靛蓝基因表达载体的构建及香石竹CHS启动子克隆研究
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摘要
在观赏植物中蓝色花普遍偏少，用作观赏的切花香石竹、郁金香、月季、玫瑰等都缺乏蓝色花系。本论文从克隆花瓣特异表达的启动子及与靛蓝色素基因连接构建表达载体等方面对蓝色花基因工程育种进行了一些研究，主要的研究内容有以下几个方面：
(1)利用Genbank公布的两个拟南芥查尔酮合成酶(Chalcone synthase，CHS)启动子序列(AF248988和AF012810)设计序列特异性的引物，从拟南芥(Arabidopsis thialiana，Columbia)基因组DNA中进行PCR得到大小分别为533bp和2046bp的两个启动子片段(其中小片段启动子是大片段启动子的3’端部分)，经过克隆测序证明与Genbank中公布的序列超过99％的同源性。将小片段启动子序列应用PlantCARE软件进行分析，表明两个序列均具有真核生物启动子普遍具有的CAAT box和TATA box等保守序列，并且具有一些CHS启动子特有的保守序列，证明两个启动子序列应该具有相应的启动子活性。
(2)通过对两个启动子序列以及载体序列进行酶切位点分析，选择合适的限制性内切酶进行表达载体的构建，以应用于以后的遗传转化研究。大、小两个启动子片段都分别与GUS基因或与Drewlo S等人从Ralstonia eutropha(真氧产碱杆菌)中克隆到的靛蓝色素基因(bec基因)相连接，目的是分别用于鉴别相应的启动子活性和在观赏植物中启动靛蓝色素基因的表达。目前，小片段启动子的两个表达载体的构建已经完成，并进行了烟草的初步转化研究；大片段启动子载体的构建正在进行。
(3)本课题的目的是得到能够在观赏植物中高效启动靛蓝色素基因表达的载体，但是考虑到拟南芥的CHS启动子启动靛蓝色素基因在观赏植物中的表达可能会受到种属特异性等一些因素的影响而不能很好的启动基因的表达，本研究还进行了从观赏植物香石竹(Dianthus caryophyllus，Feraceseo)中克隆CHS启动子的研究。由于到目前为止还没有见到有关香石竹中CHS启动子的报道，并且CHS启动子具有很强的种属特异性，在不同种属的植物之间具有很低的同源性，所以应用CHS启动子序列之间的同源性进行香石竹CHS启动子克隆的路线是不可行的。本文的研究通过CHS基因之间的同源性设计引物，从香石竹中进行PCR并经过克隆测序得到了三个CHS基因的5’端片段，经过Blast分析证明了它们具有很高的同源性。然后利用衔接头PCR的方法进行了香石竹CHS启动子克隆的初步研究，得到了约500bp和800bp的PCR产物，目前克隆测序工作正在进行。进一步的工作将进行启动子缺失分析等研究以验证启动子活性及其表达特异性。
(4)本文的研究还进行了香石竹叶片再生体系的初步试验，并得到了几个相对比较理想的培养基组合，目前进一步的研究正在进行。
With the development of mordern lives, more and more flowers are needed and that indicates a prosperous prospect of ornament market. There are some restricting factors in traditionary breeding methods, and nowadays the molecular breeding methods are well developed and have succeeded in some flowers such as chrysanthemum and lily et al.
In this paper, we introduce our study on the cloning of two petal-specific CHS (chacone synthase) promoters and the constructing of expression vectors containing of a CHS promoter and an indigo gene (bec gene) or a GUS report gene. The main contents are as follows:
(1) We have cloned two CHS promoters from Arabidopsis thialiana (Columbia ecotype) using two pairs of sequence-specific PCR primers from two homologous sequences in the Genbank (AF248988 & AF012810). The fragments are 533bp and 2046bp separately, and the cloning sequencing results indicate that the homologous between them and the Genbank sequences excess 99%. We have also analysised the two promoters' construction using PlantCARE software on the internet, the results reveal that they have the motifs that are necessary for eukaryotes promoters such as TATA box and CAAT box et al. and they also have some motifs that are specific in CHS promoters. The results prove that these two promoters should have promoter activity.
(2) By analysising the restriction endonucleases sites of the two promoter sequences and the vectors, we selected the profitable restriction endonucleases to construct the expression vectors. The two promoter fragments are ligated to a GUS report gene or a bec gene separately. The two expression vectors containing the shorter promoter have already been finished and the other two vectors containing the longer promoter are on finishing.
(3) The aim of our experiment is to get high efficient expression vetors to express the bec gene in ornaments. For fearing that the CHS promoters from Arabidopsis maybe couldn't promote the gene expression in ornaments, we tried to clone a CHS promoter from carnation. But the homologous between the CHS promoters of different plants are very low, so we designed a pare of PCR primers from carnation CHS gene sequence and have got three clones of carnation CHS gene 5' end sequences by PCR. With these three sequences we used adapter ligation PCR method to clone the promoter, and now we have got two fragments which are about 500bp and about 800bp separately, the cloning sequencing of them are on finishing. The results prove that adapter ligation PCR method is workable to clone carnation CHS promoter. And the next step we will do the deletion analysis of the promoter fragments.
