花特异启动子调控的蓝色基因表达载体构建及功能研究
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摘要
百合(LiLium spp.)花姿秀美,花茎挺拔,是点缀庭院的名贵观赏花卉,是世界上著名的五大鲜切花之一,深受世界各国人民的喜爱。自然界的百合有白色、粉红色、红色、黄色等,但唯独缺乏蓝色。通过基因工程手段培育出蓝色百合新品系是目前花卉分子育种的研究重点。花色苷作为高等植物花色形成中最具影响力的因素,其生物合成途径已经研究得很清楚,大量相关基因已被克隆鉴定。
为了探索百合蓝色花育种途径,前期工作中,我们从东方百合'Sorbonne'花瓣中克隆了查尔酮合成酶(CHS)启动子序列,并对其进行了初步验证;从风信子花瓣中克隆获得编码二氢黄酮醇4-还原酶的DFR基因序列;从蓝紫色矮牵牛花瓣中克隆获得编码类黄酮3',5'羟基化酶的Hf 1基因序列;从矮牵牛花瓣中克隆获得编码B5蛋白编码的DifF基因序列。本试验在此基础上,构建了多基因表达载体,然后通过农杆菌介导法转化模式植物矮牵牛,以期为百合蓝色花育种提供参考和依据。本研究取得的主要结果如下:
1.构建了多基因共表达载体
将花特异表达启动子PchsA与矮牵牛中编码类黄酮3',5'羟基化酶(F3'5'H)的Hf1基因融合,构建了百合花特异表达启动子调控的Hf1基因植物中间表达载体pCAM-Hf1,然后将DFR基因和DifF基因分别构建到载体pCAM-Hf1中,最终得到多基因共表达载体pCAM-Hf1-DFR和pCAM-Hf1-DifF。
2.转化模式植物矮牵牛并进行PCR检测
通过农杆菌介导的叶盘法转化矮牵牛,经潮霉素抗性筛选后获得若干阳性植株。采用CTAB法提取潮霉素阳性植株的总DNA,用查尔酮合成酶启动子、矮牵牛Hf1基因、风信子DFR基因或矮牵牛DifF基因的特异引物分别进行了PCR检测,转基因矮牵牛DNA分别扩增出了预期大小的特异PCR扩增片段,初步获得了转基因矮牵牛植株。
With the graceful posture, tall and straight flower stalk, Lily(LiLium spp.)is suitable for using in the garden. As one of the most famous cut flowers in the world, it is widely favored by the peoples from various countries. In the nature, Lily owns various colors such as white, pink, red and yellow, but lacks blue flowers. Breeding blue Lily flower is the research emphasis of flower molecular breeding via genetic engineering. As the most influential factors in the color formation of higher plants, anthocyanin biosynthetic pathway has been studied clearly, a large number of related genes have been cloned and identified.
For seeking breeding way of blue lily flower, in the previous work, we cloned chalcone synthase promoter sequences from petals of orential Lilium 'Sorbonne' and verified its functions initially; cloned DFR gene sequences coding dihydroflavonol 4-reductase from petals of Hyacinthus orientalis; cloned Hf 1gene sequences coding flavonoid 3 ', 5'- hydroxylase from royal purple petals of Petunia hybrida; cloned DifF gene sequences coding B5 protein from royal purple petals of Petunia hybrida. On this basis, our experiment have constructed two multi-gene expression vector, and transformed model plant petunia via Agrobacterium in order to reference for blue Lily flower breeding. Main results were obtained as follows:
1.Construction of multi-genes expression vectors
Inosculating flower specific promoter PchsA and Hf1 gene, we constructed intermediate plant expression vector pCAM-Hf1, after that constructed DFR and DifF genes into pCAM-Hf1, respectively. Finally, we gained two multi-genes expression vectors pCAM -Hf1-DFR and pCAM-Hf1-DifF;
2. Transformation of petunia and PCR detection of hygromycin positive plants
We transformed model plant petunia via Agrobacterium. After hygromycin resistance screening, gained some hygromycin positive plants. Extracted DNA of them using CTAB method and were carried out PCR detection using corresponding genes specific primer, transgenic petunia DNA amplified expected peculiar PCR fragments, respectively. We obtained transgenic petunia plants initially.
