PtSEP2、PtSEP3-1、PtAP1-2和PtMCP基因启动子在烟草和毛白杨中的表达特性分析
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摘要
本研究针对杨树存在的飞絮散粉问题,在已经获得毛白杨(Populus tomentosa Carr.)PtSEP3-1、PtAP1-2及PtMCP启动子的基础上,进一步从毛白杨基因组DNA中扩增获得了PtSEP2基因5′侧翼的一段序列,通过在烟草和毛白杨的转化研究了这4个启动子的组织表达特性,为基因工程定向调控毛白杨散粉飞絮性状奠定工作基础。主要研究结果如下：
1本研究利用PCR技术从毛白杨基因组DNA中扩增获得花器官发育相关的SEPALLATA2类似基因PtSEP2 5侧翼约2.3kb的一段序列,经PlantCARE序列分析表明,该序列中含有启动子特征的保守序列及多种光应答元件,初步推测其为PtSEP2基因启动子。进一步以GUS为报告基因,构建了pPtSEP2 promoter::GUS的植物表达载体,命名为PtSEP2::GUS。
2以烟草根、茎、叶、花芽为受体,通过农杆菌介导转化对这4个启动子进行瞬时表达研究,GUS组织化学染色表明：PtSEP2启动子的GUS活性只存在于花药中；PtAP1-2启动子的GUS活性在花萼及花瓣中微弱表达,在根、茎及叶中均未被检测到；PtSEP3-1启动子在烟草的根、茎、叶及花各组织中都没有检测到的GUS活性；PtMCP启动子在烟草的根、茎、叶和花芽等各组织中均有GUS活性表达,但是表达强度明显弱于35S启动子,推测其属于温和组成型启动子类型。
3将上述表达载体在毛白杨无性系TC1521和烟草中进行遗传转化,经PCR验证,共获得毛白杨阳性转化植株37株,获得烟草阳性转化植株88株。对转基因烟草的根、茎及叶进行染色,GUS染色结果与瞬时表达的染色结果一致。对转基因毛白杨小苗进行染色,结果表明,PtSEP2、PtSEP3-1和PtAP1-2启动子在整株幼苗中均未检测到GUS活性。
4对转PtMCP启动子的烟草根、茎、叶及花各组织进行GUS酶活性测定,结果表明与pBI121相比,PtMCP启动子的GUS活性明显较低,在转基因烟草的根、茎、叶及花各组织中,转PtMCP启动子的GUS活性分别为pBI121 GUS活性的21.9%、5.3%、5.3%及17.6%。野生型烟草各组织中没有检测到GUS的活性。通过上述研究分析可以得出结论：PtSEP2启动子可以驱动GUS基因只在花药中表达,初步推测其属于花药特异性启动子；PtAP1-2启动子的GUS活性只在花萼及花瓣中微弱表达,推断其可能为花组织特异性启动子；在根、茎及叶中均未被检测到；PtMCP启动子可以驱动GUS基因在烟草的根、茎、叶及花组织中均可表达,但是表达强度明显弱于35S启动子,属于温和组成型启动子类型。
本研究不仅对于杨树开花分子调控机理的研究具有重要的理论意义,而且对于通过基因工程改良杨树飞絮散粉问题具有潜在应用价值。
Considering problems of flying catkin and the shedding pollen with Populus tomentosa Carr., a 5' sequence fragment of PtSEP2 was cloned from its genomic DNA in this study, based on retrieval of promoters of PtSEP3-1, PtAP1-2 and PtMCP. Tissue expression character of this four promoters was researched by transforming tobacco and Populus tomentosa, laying a foundation for genetically modifying traits of flying catkin and shedding pollen. The main results are as follow.
1. A 5'flanking sequence of SEPALLATA like gene PtSEP2, with a length of 2.3 kb was amplified from genomic DNA of Populus tomentosa. Sequence analysis on PlantCARE showed many conserved fragment and light responded motif existing in this sequence, which was therefore predicted as PtSEP2 promoter. An expression vector was constructed using GUS as reporter gene, named PtSEP2::GUS.
2. Transient expression research was performed by Agrobacterium-mediated transformation using four constructs which containing promoter::GUS, taking root, stem, leaf, flower as the recipient. Immunohistochemical staining indicated that GUS activity only existed in anther, PtAPl-2 weakly expressed at sepal and petal, GUS activity driven by PtSEP3-1 was detected in no tissues, GUS activity driven by PtMCP was detected in every tissue, but with an obvious weaker expressed strength than that of 35S promoter, Thus inferring PtMCP as a mild and constitutive promoter.
3. The Populus tomentosa clone TC1521 and tobacco were used for genetic transformation.The positive results verified by PCR include respectively 37and 88.GUS Staining of root, stem and leaf from genetically modified tobacco showed a consistent result with that of transient expression. Staining of the whole plantlet of genetically modified Populus tomentosa suggested that PtSEP, PtSEP3-1 and PtAP1-2 all expressed in no tissues.
4. GUS activity assay on root, stem, leaf and flower of transgene tobacco indicated a weaker activity of PtMCP compared with pBI121, its activity are 21.9%,5.3%,5.3%,17.6% of GUS activity with pBI 121 respectively. Whereas, GUS activity was not detected in any tissues of wild type tobacco.
Through the above analysis, conclusions can be drawn that, PtSEP2 promoter can drive expression of GUS gene in anther only, and initially speculated that it belongs to anther-specific promoter; weak expression of GUS driven by PtAPl-2 was only found in sepals and petals only, flower tissue-specific promoter may be inferred; PtMCP promoter can drive GUS gene express in tobacco roots, stems, leaves and flower tissues, with an expression strength significantly weaker than that of 35S promoter, therefore it is a mild constitutive promoter type.
