and PIF4,regulates responses to prolonged red light by modulating phyB levels.Plant Cell,20:337-352
Li N,Zhang DS,Liu HS,et al(2006a).The rice tapetum degeneration retardation gene is required for tapetum degradation and anther development.Plant Cell,18:2999-3014
Li X,Duan X,Jiang H,et al(2006b).Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis.Plant Physiol,141:1167-1184
Liu T,Li Y,Zhang C,et al(2014).Basic helix-loop-helix transcription factor BcbHLHpol functions as a positive regulator of pollen development in non-heading Chinese cabbage.Funct Integr Genomics,14:731-739
Nakata M,Mitsuda N,Herde M,et al(2013).A bHLH-type transcription factor,ABA-INDUCIBLE BHLH-TYPETRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1,acts as a repressor to negatively regulate jasmonate signaling in Arabidopsis.Plant Cell,25:1641-1656
Pagnussat GC,Yu HJ,Ngo QA,et al(2005).Genetic and molecular identification of genes required for female gametophyte development and function in Arabidopsis.Development,132:603-614
Penfield S,Josse EM,Kannangara R,et al(2005).Cold and light control seed germination through the bHLH transcription factor SPATULA.Curr Biol,15:1998-2006
Pires N,Dolan L(2010).Origin and diversification of basic-helix-loop-helix proteins in plants.Mol Biol Evol,27:862-874
Rajani S,Sundaresan V(2001).The Arabidopsis myc/bHLHgene ALCATRAZ enables cell separation in fruit dehiscence.Curr Biol,11:1914-1922
Wang T,Liang L,Xue Y,et al(2016).A receptor heteromer mediates the male perception of female attractants in plants.Nature,531:241-244
Zhao Q,Ren YR,Wang QJ,et al(2016).Ubiquitination-related MdBT scaffold proteins target a bHLH transcription factor for iron homeostasis.Plant Physiol,172:1973-1988
Zhu E,You C,Wang S,et al(2015).The DYT1-interacting proteins bHLH010,bHLH089 and bHLH091 are redundantly required for Arabidopsis anther development and transcriptome.Plant J,83:976-990