白桦BpSPL2基因启动子的克隆及表达分析
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摘要
SPL(SQUAMOSA promoter-binding protein-like)是植物特有的转录因子,研究表明其在参与发育阶段转变、花和果实发育等方面起着重要作用。利用PCR技术从白桦基因组DNA中扩增获得BpSPL2基因上游1 960 bp启动子序列,使用PLACE和Plant CARE在线软件分析序列,发现BpSPL2基因启动子序列中含有与开花、非生物胁迫及激素响应等相关的顺式作用元件,暗示其在植物的生长发育和胁迫应答中起重要作用。进而构建了BpSPL2基因启动子驱动GUS报告基因的植物表达载体,并利用农杆菌介导将其瞬时转化至白桦和拟南芥,通过GUS组织化学染色检测BpSPL2基因启动子的组织表达特性,结果表明BpSPL2基因启动子具有启动子活性,能够驱动GUS基因在白桦和拟南芥中表达;而其表达活性在白桦的叶片、芽及根部中较强,在拟南芥的花药、雌蕊和叶片较强,为进一步研究白桦BpSPL2基因的表达调控及其功能分析提供参考。
The SPL(SQUAMOSA promoter-binding protein-like) is a plant specific transcription factor,and the study showed that it plays an important role in the transformation of developmental stage,flower and fruit development.A 1 960 bp promoter sequence of BpSPL2 gene was cloned from Betula platyphylla genomic DNA using the method of PCR.The cis-regulatory elements were analyzed by PLACE and PlantCARE web tools.Multiple flowering elements,abiotic stress response elements and hormone-responsive elements were predicted in the promoter region.It indicated that it played an important role in plant growth and development and stress response.Further,the BpSPL2 promoter was inserted to pBI121 vector under control of the 35 S promoter to generate the pBI121-BpSPL2 promoter::GUS recombinant construct,which was transient expressed in B.platyphylla and Arabidopsis thaliana seedlings by Agrobacterium tumefaciens mediated method,then investigated the expression pattern via histechemical GUS staining.BpSPL2 promoter could drive the expression of GUS gene in B.platyphylla and A.thaliana,and expressed in high level in leaves,buds,and roots of B.platyphylla while in high level in anthers,pistils,and leaves of A.thaliana.This study will provide a reference for further studies on the expression regulation and functional analysis of BpSPL2 gene in B.platyphylla.
The SPL(SQUAMOSA promoter-binding protein-like) is a plant specific transcription factor,and the study showed that it plays an important role in the transformation of developmental stage,flower and fruit development.A 1 960 bp promoter sequence of BpSPL2 gene was cloned from Betula platyphylla genomic DNA using the method of PCR.The cis-regulatory elements were analyzed by PLACE and PlantCARE web tools.Multiple flowering elements,abiotic stress response elements and hormone-responsive elements were predicted in the promoter region.It indicated that it played an important role in plant growth and development and stress response.Further,the BpSPL2 promoter was inserted to pBI121 vector under control of the 35 S promoter to generate the pBI121-BpSPL2 promoter::GUS recombinant construct,which was transient expressed in B.platyphylla and Arabidopsis thaliana seedlings by Agrobacterium tumefaciens mediated method,then investigated the expression pattern via histechemical GUS staining.BpSPL2 promoter could drive the expression of GUS gene in B.platyphylla and A.thaliana,and expressed in high level in leaves,buds,and roots of B.platyphylla while in high level in anthers,pistils,and leaves of A.thaliana.This study will provide a reference for further studies on the expression regulation and functional analysis of BpSPL2 gene in B.platyphylla.
引文
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6.Zhang Y,Schwarz S,Saedler H,et al.SPL8,a local regulator in a subset of gibberellin-mediated developmental processes in Arabidopsis[J].Plant Molecular Biology,2007,63(3):429-439.
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12.Gao R M,Gruber M Y,Amyot L,et al.SPL13 regulates shoot branching and flowering time in Medicagosativa[J].Plant Molecular Biology,2018,96(1-2):119-133.
13.Yu N,Niu Q W,Ng K H,et al.The role of mi R156/SPLs modules in Arabidopsis lateral root development[J].The Plant Journal,2015,83(4):673-685.
14.LnnenpM,Jn9nen I,H9ltt-Vuori M,et al.A new SBP-box gene Bp SPL1 in silver birch(Betula pendula)[J].Physiologia Plantarum,2004,120(3):491-500.
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16.Wei H,Meilan R,Brunner A M,et al.Transgenic sterility in Populus:expression properties of the poplar PTLF,Agrobacterium NOS and two minimal 35S promoters in vegetative tissues[J].Tree Physiology,2006,26(4):401-410.
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21.官民晓,刘雪梅,张妍,等.白桦SPL8转录因子基因的分离及转录表达分析[J].南京林业大学学报:自然科学版,2013,37(3):17-22.Guan M X,Liu X M,Zhang Y,et al.Isolation and transcription expression analysis of SPL8 transcription factors gene of Betula platyphylla[J].Journal of Nanjing Forestry University:Natural Science Edition,2013,37(3):17-22.
