葡萄VvERF3b基因和VvPDF1.2基因的启动子功能分析
摘要
在植物体内,逆境相关基因的表达在转录水平上与转录因子与基因的顺式作用元件相互作用密切相关。乙烯响应因子(Ethylene response factors, ERF)作为重要的转录因子可以调控多种植物逆境相关基因的表达。而植物防御素(PDF)基因是植物响应病虫害侵袭的重要相关基因。本文通过对这两个基因启动子的研究,推断两个基因在葡萄抗逆响应途径中的表达模式,对于了解葡萄对逆境的抵抗机理有着重要的意义。
本研究利用PCR扩增获得欧洲葡萄(Vitis vinifera)的ERF3b基因和PDF1.2基因启动子序列,并利用PLACE和PlantCARE软件分析得到的两个基因的启动子序列。在VvERF3b基因启动子序列中发现多种与植物激素相关的顺式作用元件:ABRE元件、ASF1元件、DPBF元件、DRE元件、ERE元件、GCC元件、W-box和TCA元件,而在VvPDF1.2基因启动子内发现多种与植物逆境响应相关的顺式作用元件:W-box、TGA元件、TC-rich元件、TATC元件、ERE元件、SEBF元件和MYBGAHV元件。
按照VvERF3b基因启动子顺式作用元件的分析结果设计了6个上游启动子引物分别和一个下游启动子引物配对,利用PCR扩增获得6条起始密码上游长度不同的启动子片段P1~P6,通过酶切、纯化、连接,将6条VvERF3b基因5’缺失启动子序列代替原载体的8×GCC增强子和35S启动子插入pGCC载体中,与LUC报告基因连接构建6个植物表达载体:VvERF3b-P1~VvERF3b-P6。使用PEG-Ca~(2+)介导法介导重组载体转染拟南芥原生质体,用瞬时表达方法检测LUC表达量,结果表明VvERF3b基因启动子中从-1047bp到-585bp区域对于下游基因的表达有激活的作用,而从-1324bp到-1047bp区域可能存在两个以上对下游基因的表达起抑制作用的基序,用来抑制基因本身的表达。
鉴于目前市场中没有LUC植物表达载体,该文使用PCR扩增获得LUC基因片段,通过酶切、纯化、连接等手段将LUC基因片段代替pBI221载体的GUS基因片段在35S启动子驱动下表达,构建了具有多克隆位点(MCS)的LUC植物表达载体。
The level of transcription of a gene is closely related to the interaction of transcription factors and cis-elements in the DNA sequence. Ethylene response factors (ERF), as an important transcription factor, can regulate a variety of plant genes which response to stresses. Plant defensin (PDF) can respond to biotic stress in plant. In the paper, through the investigation of the two genes, the author may discover the expression profile of the gens. It might be valuable for the enhancement of grape stress resistance.
In this thesis, the PCR was performed to obtain the promoter sequences of the VvERF3b and VvPDF1.2, and the PLACE and PlantCARE software was used to analysis the sequences. The results revealed that there were several putative cis-elements in the promoter of VvERF3b which were involved in the plant hormone responses, such as ABRE element, ASF1 element, DPBF element, DRE element, ERE element, GCC element, W-box and TCA element; and there were several putative cis-elements in the promoter of VvPDF1.2 which were involved in the plant stress responses, such as W-box, TGA element, TC-rich element, TATC element、ERE element、SEBF element and MYBGAHV element.
In according with the promoter sequence of VvERF3b gene, six forward and one reverse primers were designed. Then six different lengths of promoter fragments, that is P1, P2, P3, P4, P5 and P6 were amplified by PCR. The six amplified fragments were inserted into pGCC vector which is plant expression vector containing LUC repoter gene to substitute the 35S promoter and 8×GCC enhancer. These recombinant plasmids were named VvERF3b-P1, VvERF3b-P2, VvERF3b-P3, VvERF3b-P4, VvERF3b-P5, VvERF3b-P6. These recombinated vectors were transferred into A. thaliana protoplast cells by PEG-Ca~(2+). The results of six vectors’LUC gene transient expression indicated that the -1047 to -585 was essential for high expression of the gene, and removal of -1324 to -1185 region and -1324 to -1047 region caused increased in the expression of LUC in cells, which indicated that the region between -1324 and -1047 may contain at least two putative negative elements which caused the decrease of LUC expression.
