矮牵牛PMADS9基因的启动子克隆与功能分析
摘要
MADS-box基因是一类广泛存在于植物中的同源异型基因,在生物体不同的组织以及生长发育的不同阶段都存在MADS-box基因的表达,它属于一个古老的基因家族,是植物生长发育过程中重要的调控因子。目前对MADS-box基因调控机理的研究已成为植物分子生物学领域中的一大热点。矮牵牛PMADS9基因是MADS-box基因AGL15亚家族的成员,该亚家族基因可能具有调控开花时间、抑制花器官衰老脱落和促进体胚形成等功能。
通过RACR技术和YADE法,我们分别从矮牵牛小花蕾RNA和叶片DNA基因组中克隆得到了PMADS9基因的编码区(GenBank登录号：DQ418547)和5'端翻译起始位点(ATG)上游1053bp的启动子序列(GenBank登录号：FJ798977)。然后在两端设计引物,利用PCR技术从基因组DNA中扩增得到了全长基因序列共5861bp(GenBank登录号为EU338501)。RACE分析发现该基因至少有4个转录起始位点(TSS),2个位于编码区第一外显子内。
本研究,继续克隆了PMADS9基因上游未知的启动子区域,获得了更长的800bp序列,并对该启动子的功能进行了初步地研究,这对于进一步了解PMADS9基因表达调控的特点,以及更深入研究矮牵牛花器官发育的分子机理都有着重要的意义。
本文所取得的主要实验结论如下：
1.在已获1053bp启动子序列的基础上,利用hiTAIL-PCR技术对矮牵牛PMADS9基因上游未知启动子序列进行了再扩增,获得了更长的800bp启动子片段,用SeqMan软件对新获取的序列与原有1053bp序列进行了拼接,最终获得了PMADS9基因全长启动子序列1853bp。
2.运用PLACE和PlantCare等在线分析软件对该序列进行了预测分析,该启动子除了具有TATA-box和CAAT-box等基本转录元件外,还富含花粉和种子发育过程中特异表达所需的调控元件,以及与环境应答相关的各种顺式调控元件。FootPrinter分析表明,AGL15同源基因启动子之间存在非常保守的RY-repeat元件,启动子的保守性与物种的遗传距离不一致；推测PMADS9基因翻译起始位点(ATG)上游200～400bp和800～1000bp区域具有重要的功能。
3.应用PCR技术对启动子序列进行5'缺失；用各缺失片段替换表达载体pCAMBIA1305.1中的CaMV35S组成型启动子序列,与GUS报告基因连接,成功构建了所有缺失重组表达载pCAMBIA1305.1空载为阳性对照,转化矮牵牛V1。GUS染色与PCR鉴定表明,成功实现了各表达载体对矮牵牛的稳定遗传转化,获得了阳性转基因植株。
4.待移栽后的转基因植株开花后,对其进行GUS组织化学染色分析,结果表明全长启动子序列(1853bp)转化后的阳性植株,在茎、营养叶、苞叶以及花萼中几乎检测不到GUS活性；GUS基因在雄蕊、雌蕊、子房中有较高的表达丰度,在花瓣中也有一定丰度的表达,这与之前PMADS9基因表达分析的结果基本一致。在转基因植株内,各缺失表达载体的表达模式无明显差异,最短的启动子缺失片段(298bp)就能启动报告基因在转基因植物体内表达,说明其已具备启动子所需的各种基本调控元件。
MADS-box genes are a class of homeotic genes widely distributed in plants and the expression of MADS-box genes are discoveried in different tissues, organisms, stages of growth and development.They belong to an ancient gene family and are a kind of important regulatory factors during the growth and development of plant. At present,the research of MADS-box genes regulatory mechanism has become a hot topic in the field of plant molecular biology. PMADS9 gene of Petunia is a member of AGL15 subfamily which might regulate flowering time, inhibit floral organ senescence,and promote the formation of somatic embryos.
By RACE technology and YADE method,we had cloned coding region of PMADS9(GenBank accession number:DQ418547) from RNA Petunia flower buds and obtained 1053bp promoter sequence (GenBank accession number:FJ798977) upstream the 5'translation start site(ATG) from DNA of leaves. Then designing primers at both ends of the sequence and using PCR method, we got the full-length sequences of PMADS9 gene,5861bp (GenBank accession number EU338501). RACE analysis revealed that the gene has at least four transcription start sites(TSS),two of which located within the first exon of the coding region.
