白桦BpGT14基因启动子克隆及表达活性分析
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摘要
本文利用Site Finding-PCR方法克隆了白桦BpGT14基因起始密码子ATG上游2 169 bp序列,并通过PLACE启动子预测工具对其进行元件分析。结果表明,该启动子片段含有启动子核心元件及多种逆境及激素响应元件,同时具有植物苯丙烷及木质素生物合成的MYB类转录因子的重要结合基序。研究选取了其中含有启动子核心元件的1 156 bp片段构建了pBpGT14∷GUS植物表达载体,利用农杆菌侵染的方法将pBpGT14∷GUS报告基因瞬时转化烟草植株,鉴定该启动子在烟草中的表达活性及对非生物胁迫和激素的响应模式。对转基因烟草植株进行GUS染色,结果表明该启动子具有启动活性,且在茎段处活性较高;进一步分析非生物胁迫对烟草中GUS酶活性的影响,表明该启动子对ABA、NaCl、PEG及高温处理均有明显响应,且对于NaCl及PEG处理响应迅速。为了更好的鉴定白桦BpGT14基因启动子在白桦细胞中的启动活性及响应模式,本文构建了pBpGT14∷GFP载体并瞬时转化白桦茎段悬浮细胞,进行研究。GFP转录水平分析结果与GUS酶活性结果基本一致,但其中部分时间点仍存在差异。选取PEG处理3、6、12及24 h的转GFP基因白桦茎段悬浮细胞,在显微镜下观察其绿色荧光蛋白,以此揭示该启动子对干旱的响应模式。结果表明,该启动子在白桦茎段悬浮细胞中启动了GFP的表达,在处理初期(3 h),荧光效果明显;随着处理时间的增加,细胞脱水明显,且在细胞壁表现高亮度荧光。
We cloned a 2 169 bp promoter sequence of BpGT14 gene from birch genomic DNA using the method of Site Finding-PCR. The promoter sequence was analyzed by PLACE,and the result showed that this fragment contained promoter core elements and some elements which can respond to abiotic stress and hormones. Meanwhile, two important MYB transcription factor binding elements were found which regulate phenylpropanoid and lignin biosynthesis. To study the promoter activity,a 1 156 bp fragment was chosen to construct p BpGT14∷GUS plant expression vector and transformed into tobacco. GUS staining proved that the promoter had high activity in stem segments. When the tobacco was treated with GA and H2O2 at 4 ℃,the promoter had no significant response and the enzyme activity had a downward trend. In contrast,the promoter activity was significantly increased by ABA,Na Cl,PEG and 37 ℃ treatment.Further transformation of birch cells using p BpGT14 ∷ GFP plant expression vector indicated that the promoter had a similar response pattern to that of tobacco treated with abiotic stress and hormone except for a few time points. As the promoter was significantly and quickly responsive to drought stress,we have observed the GFP fluorescence protein in birch stem segments suspension cells transformed by GFP for 3,6,12 and 24 h with PEG treatment. The results showed that BpGT14 promoter had activity in birch stem segments suspension cells and fluorescence can be observed in the suspension cells,especially in cell walls. Successful implementation of this study has important significance for analysis of gene regulation and function. Meanwhile,it provides a theoretical basis for gene promoter function studies of other woody plants.
