PtrDREB28转录因子的克隆及抗逆性
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摘要
DREB/CBF转录因子响应植物干旱、高盐和低温等非生物胁迫应答是通过特异结合DRE/CRT顺式作用元件。利用RT-PCR技术从毛果杨克隆PtrDREB28转录因子基因,用基因枪法对其进行亚细胞定位分析,结果表明其定位在细胞核中;利用农杆菌介导法对野生型大青杨进行遗传转化,并对转基因株系进行抗逆性分析,结果显示经高盐胁迫处理的转基因株系的耐受性明显高于野生型植株;通过测定和比较经盐胁迫处理后转基因株系和野生型植株的丙二醛质量摩尔浓度和相对电导率,发现转基因株系的丙二醛质量摩尔浓度和相对电导率均显著低于野生型植株,表明PtrDREB28基因的过表达可以显著提高杨树耐盐性,由此推测PtrDREB28转录因子可能参与杨树逆境胁迫应答过程。
DREB/CBF transcription factors can respond to abiotic stresses,such as drought,high salinity and low temperature,through specifically combined with DRE/CRT cis acting elements. A Ptr DREB28 transcription factor gene was cloned from Populus trichocarpa by RT-PCR. The analysis of subcellular localization by particle bombardment showed that Ptr DREB28 was in the nucleus. To conduct genetic transformation in P. ussuriensis by agrobacterium mediated genetic transformation and the transgenic plants were treated with stress resistance analysis to text resistance. The tolerance of transgenic plants treated with high salt stress was significantly higher than that of wild type. Through the determination and comparison with the content of malondialdehyde and relative conductivity of transgenic plants and wild type plants after salt stress treatment,the content of malondialdehyde and relative conductivity of transgenic plants were significantly lower than that of wild plant,and it is inferred that the functions of Ptr DREB28 transcription factor may be related to stress responses in Populus.
DREB/CBF transcription factors can respond to abiotic stresses,such as drought,high salinity and low temperature,through specifically combined with DRE/CRT cis acting elements. A Ptr DREB28 transcription factor gene was cloned from Populus trichocarpa by RT-PCR. The analysis of subcellular localization by particle bombardment showed that Ptr DREB28 was in the nucleus. To conduct genetic transformation in P. ussuriensis by agrobacterium mediated genetic transformation and the transgenic plants were treated with stress resistance analysis to text resistance. The tolerance of transgenic plants treated with high salt stress was significantly higher than that of wild type. Through the determination and comparison with the content of malondialdehyde and relative conductivity of transgenic plants and wild type plants after salt stress treatment,the content of malondialdehyde and relative conductivity of transgenic plants were significantly lower than that of wild plant,and it is inferred that the functions of Ptr DREB28 transcription factor may be related to stress responses in Populus.
引文
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[11]GILMOUR S J,ZARKA D G,STOCKINGER E J,et al.Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression[J].Plant J,1998,16(4):433-442.
[12]梁欣欣.利用DREB转录因子改良小麦耐盐性的研究[D].北京:中国农业科学研究院,2004.
[13]周伟,吴宪,贾承国,等.山葡萄转录因子基因Va DREB的克隆及其抗逆功能分析[J].植物生理学报,2014,50(10):1563-1573.
[14]LIU Q,KASUGA M,SAKUMA Y,et al.Two transcription factors,DREB1 and DREB2,with EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression,respectively,in Arabidopsis[J].Plant Cell,1998,10(8):1391-1406.
[15]DUBOUZET J G,SAKUMA Y,ITO Y,et al.Os DREB genes in rice,Oryza sativa L.,encode transcription activators that function in drought-,high-salt-and cold-responsive gene expression[J].Plant J,2003,21(4):751-763.
[2]陈金焕,夏新莉,尹伟伦.植物DREB转录因子及其转基因研究进展[J].分子植物育种,2007,5(6):29-35.
[3]LI X,ZHANG D,GAO B,et al.Transcriptome-wide identification,classification,and characterization of AP2/ERF family genes in the desert moss Syntrichia caninervis[J].Front Plant Sci,2017,8(47):262-268.
[4]ALLEN M D,YAMASAKI K,OHME-TAKAGI M,et al.A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[J].EMBO J,1998,17(18):5484-5496.
[5]AGARWAL P K,GUPTA K,LOPATO S,et al.Dehydration responsive element binding transcription factors and their applications for the engineering of stress tolerance[J].J Exp Bot,2017,68(9):2135-2148.
[6]李志兰,杨敏生,王进茂,等.杨树基因工程育种研究进展[J].河北农业大学学报,2002,25(z1):145-148.
[7]NOVILLO F,ALONSO J M,ECKER J R,et al.CBF2/DREB1C is a negative regulator of CBF1/DREB1B and CBF3/DREB1A expression and plays a central role in stress tolerance in Arabidopsis[J].Proc Natl Acad Sci U S A,2004,101(11):3985-3990.
[8]DHINDSA R S,PLUMB-DHINSA P,THORPE T A.Leaf senecence:corelated with increased leaves of membrane permeability and lipid per oxidation,and decreased levels of superoxide dismutase and catalase[J].J Exp Bot,1981,36(4):93-101.
[9]李小双,梁玉青,高贝,等.植物抗逆相关转录因子基因DREB的研究展望[J].分子植物育种,2017,15(7):2612-2622.
[10]OSAKI Y,KODAMA Y.Particle bombardment and subcellular protein localization analysis in the aquatic plant Egeria densa[J].Peer J,2017,5(9):37-43.
[11]GILMOUR S J,ZARKA D G,STOCKINGER E J,et al.Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression[J].Plant J,1998,16(4):433-442.
[12]梁欣欣.利用DREB转录因子改良小麦耐盐性的研究[D].北京:中国农业科学研究院,2004.
[13]周伟,吴宪,贾承国,等.山葡萄转录因子基因Va DREB的克隆及其抗逆功能分析[J].植物生理学报,2014,50(10):1563-1573.
[14]LIU Q,KASUGA M,SAKUMA Y,et al.Two transcription factors,DREB1 and DREB2,with EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression,respectively,in Arabidopsis[J].Plant Cell,1998,10(8):1391-1406.
[15]DUBOUZET J G,SAKUMA Y,ITO Y,et al.Os DREB genes in rice,Oryza sativa L.,encode transcription activators that function in drought-,high-salt-and cold-responsive gene expression[J].Plant J,2003,21(4):751-763.