In this study,we continued to amplify the upstream unknown promoter region of PMADS9 gene,and obtained a longer 800bp sequence.The preliminary studies of the promoter functions are great significant for understanding the characteristics of expression regulation of PMADS9 gene,as well as more in-depth studying the molecular mechanisms of floral organ development. In this paper,the main experimental results are as follows:
1.On this basis of 1053bp promoter sequence,we used hiTAIL-PCR technology to amplify the unstream unknown promoter sequence and obtained a longer 800bp fragment.Through the comparion and splice of the new sequence and the original 1053bp by SeqMan software, we ultimately acquired the 1853bp promoter sequence of PMADS9 gene.
2.Cis-regulatory elements of the PMADS9 promoter sequence predicted by PLACE and PlantCARE online analysis software are related with seed and pollen development and environmental response.Analysis of promoter sequences from AGL15-clade MADS-box genes by FootPrinter,showed that very conserved RY-repeat motifs were exist amony them,and the conversation of promoters between So lanaceae and the selected 5 species of Rosids is higher than that between Brassicaceae and the same selected species even though So lanaceae is less closely related to Rosids than Brassicaceae.Furthermore,the results also suggested that regions of 200～400bp and 800～1000bp upstream of the ATG were functionally important.
3.According to the cis-regulatory elements locations on the promoter,we designed different primers and used PCR method to obtain the 5'end deletion fragments of PMADS9 promoter.New restructuring expression vectors were constructed by replacing the CaMV35S promoter of pCAMBIA1305.1 with the 6 deletion fragments respectively to drive the expression of the reporter gene GUSpus..The new vector was transferred into Agrobacterium to infect leaf disks of Petunia.The results of GUS staining and PCR identification indicated that the 6 promoter fragments were successfully transferred into the receptor plants.
4.After transgenic plants flowering,the analysis results of GUS histochemical staining showed that the full-length promoter sequence(1853bp) can start GUS gene high express in stamens,pistils and ovarys.On the contrary,GUS activity was not detected in the stems,leaves,bracts and calyxes.This is consistent with the previous results of PMADS9 gene expression analysis.Among the different deletion fragments,there no significant difference in expression patterns.The shortest deletion promoter fragment(298bp) was able to start the GUS reporter gene expression in transgenic plants,indicating that it contained a variety of basic regulatory elements required for promoter.
通过RACR技术和YADE法,我们分别从矮牵牛小花蕾RNA和叶片DNA基因组中克隆得到了PMADS9基因的编码区(GenBank登录号：DQ418547)和5'端翻译起始位点(ATG)上游1053bp的启动子序列(GenBank登录号：FJ798977)。然后在两端设计引物,利用PCR技术从基因组DNA中扩增得到了全长基因序列共5861bp(GenBank登录号为EU338501)。RACE分析发现该基因至少有4个转录起始位点(TSS),2个位于编码区第一外显子内。
本研究,继续克隆了PMADS9基因上游未知的启动子区域,获得了更长的800bp序列,并对该启动子的功能进行了初步地研究,这对于进一步了解PMADS9基因表达调控的特点,以及更深入研究矮牵牛花器官发育的分子机理都有着重要的意义。
本文所取得的主要实验结论如下：
1.在已获1053bp启动子序列的基础上,利用hiTAIL-PCR技术对矮牵牛PMADS9基因上游未知启动子序列进行了再扩增,获得了更长的800bp启动子片段,用SeqMan软件对新获取的序列与原有1053bp序列进行了拼接,最终获得了PMADS9基因全长启动子序列1853bp。
2.运用PLACE和PlantCare等在线分析软件对该序列进行了预测分析,该启动子除了具有TATA-box和CAAT-box等基本转录元件外,还富含花粉和种子发育过程中特异表达所需的调控元件,以及与环境应答相关的各种顺式调控元件。FootPrinter分析表明,AGL15同源基因启动子之间存在非常保守的RY-repeat元件,启动子的保守性与物种的遗传距离不一致；推测PMADS9基因翻译起始位点(ATG)上游200～400bp和800～1000bp区域具有重要的功能。
3.应用PCR技术对启动子序列进行5'缺失；用各缺失片段替换表达载体pCAMBIA1305.1中的CaMV35S组成型启动子序列,与GUS报告基因连接,成功构建了所有缺失重组表达载pCAMBIA1305.1空载为阳性对照,转化矮牵牛V1。GUS染色与PCR鉴定表明,成功实现了各表达载体对矮牵牛的稳定遗传转化,获得了阳性转基因植株。
4.待移栽后的转基因植株开花后,对其进行GUS组织化学染色分析,结果表明全长启动子序列(1853bp)转化后的阳性植株,在茎、营养叶、苞叶以及花萼中几乎检测不到GUS活性；GUS基因在雄蕊、雌蕊、子房中有较高的表达丰度,在花瓣中也有一定丰度的表达,这与之前PMADS9基因表达分析的结果基本一致。在转基因植株内,各缺失表达载体的表达模式无明显差异,最短的启动子缺失片段(298bp)就能启动报告基因在转基因植物体内表达,说明其已具备启动子所需的各种基本调控元件。