We cloned a 2 169 bp promoter sequence of BpGT14 gene from birch genomic DNA using the method of Site Finding-PCR. The promoter sequence was analyzed by PLACE,and the result showed that this fragment contained promoter core elements and some elements which can respond to abiotic stress and hormones. Meanwhile, two important MYB transcription factor binding elements were found which regulate phenylpropanoid and lignin biosynthesis. To study the promoter activity,a 1 156 bp fragment was chosen to construct p BpGT14∷GUS plant expression vector and transformed into tobacco. GUS staining proved that the promoter had high activity in stem segments. When the tobacco was treated with GA and H2O2 at 4 ℃,the promoter had no significant response and the enzyme activity had a downward trend. In contrast,the promoter activity was significantly increased by ABA,Na Cl,PEG and 37 ℃ treatment.Further transformation of birch cells using p BpGT14 ∷ GFP plant expression vector indicated that the promoter had a similar response pattern to that of tobacco treated with abiotic stress and hormone except for a few time points. As the promoter was significantly and quickly responsive to drought stress,we have observed the GFP fluorescence protein in birch stem segments suspension cells transformed by GFP for 3,6,12 and 24 h with PEG treatment. The results showed that BpGT14 promoter had activity in birch stem segments suspension cells and fluorescence can be observed in the suspension cells,especially in cell walls. Successful implementation of this study has important significance for analysis of gene regulation and function. Meanwhile,it provides a theoretical basis for gene promoter function studies of other woody plants.
引文
[1]PINO M T,SKINNER J S,PARK E J,et al.Use of a stress inducible promoter to drive ectopic At CBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield[J].Plant Biotechnology Journal,2007,5(5):591--604.
[2]BUTLER J E F,KADONAGA J T.The RNA polymerase II core promoter:a key component in the regulation of gene expression[J].Genes&Development,2002,16(20):2583-2592.
[3]尹辉,李丹,张毅,等.植物基因启动子的克隆方法及其应用[J].分子植物育种,2008(增刊1):85-91.YIN H,LI D,ZHANG Y,et al.Cloning methods of plant gene promoters and their applications[J].Molecular Plant Breeding,2008,17(Suppl.1):85-91.
[4]TAN G,GAO Y,SHI M,et al.Site Finding-PCR:a simple and efficient PCR method for chromosome walking[J].Nucleic Acids Research,2005,33(13):122--122.
[5]LIM E K,BOWLES D J.A class of plant glycosyltransferases involved in cellular homeostasis[J].The EMBO Journal,2004,23(15):2915-2922.
[6]DOBLIN M S,PETTOLINO F,BACIC A.Evans review:plant cell walls:the skeleton of the plant world[J].Functional Plant Biology,2010,37(5):357--381.
[7]SADO P E,TESSIER D,VASSEUR M,et al.Integrating genes and phenotype:a wheat-Arabidopsis-rice glycosyltransferase database for candidate gene analyses[J].Functional&Integrative Genomics,2009,9(1):43--58.
[8]KIM I A,HEO J O,CHANG K S,et al.Overexpression and inactivation of UGT73B2 modulate tolerance to oxidative stress in Arabidopsis[J].Journal of Plant Biology,2010,53(3):233-239.
[9]TOGNETTI V B,VAN AKEN O,MORREEL K,et al.Perturbation of indole-3-butyric acid homeostasis by the UDPglucosyltransferase UGT74E2 modulates Arabidopsis architecture and water stress tolerance[J].The Plant Cell Online,2010,22(8):2660-2679.
[10]LIU X,WANG Q,CHEN P,et al.Four novel cellulose synthase(CESA)genes from Birch(Betula platyphylla Suk.)involved in primary and secondary cell wall biosynthesis[J].International Journal of Molecular Sciences,2012,13(10):12195-12212.
[11]詹亚光,曾凡锁.富含多糖的白桦成熟叶片DNA的提取方法[J].东北林业大学学报,2005,33(3):24--25.ZHAN Y G,ZENG F S.A method for DNA extraction from mature birch leaves rich in polysaccharide[J].Journal of Northeast Forestry University,2005,33(3):24--25.
[12]CHEN S,SONGKUMARN P,LIU J,et al.A versatile zero background T-vector system for gene cloning and functional genomics[J].Plant Physiology,2009,150(3):1111-1121.
[13]徐威,朱春宝,朱宝泉,等.利用电转化和三亲杂交方法高效转化根癌农杆菌[J].沈阳医科大学学报,2003,20(3):451--454.XU W,ZHU C B,ZHU B Q,et al.Highly efficient gene transfer in Agrobacterium tumefaciens LBA4404 by tri-parental mating and electroporation[J].Journal of Shenyang Pharmaceutical University,2003,20(3):451-454.