There is no plant expression vector which can expression LUC gene in the current market. So the sequence of LUC gene is amplified by PCR in the research, and inserted into pBI221 vector to substitute the GUS gene in the vector. We named the recombinated plasmid as pBI221-LUC.
本研究利用PCR扩增获得欧洲葡萄(Vitis vinifera)的ERF3b基因和PDF1.2基因启动子序列,并利用PLACE和PlantCARE软件分析得到的两个基因的启动子序列。在VvERF3b基因启动子序列中发现多种与植物激素相关的顺式作用元件:ABRE元件、ASF1元件、DPBF元件、DRE元件、ERE元件、GCC元件、W-box和TCA元件,而在VvPDF1.2基因启动子内发现多种与植物逆境响应相关的顺式作用元件:W-box、TGA元件、TC-rich元件、TATC元件、ERE元件、SEBF元件和MYBGAHV元件。
按照VvERF3b基因启动子顺式作用元件的分析结果设计了6个上游启动子引物分别和一个下游启动子引物配对,利用PCR扩增获得6条起始密码上游长度不同的启动子片段P1~P6,通过酶切、纯化、连接,将6条VvERF3b基因5’缺失启动子序列代替原载体的8×GCC增强子和35S启动子插入pGCC载体中,与LUC报告基因连接构建6个植物表达载体:VvERF3b-P1~VvERF3b-P6。使用PEG-Ca~(2+)介导法介导重组载体转染拟南芥原生质体,用瞬时表达方法检测LUC表达量,结果表明VvERF3b基因启动子中从-1047bp到-585bp区域对于下游基因的表达有激活的作用,而从-1324bp到-1047bp区域可能存在两个以上对下游基因的表达起抑制作用的基序,用来抑制基因本身的表达。
鉴于目前市场中没有LUC植物表达载体,该文使用PCR扩增获得LUC基因片段,通过酶切、纯化、连接等手段将LUC基因片段代替pBI221载体的GUS基因片段在35S启动子驱动下表达,构建了具有多克隆位点(MCS)的LUC植物表达载体。
The level of transcription of a gene is closely related to the interaction of transcription factors and cis-elements in the DNA sequence. Ethylene response factors (ERF), as an important transcription factor, can regulate a variety of plant genes which response to stresses. Plant defensin (PDF) can respond to biotic stress in plant. In the paper, through the investigation of the two genes, the author may discover the expression profile of the gens. It might be valuable for the enhancement of grape stress resistance.
In this thesis, the PCR was performed to obtain the promoter sequences of the VvERF3b and VvPDF1.2, and the PLACE and PlantCARE software was used to analysis the sequences. The results revealed that there were several putative cis-elements in the promoter of VvERF3b which were involved in the plant hormone responses, such as ABRE element, ASF1 element, DPBF element, DRE element, ERE element, GCC element, W-box and TCA element; and there were several putative cis-elements in the promoter of VvPDF1.2 which were involved in the plant stress responses, such as W-box, TGA element, TC-rich element, TATC element、ERE element、SEBF element and MYBGAHV element.
In according with the promoter sequence of VvERF3b gene, six forward and one reverse primers were designed. Then six different lengths of promoter fragments, that is P1, P2, P3, P4, P5 and P6 were amplified by PCR. The six amplified fragments were inserted into pGCC vector which is plant expression vector containing LUC repoter gene to substitute the 35S promoter and 8×GCC enhancer. These recombinant plasmids were named VvERF3b-P1, VvERF3b-P2, VvERF3b-P3, VvERF3b-P4, VvERF3b-P5, VvERF3b-P6. These recombinated vectors were transferred into A. thaliana protoplast cells by PEG-Ca~(2+). The results of six vectors’LUC gene transient expression indicated that the -1047 to -585 was essential for high expression of the gene, and removal of -1324 to -1185 region and -1324 to -1047 region caused increased in the expression of LUC in cells, which indicated that the region between -1324 and -1047 may contain at least two putative negative elements which caused the decrease of LUC expression.
There is no plant expression vector which can expression LUC gene in the current market. So the sequence of LUC gene is amplified by PCR in the research, and inserted into pBI221 vector to substitute the GUS gene in the vector. We named the recombinated plasmid as pBI221-LUC.
引文
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