22.郭勇,王玉成,王智博.一种基于农杆菌介导的拟南芥瞬时转化技术优化[J].东北林业大学学报,2016,44(6):41-44,83.Guo Y,Wang Y C,Wang Z B.Optimizing transient genetic transformation method on Arabidopsis plants mediated by Agrobacterium tumefaciens[J].Journal of Northeast Forestry University,2016,44(6):41-44,83.
23.李萌,哈努拉·塔斯肯,陈卓,等.白桦4CL基因启动子克隆及表达分析[J].中国农学通报,2017,33(5):29-34.Li M,Tasiken H,Chen Z,et al.Cloning and expression analysis of 4CL gene promoter of Betula platyphylla[J].Chinese Agricultural Science Bulletin,2017,33(5):29-34.
24.Padmanabhan M S,Ma S S,Burch-Smith T M,et al.Novel positive regulatory role for the SPL6 transcription factor in the N TIR-NB-LRR receptor-mediated plant innate immunity[J].PLo S Pathogens,2013,9(3):e1003235.
25.Xing S P,Salinas M,H9hmann S,et al.mi R156-targeted and nontargeted SBP-box transcription factors act in concert to secure male fertility in Arabidopsis[J].The Plant Cell,2010,22(12):3935-3950.
26.Li X Y,Lin E P,Huang H H,et al.Molecular characterization of SQUAMOSA PROMOTERBINDING PROTEIN-LIKE(SPL)gene family in Betula luminifera[J].Frontiers in Plant Science,2018,9:608.
27.杨洋,王子佳,李慧玉,等.白桦Bp MADS12基因启动子的克隆及表达分析[J].西南林业大学学报,2016,36(1):9-15.Yang Y,Wang Z J,Li H Y,et al.Cloning and expression analysis of Bp MADS12 promoter from Betula platyphylla[J].Journalof Southwest Forestry University,2016,36(1):9-15.
28.王遂.欧洲白桦Bp CUC2基因及其启动子的克隆与功能研究[D].哈尔滨:东北林业大学,2015.Wang S.Isolation and functional analysis of the Bp CUC2gene and its promoter in Betula pendula[D].Harbin:Northeast Forestry University,2015.
29.Arshad M,Feyissa B A,Amyot L,et al.MicroRNA156 improves drought stress tolerance in alfalfa(Medicago sativa)by silencing SPL13[J].Plant Science,2017,258:122-136.
2.Usami T,Horiguchi G,Yano S,et al.The more and smaller cells mutants of Arabidopsis thaliana identify novel roles for SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes in the control of heteroblasty[J].Development(Cambridge,England),2009,136(6):955-964.
3.Manning K,T9r M,Poole M,et al.A naturally occurring epigenetic mutation in a gene encoding an SBP-box transcription factor inhibits tomato fruit ripening[J].Nature Genetics,2006,38(8):948-952.
4.Jiao Y Q,Wang Y H,Xue D W,et al.Regulation of Os S-PL14 by Osmi R156 defines ideal plant architecture in rice[J].Nature Genetics,2010,42(6):541-544.
5.Unte U S,Sorensen A M,Pesaresi P,et al.SPL8,an SBP-box gene that affects pollen sac development in Arabidopsis[J].The Plant Cell,2003,15(4):1009-1019.
6.Zhang Y,Schwarz S,Saedler H,et al.SPL8,a local regulator in a subset of gibberellin-mediated developmental processes in Arabidopsis[J].Plant Molecular Biology,2007,63(3):429-439.
7.Becraft P W,Bongard-Pierce D K,Sylvester A W,et al.The liguleless-1 gene acts tissue specifically in maize leaf development[J].Developmental Biology,1990,141(1):220-232.
8.Wang Y,Hu Z L,Yang Y X,et al.Function annotation of an SBP-box gene in Arabidopsis based on analysis of co-expression networks and promoters[J].International Journal of Molecular Sciences,2009,10(1):116-132.
9.Wu G,Poethig R S.Temporal regulation of shoot development in Arabidopsis thaliana by mir156 and its target SPL3[J].Development(Cambridge,England),2006,133(18):3539-3547.
10.Yamaguchi A,Wu M F,Yang L,et al.The Micro RNA-regulated SBP-Box transcription factor SPL3 is a direct upstream activator of LEAFY,FRUITFULL and APETALA1[J].Developmental Cell,2009,17(2):268-278.
11.Stone J M,Liang X W,Nekl E R,et al.Arabidopsis AtSPL14,a plant-specific SBP-domain transcription factor,participates in plant development and sensitivity to fumonisin B1[J].The Plant Journal,2005,41(5):744-754.
12.Gao R M,Gruber M Y,Amyot L,et al.SPL13 regulates shoot branching and flowering time in Medicagosativa[J].Plant Molecular Biology,2018,96(1-2):119-133.
