紫花苜蓿诱导表达启动子MsZPP的克隆及功能分析
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摘要
启动子是一段位于结构基因5’端上游的DNA序列，能够活化RNA聚合酶，使之与模板DNA准确结合并起始转录。目前，植物启动子已经被大量的分离并研究。但是还未见紫花苜蓿启动子研究的相关报道。
本研究以紫花苜蓿中苜1号为材料，研究编码锌指蛋白转录因子基因的启动子，旨在阐明该启动子不同区域发挥的功能，及其响应的诱导条件。主要试验结果如下：
1.克隆长1272bp的MsZFN基因启动子，命名为MsZPP（登录号：FJ161979.2）。研究确定了转录起始位点位于翻译起始位点上游69bp处。生物信息学分析预测，这段序列存在TATA-box和多个CAAT-box。此外，该序列存在多个光响应作用元件，激素响应作用元件，以及一些响应生物或非生物胁迫诱导的作用元件。
2.构建缺失启动子及全长启动子表达载体，并采用农杆菌介导法转化烟草。成功获得阳性转化再生烟草植株TP1、TP2、TP3、TP4、TP5和TP6，为后续研究启动子功能提供试验材料。
3.经过转化植株GUS蛋白组织化学染色及GUS酶活性定量分析，结果表明：不同长度的启动子片段调控功能存在差别。除种子外，转化植株根，茎，叶，花中都可以检测到gus基因的表达，并且随着启动子长度增加，GUS酶活性也相应增强。TP1、TP2与TP3、TP4、TP5和TP6相比，调控gus基因表达能力较弱。启动子长度为623bp时，就能够有效调控下游基因表达，所以该启动子的核心区域约为623bp。长度为460bp的启动子可以调控gus基因在气孔保卫细胞表达，初步说明在该区域存在能够特异性定位基因表达的顺式作用元件。
4.测定不同诱导条件下各种转化植株的GUS酶活性，结果表明：黑暗处理后，GUS酶活性显著增加，但不同缺失启动子之间差异不显著。200μM·L~(-1)MeJA处理后，只有转化植株TP6的GUS酶活性显著增加，是对照的2.96倍，其他差异不显著。2.85μM·L~(-1)IAA处理后，启动子长度大于460bp的转化植株GUS酶活性是对照的1.8倍左右，小于460bp的转化植株与对照相比没有明显变化。在70μM·L~(-1)GA3、50μM·L~(-1)ABA、25μM·L~(-1)PEG6000以及50mM·L~(-1)NaCl处理条件下，转化植株中GUS酶活性与对照相比没有显著差异。由此表明，启动子MsZPP可以应答黑暗、IAA及MeJA的诱导，但不响应另外四种诱导。
5.克隆MsZFN基因上游1340bp的片段，其中包括基因5’非编码区与全长启动子，构建表达载体，并成功转化烟草。与含有全长启动子的阳性转化植株比较，结果发现，两者在组织特异性表达和GUS酶活性分析上没有差别。由此可知，MsZFN基因的5’-UTR对启动子在转录调控水平没有影响。
Promoter is a DNA sequence in upstream of functional gene, which can recognize and combined toRNA polymerase to start gene transcription. In recently, there were many promoters have been clonedand analyzed, but there was still no report about alfalfa promoter research.
The research is meanly about a promoter of zinc finger protein transcription factor gene. In order tofind this gene was regulated by which inducement and the different functions of different fragments, wecloned the promoter of MsZFN gene and deletion analysis this promoter. The mainly research results areas follows:
1. According to the known MsZPP promoter sequence, Genbank number: FJ161979.2, we clonedthe peomoter fragment, it was1272bp. The transcription start site was found by5’-SMART-RACEmethod, it was at the upstream69bp of translation start site. In addition, there are many light-responsivecis-acting elements, some hormone response elements and many biological or abiotic stress-inducedelements.
2. In order to analyze the promoter function, we constructed the deletion promoter and full-lengthpromoter expression vector and transformed tobacco with the Agrobacterium mediat method. In thisresearch, we got transgenic tobacco successfully, this result provide test material for the follow-up studyof promoter function.