(4) We have also done some experiments on establishing the regeneration system of carnation, and have selected several kinds of comparativly ideal cultures. The following works are on proceeding.
(1)利用Genbank公布的两个拟南芥查尔酮合成酶(Chalcone synthase，CHS)启动子序列(AF248988和AF012810)设计序列特异性的引物，从拟南芥(Arabidopsis thialiana，Columbia)基因组DNA中进行PCR得到大小分别为533bp和2046bp的两个启动子片段(其中小片段启动子是大片段启动子的3’端部分)，经过克隆测序证明与Genbank中公布的序列超过99％的同源性。将小片段启动子序列应用PlantCARE软件进行分析，表明两个序列均具有真核生物启动子普遍具有的CAAT box和TATA box等保守序列，并且具有一些CHS启动子特有的保守序列，证明两个启动子序列应该具有相应的启动子活性。
(2)通过对两个启动子序列以及载体序列进行酶切位点分析，选择合适的限制性内切酶进行表达载体的构建，以应用于以后的遗传转化研究。大、小两个启动子片段都分别与GUS基因或与Drewlo S等人从Ralstonia eutropha(真氧产碱杆菌)中克隆到的靛蓝色素基因(bec基因)相连接，目的是分别用于鉴别相应的启动子活性和在观赏植物中启动靛蓝色素基因的表达。目前，小片段启动子的两个表达载体的构建已经完成，并进行了烟草的初步转化研究；大片段启动子载体的构建正在进行。
(3)本课题的目的是得到能够在观赏植物中高效启动靛蓝色素基因表达的载体，但是考虑到拟南芥的CHS启动子启动靛蓝色素基因在观赏植物中的表达可能会受到种属特异性等一些因素的影响而不能很好的启动基因的表达，本研究还进行了从观赏植物香石竹(Dianthus caryophyllus，Feraceseo)中克隆CHS启动子的研究。由于到目前为止还没有见到有关香石竹中CHS启动子的报道，并且CHS启动子具有很强的种属特异性，在不同种属的植物之间具有很低的同源性，所以应用CHS启动子序列之间的同源性进行香石竹CHS启动子克隆的路线是不可行的。本文的研究通过CHS基因之间的同源性设计引物，从香石竹中进行PCR并经过克隆测序得到了三个CHS基因的5’端片段，经过Blast分析证明了它们具有很高的同源性。然后利用衔接头PCR的方法进行了香石竹CHS启动子克隆的初步研究，得到了约500bp和800bp的PCR产物，目前克隆测序工作正在进行。进一步的工作将进行启动子缺失分析等研究以验证启动子活性及其表达特异性。
(4)本文的研究还进行了香石竹叶片再生体系的初步试验，并得到了几个相对比较理想的培养基组合，目前进一步的研究正在进行。
With the development of mordern lives, more and more flowers are needed and that indicates a prosperous prospect of ornament market. There are some restricting factors in traditionary breeding methods, and nowadays the molecular breeding methods are well developed and have succeeded in some flowers such as chrysanthemum and lily et al.
In this paper, we introduce our study on the cloning of two petal-specific CHS (chacone synthase) promoters and the constructing of expression vectors containing of a CHS promoter and an indigo gene (bec gene) or a GUS report gene. The main contents are as follows:
(1) We have cloned two CHS promoters from Arabidopsis thialiana (Columbia ecotype) using two pairs of sequence-specific PCR primers from two homologous sequences in the Genbank (AF248988 & AF012810). The fragments are 533bp and 2046bp separately, and the cloning sequencing results indicate that the homologous between them and the Genbank sequences excess 99%. We have also analysised the two promoters' construction using PlantCARE software on the internet, the results reveal that they have the motifs that are necessary for eukaryotes promoters such as TATA box and CAAT box et al. and they also have some motifs that are specific in CHS promoters. The results prove that these two promoters should have promoter activity.
(2) By analysising the restriction endonucleases sites of the two promoter sequences and the vectors, we selected the profitable restriction endonucleases to construct the expression vectors. The two promoter fragments are ligated to a GUS report gene or a bec gene separately. The two expression vectors containing the shorter promoter have already been finished and the other two vectors containing the longer promoter are on finishing.
(3) The aim of our experiment is to get high efficient expression vetors to express the bec gene in ornaments. For fearing that the CHS promoters from Arabidopsis maybe couldn't promote the gene expression in ornaments, we tried to clone a CHS promoter from carnation. But the homologous between the CHS promoters of different plants are very low, so we designed a pare of PCR primers from carnation CHS gene sequence and have got three clones of carnation CHS gene 5' end sequences by PCR. With these three sequences we used adapter ligation PCR method to clone the promoter, and now we have got two fragments which are about 500bp and about 800bp separately, the cloning sequencing of them are on finishing. The results prove that adapter ligation PCR method is workable to clone carnation CHS promoter. And the next step we will do the deletion analysis of the promoter fragments.
(4) We have also done some experiments on establishing the regeneration system of carnation, and have selected several kinds of comparativly ideal cultures. The following works are on proceeding.
引文
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