[14]刘志钦.Ca WRKY5启动子分离及其在烟草瞬间表达系统中分析[D].福州:福建农林大学,2010.LIU Z Q.Isolation and expression analysis of the promoter of Ca WRKY5 in tobacco transient expression system[D].Fuzhou:Fujian Agriculture and Forest University,2010.
[15]曾凡锁,钱晶晶,康君,等.转基因白桦中GUS基因表达的定量分析[J].植物学报,2009,44(4):484-490.ZENG F S,QIAN J J,KANG J,et al.Histochemical study ofβ-glucuronidase activity in transgenic birch[J].Chinese Bulletin of Botany,2009,44(4):484--490.
[16]ZENG F S,ZHAN Y G,ZHAO H C,et al.Molecular characterization of T-DNA integration sites in transgenic birch[J].Trees,2010,24(4):753--762.
[17]TAMAGNONE L,MERIDA A,PARR A,et al.The Am MYB308and Am MYB330 transcription factors from Antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco[J].The Plant Cell,1998,10(2):135-154.
[18]HENRISSAT B,DAVIES G J.Glycoside hydrolases and glycosyltransferases.Families,modules,and implications for genomics[J].Plant Physiology,2000,124(4):1515--1519.
[19]李燕.紫花苜蓿诱导表达启动子Ms ZPP的克隆及功能分析[D].北京:中国农业科学院,2012.LI Y.Cloning and analysis of an inducement-responsive promoter Ms ZPP of Medicago sativa L[D].Beijing:Chinese Academy of Agricultural Sciences,2012.
[20]ONO A,IZAWA T,CHUA N H,et al.The rab16B promoter of rice contains two distinct abscisic acid-responsive elements[J].Plant Physiology,1996,112(2):483--491.
[21]TAYLOR-TEEPLES M,LIN L,DE LUCAS M,et al.An Arabidopsis gene regulatory network for secondary cell wall synthesis[J].Nature,2015,517:571--575.
[22]裴惠娟,张满效,安黎哲.非生物胁迫下植物细胞壁组分变化[J].生态学杂志,2011,30(6):1279-1286.PEI H J,ZHANG M X,AN L Z.Changes of plant cell wall components under abiotic stresses[J].Chinese Journal of Ecology,2011,30(6):1279--1286.
[2]BUTLER J E F,KADONAGA J T.The RNA polymerase II core promoter:a key component in the regulation of gene expression[J].Genes&Development,2002,16(20):2583-2592.
[3]尹辉,李丹,张毅,等.植物基因启动子的克隆方法及其应用[J].分子植物育种,2008(增刊1):85-91.YIN H,LI D,ZHANG Y,et al.Cloning methods of plant gene promoters and their applications[J].Molecular Plant Breeding,2008,17(Suppl.1):85-91.
[4]TAN G,GAO Y,SHI M,et al.Site Finding-PCR:a simple and efficient PCR method for chromosome walking[J].Nucleic Acids Research,2005,33(13):122--122.
[5]LIM E K,BOWLES D J.A class of plant glycosyltransferases involved in cellular homeostasis[J].The EMBO Journal,2004,23(15):2915-2922.
[6]DOBLIN M S,PETTOLINO F,BACIC A.Evans review:plant cell walls:the skeleton of the plant world[J].Functional Plant Biology,2010,37(5):357--381.
[7]SADO P E,TESSIER D,VASSEUR M,et al.Integrating genes and phenotype:a wheat-Arabidopsis-rice glycosyltransferase database for candidate gene analyses[J].Functional&Integrative Genomics,2009,9(1):43--58.
[8]KIM I A,HEO J O,CHANG K S,et al.Overexpression and inactivation of UGT73B2 modulate tolerance to oxidative stress in Arabidopsis[J].Journal of Plant Biology,2010,53(3):233-239.