3. Histochemical assay and fluorometric assay of GUS reporter gene activity in transgenic tobaccoshowed that the regulate function were different in the different length promoter. All of the organs couldmeasure the GUS activity transgenic tobacco without the seed. The GUS expression in TP1and TP2root,stems, leaves and flowers were weak contrast with TP3, TP4, TP5and TP6. We can observe the gus geneexpressed in stomatal cell of TP3tobacco. Therefore, with the increase of promoter length, the ability ofregulate downstream gene expression increased too. The623bp promoter has could regulate downstreamgene expressed strongly. The460bp promoter can regulate gus reporter gene expressed in stomatal cell,indicating this region may have cis-acting element of the specific localization of gene expression, thein–depth studies need future experiments to confirm.
4. We used the transgenic tobacco which with different length of promoter as test material, aftertreated with different inducements; we measured the GUS activity to find the promoter was responsive towhich inducement. The result showed the GUS activity was increased after dark treatment, but there wasno significant difference in deletion promoters. After treated with200μM·L~(-1) MeJA, the GUS activity inTP6tobacco was2.96fold then control. After treated with2.85μM·L~(-1) IAA, the GUS activity in tobaccoTP4, TP5and TP6were1.8fold then control, there was no increase in transgenic plants TP1, TP2andTP3. After treat with70μM·L~(-1) GA3,50μM·L~(-1) ABA,25μM·L~(-1) PEG6000,50mM·L~(-1) NaClrespectively, the GUS activity didn’t change compared with control. Therefore, these results showed thepromoter MsZPP was respond to dark,200μM·L~(-1) MeJA and2.85μM·L~(-1) IAA, but didn’t respond toother four induce. Maybe the promoter respond to these inducements need cooperate with othercis-acting element.
5. We design a pair of primers to clone the promoter fragment with5’-UTR, and then transformedtobacco. Histochemical assay and fluorometric assay of gus reporter gene activity in transgenic tobaccoshowed that there was no different with the transgenic tobacco contained full-length promoter. Therefore,the5’-UTR of downstream gene didn’t affect MsZPP promoter function in transcription regulation.
本研究以紫花苜蓿中苜1号为材料，研究编码锌指蛋白转录因子基因的启动子，旨在阐明该启动子不同区域发挥的功能，及其响应的诱导条件。主要试验结果如下：
1.克隆长1272bp的MsZFN基因启动子，命名为MsZPP（登录号：FJ161979.2）。研究确定了转录起始位点位于翻译起始位点上游69bp处。生物信息学分析预测，这段序列存在TATA-box和多个CAAT-box。此外，该序列存在多个光响应作用元件，激素响应作用元件，以及一些响应生物或非生物胁迫诱导的作用元件。
2.构建缺失启动子及全长启动子表达载体，并采用农杆菌介导法转化烟草。成功获得阳性转化再生烟草植株TP1、TP2、TP3、TP4、TP5和TP6，为后续研究启动子功能提供试验材料。
3.经过转化植株GUS蛋白组织化学染色及GUS酶活性定量分析，结果表明：不同长度的启动子片段调控功能存在差别。除种子外，转化植株根，茎，叶，花中都可以检测到gus基因的表达，并且随着启动子长度增加，GUS酶活性也相应增强。TP1、TP2与TP3、TP4、TP5和TP6相比，调控gus基因表达能力较弱。启动子长度为623bp时，就能够有效调控下游基因表达，所以该启动子的核心区域约为623bp。长度为460bp的启动子可以调控gus基因在气孔保卫细胞表达，初步说明在该区域存在能够特异性定位基因表达的顺式作用元件。
4.测定不同诱导条件下各种转化植株的GUS酶活性，结果表明：黑暗处理后，GUS酶活性显著增加，但不同缺失启动子之间差异不显著。200μM·L~(-1)MeJA处理后，只有转化植株TP6的GUS酶活性显著增加，是对照的2.96倍，其他差异不显著。2.85μM·L~(-1)IAA处理后，启动子长度大于460bp的转化植株GUS酶活性是对照的1.8倍左右，小于460bp的转化植株与对照相比没有明显变化。在70μM·L~(-1)GA3、50μM·L~(-1)ABA、25μM·L~(-1)PEG6000以及50mM·L~(-1)NaCl处理条件下，转化植株中GUS酶活性与对照相比没有显著差异。由此表明，启动子MsZPP可以应答黑暗、IAA及MeJA的诱导，但不响应另外四种诱导。
5.克隆MsZFN基因上游1340bp的片段，其中包括基因5’非编码区与全长启动子，构建表达载体，并成功转化烟草。与含有全长启动子的阳性转化植株比较，结果发现，两者在组织特异性表达和GUS酶活性分析上没有差别。由此可知，MsZFN基因的5’-UTR对启动子在转录调控水平没有影响。
Promoter is a DNA sequence in upstream of functional gene, which can recognize and combined toRNA polymerase to start gene transcription. In recently, there were many promoters have been clonedand analyzed, but there was still no report about alfalfa promoter research.