13.Yu N,Niu Q W,Ng K H,et al.The role of mi R156/SPLs modules in Arabidopsis lateral root development[J].The Plant Journal,2015,83(4):673-685.
14.LnnenpM,Jn9nen I,H9ltt-Vuori M,et al.A new SBP-box gene Bp SPL1 in silver birch(Betula pendula)[J].Physiologia Plantarum,2004,120(3):491-500.
15.Higo K,Ugawa Y,Iwamoto M,et al.Plant cis-acting regulatory DNA elements(PLACE)database:1999[J].Nucleic Acids Research,1998,27(1):297-300.
16.Wei H,Meilan R,Brunner A M,et al.Transgenic sterility in Populus:expression properties of the poplar PTLF,Agrobacterium NOS and two minimal 35S promoters in vegetative tissues[J].Tree Physiology,2006,26(4):401-410.
17.陈聪.茶树花特异表达启动子---花粉壁蛋白基因启动子表达研究[D].合肥:安徽农业大学,2009.Chen C.Study on Cpcp(pollen coat protein gene)promoter expression of Tea Plant(Camellia sinensis)[D].Hefei:Anhui Agriculture University,2009.
18.郑万钧.中国树木志[M].北京:中国林业出版社,1985.Zheng W J.Records of Chinese Trees[M].Beijing:China Forestry Publishing House,1985.
19.刘雪梅,杨传平.白桦雌雄花发育周期的时序特征[J].林业科学,2006,42(12):28-32.Liu X M,Yang C P.Temporal characteristics of developmental cyclesoffemale andmale flowers in Betula platyphylla in Northeastern China[J].Scientia Silvae Sinicae,2006,42(12):28-32.
20.许思佳,颜斌,董京祥,等.白桦Bp BEE1基因启动子的表达特性分析[J].植物研究,2018,38(2):247-253.Xu S J,Yan B,Dong J X,et al.Analysis of Bp BEE1 gene promoter in Betula platyphylla[J].Bulletin of Botanical Research,2018,38(2):247-253.
21.官民晓,刘雪梅,张妍,等.白桦SPL8转录因子基因的分离及转录表达分析[J].南京林业大学学报:自然科学版,2013,37(3):17-22.Guan M X,Liu X M,Zhang Y,et al.Isolation and transcription expression analysis of SPL8 transcription factors gene of Betula platyphylla[J].Journal of Nanjing Forestry University:Natural Science Edition,2013,37(3):17-22.
22.郭勇,王玉成,王智博.一种基于农杆菌介导的拟南芥瞬时转化技术优化[J].东北林业大学学报,2016,44(6):41-44,83.Guo Y,Wang Y C,Wang Z B.Optimizing transient genetic transformation method on Arabidopsis plants mediated by Agrobacterium tumefaciens[J].Journal of Northeast Forestry University,2016,44(6):41-44,83.
23.李萌,哈努拉·塔斯肯,陈卓,等.白桦4CL基因启动子克隆及表达分析[J].中国农学通报,2017,33(5):29-34.Li M,Tasiken H,Chen Z,et al.Cloning and expression analysis of 4CL gene promoter of Betula platyphylla[J].Chinese Agricultural Science Bulletin,2017,33(5):29-34.
24.Padmanabhan M S,Ma S S,Burch-Smith T M,et al.Novel positive regulatory role for the SPL6 transcription factor in the N TIR-NB-LRR receptor-mediated plant innate immunity[J].PLo S Pathogens,2013,9(3):e1003235.
25.Xing S P,Salinas M,H9hmann S,et al.mi R156-targeted and nontargeted SBP-box transcription factors act in concert to secure male fertility in Arabidopsis[J].The Plant Cell,2010,22(12):3935-3950.
26.Li X Y,Lin E P,Huang H H,et al.Molecular characterization of SQUAMOSA PROMOTERBINDING PROTEIN-LIKE(SPL)gene family in Betula luminifera[J].Frontiers in Plant Science,2018,9:608.
27.杨洋,王子佳,李慧玉,等.白桦Bp MADS12基因启动子的克隆及表达分析[J].西南林业大学学报,2016,36(1):9-15.Yang Y,Wang Z J,Li H Y,et al.Cloning and expression analysis of Bp MADS12 promoter from Betula platyphylla[J].Journalof Southwest Forestry University,2016,36(1):9-15.
28.王遂.欧洲白桦Bp CUC2基因及其启动子的克隆与功能研究[D].哈尔滨:东北林业大学,2015.Wang S.Isolation and functional analysis of the Bp CUC2gene and its promoter in Betula pendula[D].Harbin:Northeast Forestry University,2015.
29.Arshad M,Feyissa B A,Amyot L,et al.MicroRNA156 improves drought stress tolerance in alfalfa(Medicago sativa)by silencing SPL13[J].Plant Science,2017,258:122-136.