为了探索百合蓝色花育种途径,前期工作中,我们从东方百合'Sorbonne'花瓣中克隆了查尔酮合成酶(CHS)启动子序列,并对其进行了初步验证;从风信子花瓣中克隆获得编码二氢黄酮醇4-还原酶的DFR基因序列;从蓝紫色矮牵牛花瓣中克隆获得编码类黄酮3',5'羟基化酶的Hf 1基因序列;从矮牵牛花瓣中克隆获得编码B5蛋白编码的DifF基因序列。本试验在此基础上,构建了多基因表达载体,然后通过农杆菌介导法转化模式植物矮牵牛,以期为百合蓝色花育种提供参考和依据。本研究取得的主要结果如下:
1.构建了多基因共表达载体
将花特异表达启动子PchsA与矮牵牛中编码类黄酮3',5'羟基化酶(F3'5'H)的Hf1基因融合,构建了百合花特异表达启动子调控的Hf1基因植物中间表达载体pCAM-Hf1,然后将DFR基因和DifF基因分别构建到载体pCAM-Hf1中,最终得到多基因共表达载体pCAM-Hf1-DFR和pCAM-Hf1-DifF。
2.转化模式植物矮牵牛并进行PCR检测
通过农杆菌介导的叶盘法转化矮牵牛,经潮霉素抗性筛选后获得若干阳性植株。采用CTAB法提取潮霉素阳性植株的总DNA,用查尔酮合成酶启动子、矮牵牛Hf1基因、风信子DFR基因或矮牵牛DifF基因的特异引物分别进行了PCR检测,转基因矮牵牛DNA分别扩增出了预期大小的特异PCR扩增片段,初步获得了转基因矮牵牛植株。
With the graceful posture, tall and straight flower stalk, Lily(LiLium spp.)is suitable for using in the garden. As one of the most famous cut flowers in the world, it is widely favored by the peoples from various countries. In the nature, Lily owns various colors such as white, pink, red and yellow, but lacks blue flowers. Breeding blue Lily flower is the research emphasis of flower molecular breeding via genetic engineering. As the most influential factors in the color formation of higher plants, anthocyanin biosynthetic pathway has been studied clearly, a large number of related genes have been cloned and identified.
For seeking breeding way of blue lily flower, in the previous work, we cloned chalcone synthase promoter sequences from petals of orential Lilium 'Sorbonne' and verified its functions initially; cloned DFR gene sequences coding dihydroflavonol 4-reductase from petals of Hyacinthus orientalis; cloned Hf 1gene sequences coding flavonoid 3 ', 5'- hydroxylase from royal purple petals of Petunia hybrida; cloned DifF gene sequences coding B5 protein from royal purple petals of Petunia hybrida. On this basis, our experiment have constructed two multi-gene expression vector, and transformed model plant petunia via Agrobacterium in order to reference for blue Lily flower breeding. Main results were obtained as follows:
1.Construction of multi-genes expression vectors
Inosculating flower specific promoter PchsA and Hf1 gene, we constructed intermediate plant expression vector pCAM-Hf1, after that constructed DFR and DifF genes into pCAM-Hf1, respectively. Finally, we gained two multi-genes expression vectors pCAM -Hf1-DFR and pCAM-Hf1-DifF;
2. Transformation of petunia and PCR detection of hygromycin positive plants
We transformed model plant petunia via Agrobacterium. After hygromycin resistance screening, gained some hygromycin positive plants. Extracted DNA of them using CTAB method and were carried out PCR detection using corresponding genes specific primer, transgenic petunia DNA amplified expected peculiar PCR fragments, respectively. We obtained transgenic petunia plants initially.
引文
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