The research is meanly about a promoter of zinc finger protein transcription factor gene. In order tofind this gene was regulated by which inducement and the different functions of different fragments, wecloned the promoter of MsZFN gene and deletion analysis this promoter. The mainly research results areas follows:
1. According to the known MsZPP promoter sequence, Genbank number: FJ161979.2, we clonedthe peomoter fragment, it was1272bp. The transcription start site was found by5’-SMART-RACEmethod, it was at the upstream69bp of translation start site. In addition, there are many light-responsivecis-acting elements, some hormone response elements and many biological or abiotic stress-inducedelements.
2. In order to analyze the promoter function, we constructed the deletion promoter and full-lengthpromoter expression vector and transformed tobacco with the Agrobacterium mediat method. In thisresearch, we got transgenic tobacco successfully, this result provide test material for the follow-up studyof promoter function.
3. Histochemical assay and fluorometric assay of GUS reporter gene activity in transgenic tobaccoshowed that the regulate function were different in the different length promoter. All of the organs couldmeasure the GUS activity transgenic tobacco without the seed. The GUS expression in TP1and TP2root,stems, leaves and flowers were weak contrast with TP3, TP4, TP5and TP6. We can observe the gus geneexpressed in stomatal cell of TP3tobacco. Therefore, with the increase of promoter length, the ability ofregulate downstream gene expression increased too. The623bp promoter has could regulate downstreamgene expressed strongly. The460bp promoter can regulate gus reporter gene expressed in stomatal cell,indicating this region may have cis-acting element of the specific localization of gene expression, thein–depth studies need future experiments to confirm.
4. We used the transgenic tobacco which with different length of promoter as test material, aftertreated with different inducements; we measured the GUS activity to find the promoter was responsive towhich inducement. The result showed the GUS activity was increased after dark treatment, but there wasno significant difference in deletion promoters. After treated with200μM·L~(-1) MeJA, the GUS activity inTP6tobacco was2.96fold then control. After treated with2.85μM·L~(-1) IAA, the GUS activity in tobaccoTP4, TP5and TP6were1.8fold then control, there was no increase in transgenic plants TP1, TP2andTP3. After treat with70μM·L~(-1) GA3,50μM·L~(-1) ABA,25μM·L~(-1) PEG6000,50mM·L~(-1) NaClrespectively, the GUS activity didn’t change compared with control. Therefore, these results showed thepromoter MsZPP was respond to dark,200μM·L~(-1) MeJA and2.85μM·L~(-1) IAA, but didn’t respond toother four induce. Maybe the promoter respond to these inducements need cooperate with othercis-acting element.
5. We design a pair of primers to clone the promoter fragment with5’-UTR, and then transformedtobacco. Histochemical assay and fluorometric assay of gus reporter gene activity in transgenic tobaccoshowed that there was no different with the transgenic tobacco contained full-length promoter. Therefore,the5’-UTR of downstream gene didn’t affect MsZPP promoter function in transcription regulation.
引文
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