MADS-box genes are a class of homeotic genes widely distributed in plants and the expression of MADS-box genes are discoveried in different tissues, organisms, stages of growth and development.They belong to an ancient gene family and are a kind of important regulatory factors during the growth and development of plant. At present,the research of MADS-box genes regulatory mechanism has become a hot topic in the field of plant molecular biology. PMADS9 gene of Petunia is a member of AGL15 subfamily which might regulate flowering time, inhibit floral organ senescence,and promote the formation of somatic embryos.
By RACE technology and YADE method,we had cloned coding region of PMADS9(GenBank accession number:DQ418547) from RNA Petunia flower buds and obtained 1053bp promoter sequence (GenBank accession number:FJ798977) upstream the 5'translation start site(ATG) from DNA of leaves. Then designing primers at both ends of the sequence and using PCR method, we got the full-length sequences of PMADS9 gene,5861bp (GenBank accession number EU338501). RACE analysis revealed that the gene has at least four transcription start sites(TSS),two of which located within the first exon of the coding region.
In this study,we continued to amplify the upstream unknown promoter region of PMADS9 gene,and obtained a longer 800bp sequence.The preliminary studies of the promoter functions are great significant for understanding the characteristics of expression regulation of PMADS9 gene,as well as more in-depth studying the molecular mechanisms of floral organ development. In this paper,the main experimental results are as follows:
1.On this basis of 1053bp promoter sequence,we used hiTAIL-PCR technology to amplify the unstream unknown promoter sequence and obtained a longer 800bp fragment.Through the comparion and splice of the new sequence and the original 1053bp by SeqMan software, we ultimately acquired the 1853bp promoter sequence of PMADS9 gene.
2.Cis-regulatory elements of the PMADS9 promoter sequence predicted by PLACE and PlantCARE online analysis software are related with seed and pollen development and environmental response.Analysis of promoter sequences from AGL15-clade MADS-box genes by FootPrinter,showed that very conserved RY-repeat motifs were exist amony them,and the conversation of promoters between So lanaceae and the selected 5 species of Rosids is higher than that between Brassicaceae and the same selected species even though So lanaceae is less closely related to Rosids than Brassicaceae.Furthermore,the results also suggested that regions of 200～400bp and 800～1000bp upstream of the ATG were functionally important.
3.According to the cis-regulatory elements locations on the promoter,we designed different primers and used PCR method to obtain the 5'end deletion fragments of PMADS9 promoter.New restructuring expression vectors were constructed by replacing the CaMV35S promoter of pCAMBIA1305.1 with the 6 deletion fragments respectively to drive the expression of the reporter gene GUSpus..The new vector was transferred into Agrobacterium to infect leaf disks of Petunia.The results of GUS staining and PCR identification indicated that the 6 promoter fragments were successfully transferred into the receptor plants.
4.After transgenic plants flowering,the analysis results of GUS histochemical staining showed that the full-length promoter sequence(1853bp) can start GUS gene high express in stamens,pistils and ovarys.On the contrary,GUS activity was not detected in the stems,leaves,bracts and calyxes.This is consistent with the previous results of PMADS9 gene expression analysis.Among the different deletion fragments,there no significant difference in expression patterns.The shortest deletion promoter fragment(298bp) was able to start the GUS reporter gene expression in transgenic plants,indicating that it contained a variety of basic regulatory elements required for promoter.
引文
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