[9]TOGNETTI V B,VAN AKEN O,MORREEL K,et al.Perturbation of indole-3-butyric acid homeostasis by the UDPglucosyltransferase UGT74E2 modulates Arabidopsis architecture and water stress tolerance[J].The Plant Cell Online,2010,22(8):2660-2679.
[10]LIU X,WANG Q,CHEN P,et al.Four novel cellulose synthase(CESA)genes from Birch(Betula platyphylla Suk.)involved in primary and secondary cell wall biosynthesis[J].International Journal of Molecular Sciences,2012,13(10):12195-12212.
[11]詹亚光,曾凡锁.富含多糖的白桦成熟叶片DNA的提取方法[J].东北林业大学学报,2005,33(3):24--25.ZHAN Y G,ZENG F S.A method for DNA extraction from mature birch leaves rich in polysaccharide[J].Journal of Northeast Forestry University,2005,33(3):24--25.
[12]CHEN S,SONGKUMARN P,LIU J,et al.A versatile zero background T-vector system for gene cloning and functional genomics[J].Plant Physiology,2009,150(3):1111-1121.
[13]徐威,朱春宝,朱宝泉,等.利用电转化和三亲杂交方法高效转化根癌农杆菌[J].沈阳医科大学学报,2003,20(3):451--454.XU W,ZHU C B,ZHU B Q,et al.Highly efficient gene transfer in Agrobacterium tumefaciens LBA4404 by tri-parental mating and electroporation[J].Journal of Shenyang Pharmaceutical University,2003,20(3):451-454.
[14]刘志钦.Ca WRKY5启动子分离及其在烟草瞬间表达系统中分析[D].福州:福建农林大学,2010.LIU Z Q.Isolation and expression analysis of the promoter of Ca WRKY5 in tobacco transient expression system[D].Fuzhou:Fujian Agriculture and Forest University,2010.
[15]曾凡锁,钱晶晶,康君,等.转基因白桦中GUS基因表达的定量分析[J].植物学报,2009,44(4):484-490.ZENG F S,QIAN J J,KANG J,et al.Histochemical study ofβ-glucuronidase activity in transgenic birch[J].Chinese Bulletin of Botany,2009,44(4):484--490.
[16]ZENG F S,ZHAN Y G,ZHAO H C,et al.Molecular characterization of T-DNA integration sites in transgenic birch[J].Trees,2010,24(4):753--762.
[17]TAMAGNONE L,MERIDA A,PARR A,et al.The Am MYB308and Am MYB330 transcription factors from Antirrhinum regulate phenylpropanoid and lignin biosynthesis in transgenic tobacco[J].The Plant Cell,1998,10(2):135-154.
[18]HENRISSAT B,DAVIES G J.Glycoside hydrolases and glycosyltransferases.Families,modules,and implications for genomics[J].Plant Physiology,2000,124(4):1515--1519.
[19]李燕.紫花苜蓿诱导表达启动子Ms ZPP的克隆及功能分析[D].北京:中国农业科学院,2012.LI Y.Cloning and analysis of an inducement-responsive promoter Ms ZPP of Medicago sativa L[D].Beijing:Chinese Academy of Agricultural Sciences,2012.
[20]ONO A,IZAWA T,CHUA N H,et al.The rab16B promoter of rice contains two distinct abscisic acid-responsive elements[J].Plant Physiology,1996,112(2):483--491.
[21]TAYLOR-TEEPLES M,LIN L,DE LUCAS M,et al.An Arabidopsis gene regulatory network for secondary cell wall synthesis[J].Nature,2015,517:571--575.
[22]裴惠娟,张满效,安黎哲.非生物胁迫下植物细胞壁组分变化[J].生态学杂志,2011,30(6):1279-1286.PEI H J,ZHANG M X,AN L Z.Changes of plant cell wall components under abiotic stresses[J].Chinese Journal of Ecology,2011,30(6):1279--1286.