The above-mentioned results do not only make a profound theoretical sense for illuminating molecular mechanism of poplar flowering, but also provide potential practical value for gene engineering addressing the flying catkin-caused problem.
1本研究利用PCR技术从毛白杨基因组DNA中扩增获得花器官发育相关的SEPALLATA2类似基因PtSEP2 5侧翼约2.3kb的一段序列,经PlantCARE序列分析表明,该序列中含有启动子特征的保守序列及多种光应答元件,初步推测其为PtSEP2基因启动子。进一步以GUS为报告基因,构建了pPtSEP2 promoter::GUS的植物表达载体,命名为PtSEP2::GUS。
2以烟草根、茎、叶、花芽为受体,通过农杆菌介导转化对这4个启动子进行瞬时表达研究,GUS组织化学染色表明：PtSEP2启动子的GUS活性只存在于花药中；PtAP1-2启动子的GUS活性在花萼及花瓣中微弱表达,在根、茎及叶中均未被检测到；PtSEP3-1启动子在烟草的根、茎、叶及花各组织中都没有检测到的GUS活性；PtMCP启动子在烟草的根、茎、叶和花芽等各组织中均有GUS活性表达,但是表达强度明显弱于35S启动子,推测其属于温和组成型启动子类型。
3将上述表达载体在毛白杨无性系TC1521和烟草中进行遗传转化,经PCR验证,共获得毛白杨阳性转化植株37株,获得烟草阳性转化植株88株。对转基因烟草的根、茎及叶进行染色,GUS染色结果与瞬时表达的染色结果一致。对转基因毛白杨小苗进行染色,结果表明,PtSEP2、PtSEP3-1和PtAP1-2启动子在整株幼苗中均未检测到GUS活性。
4对转PtMCP启动子的烟草根、茎、叶及花各组织进行GUS酶活性测定,结果表明与pBI121相比,PtMCP启动子的GUS活性明显较低,在转基因烟草的根、茎、叶及花各组织中,转PtMCP启动子的GUS活性分别为pBI121 GUS活性的21.9%、5.3%、5.3%及17.6%。野生型烟草各组织中没有检测到GUS的活性。通过上述研究分析可以得出结论：PtSEP2启动子可以驱动GUS基因只在花药中表达,初步推测其属于花药特异性启动子；PtAP1-2启动子的GUS活性只在花萼及花瓣中微弱表达,推断其可能为花组织特异性启动子；在根、茎及叶中均未被检测到；PtMCP启动子可以驱动GUS基因在烟草的根、茎、叶及花组织中均可表达,但是表达强度明显弱于35S启动子,属于温和组成型启动子类型。
本研究不仅对于杨树开花分子调控机理的研究具有重要的理论意义,而且对于通过基因工程改良杨树飞絮散粉问题具有潜在应用价值。
Considering problems of flying catkin and the shedding pollen with Populus tomentosa Carr., a 5' sequence fragment of PtSEP2 was cloned from its genomic DNA in this study, based on retrieval of promoters of PtSEP3-1, PtAP1-2 and PtMCP. Tissue expression character of this four promoters was researched by transforming tobacco and Populus tomentosa, laying a foundation for genetically modifying traits of flying catkin and shedding pollen. The main results are as follow.
1. A 5'flanking sequence of SEPALLATA like gene PtSEP2, with a length of 2.3 kb was amplified from genomic DNA of Populus tomentosa. Sequence analysis on PlantCARE showed many conserved fragment and light responded motif existing in this sequence, which was therefore predicted as PtSEP2 promoter. An expression vector was constructed using GUS as reporter gene, named PtSEP2::GUS.
2. Transient expression research was performed by Agrobacterium-mediated transformation using four constructs which containing promoter::GUS, taking root, stem, leaf, flower as the recipient. Immunohistochemical staining indicated that GUS activity only existed in anther, PtAPl-2 weakly expressed at sepal and petal, GUS activity driven by PtSEP3-1 was detected in no tissues, GUS activity driven by PtMCP was detected in every tissue, but with an obvious weaker expressed strength than that of 35S promoter, Thus inferring PtMCP as a mild and constitutive promoter.
3. The Populus tomentosa clone TC1521 and tobacco were used for genetic transformation.The positive results verified by PCR include respectively 37and 88.GUS Staining of root, stem and leaf from genetically modified tobacco showed a consistent result with that of transient expression. Staining of the whole plantlet of genetically modified Populus tomentosa suggested that PtSEP, PtSEP3-1 and PtAP1-2 all expressed in no tissues.
4. GUS activity assay on root, stem, leaf and flower of transgene tobacco indicated a weaker activity of PtMCP compared with pBI121, its activity are 21.9%,5.3%,5.3%,17.6% of GUS activity with pBI 121 respectively. Whereas, GUS activity was not detected in any tissues of wild type tobacco.
Through the above analysis, conclusions can be drawn that, PtSEP2 promoter can drive expression of GUS gene in anther only, and initially speculated that it belongs to anther-specific promoter; weak expression of GUS driven by PtAPl-2 was only found in sepals and petals only, flower tissue-specific promoter may be inferred; PtMCP promoter can drive GUS gene express in tobacco roots, stems, leaves and flower tissues, with an expression strength significantly weaker than that of 35S promoter, therefore it is a mild constitutive promoter type.
The above-mentioned results do not only make a profound theoretical sense for illuminating molecular mechanism of poplar flowering, but also provide potential practical value for gene engineering addressing the flying catkin-caused problem.
引文
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