棉花生长素应答因子的克隆及其功能初步分析
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摘要
棉花是一种重要的全球性的经济作物。棉花纤维以其好的保暖性、保湿性、透气性等不可为其它纤维代替的优点,越来越受到世界各国的重视。同时人们对纤维品质的追求,也越来越高。随着分子生物学的发展,利用基因工程改良棉纤维品质展现了广阔的前景。如通过分离鉴定棉纤维发育相关的基因,并导入棉花中,使其过量或缺失表达,影响纤维的发育。虽然对棉花纤维的改良已有很多成功的例子,但是还未见从生长素响应因子(ARF,auxin response factor)方面的报道。
生长素在植物的生长和发育过程中起着关键的作用。据报道,生长素促进纤维细胞的伸长。纤维原始细胞在开花前3天到前2天,可以由于IAA的刺激从而获得细胞延伸的生理能力。开花前后可以由于外施IAA,明显增加胚珠的纤维数量。ARF是生长素信号转导途径中的关键因子,它可以接受生长素传递的信号,启动或是抑制下游基因的表达。
本研究通过电子克隆技术和生物信息学知识,结合RACE技术,克隆到了一个棉花中的新ARF基因,命名为ARF3(GENBANK登录号为:EF467605),并根据已知的序列,通过RT-PCR的方法获得了棉花中ARF10基因含完整读码框的cDNA序列。将以上获得的cDNA序列克隆到pCAMBIA1301表达载体上,构建了pCAMBIA1301-ARF3和pCAMBIA1301-ARF10植物表达载体。在农杆菌GV3101介导下,通过真空渗透转化技术转化模式植物拟南芥,得到了批量的转基因植株。ARF3转基因植株经抗性筛选,得到了12株T1代转基因植株;ARF10转基因植株经抗性筛选,得到11株T1代转基因植株。PCR检测证明,目的基因已成功导入模式植物拟南芥中;RT-PCR检测目的基因在转基因植株中得到表达。对转基因植株表型的观察,在得到转基因植株纯合子之后进行。
Cotton is an important global economic crop. The cotton fiber is the main product, which has become more and more important for its advantages. Compared to other fibers, it has better traping body heat,better keeping moisture and permeable. In view of these nature advantages, the world has paid more and more attention to the quality of the cotton fiber. As the development of molecular biology,the use of genetic engineering to improve the quality of the cotton fiber has a good prospect of application and extension. For example, through seperating the aim sequence from the cotton genome and conducting it into the cotton, we find the fiber of the transgenic cottons is influenced. Though there have been many successful examples of cotton fiber improvement using genetic engineering technology, the report of ARFs influence on cotton fiber is still not reported recently.
The auxin plays an important role in the process of plant upgrowth. It is reported auxin can promote the elongation of cotton fiber cell three days or two days before blossoming, the fiber cell can get elonggation ability throungh IAA-stimulation. Adding to IAA before blooming previously increases the cotton fiber quantity. ARF is the key factor in auxin signal transduction pathway, which can receive the signals caused by auxin and activate or repress the expression of the backward genes.
In this research,through electronic cloning technology and bioinformation knowledge, we have seperated a new ARF gene by RACE technology, named ARF3. The GenBank accession number is EF467605. We have also got the whole cDNA sequence of ARF10 by RT-PCR method. We connect the two cDNA sequences to plant expression vector pCAMBIA1301, named pCAMBIA1301-ARF3 and pCAMBIA1301-ARF10. By vacuum infiltration technology, the two plant expression vectors have been transmitted into Arabidoposis thaliana. We have got many transgenic plants. The PCR detection proves the sequnece has successfully recombined with the genome of Arabidoposis thaliana. We have also proved its expression by the method of RT-PCR.We will take note of the phenotype of the transgenic plants after we get homozygotes.
生长素在植物的生长和发育过程中起着关键的作用。据报道,生长素促进纤维细胞的伸长。纤维原始细胞在开花前3天到前2天,可以由于IAA的刺激从而获得细胞延伸的生理能力。开花前后可以由于外施IAA,明显增加胚珠的纤维数量。ARF是生长素信号转导途径中的关键因子,它可以接受生长素传递的信号,启动或是抑制下游基因的表达。
本研究通过电子克隆技术和生物信息学知识,结合RACE技术,克隆到了一个棉花中的新ARF基因,命名为ARF3(GENBANK登录号为:EF467605),并根据已知的序列,通过RT-PCR的方法获得了棉花中ARF10基因含完整读码框的cDNA序列。将以上获得的cDNA序列克隆到pCAMBIA1301表达载体上,构建了pCAMBIA1301-ARF3和pCAMBIA1301-ARF10植物表达载体。在农杆菌GV3101介导下,通过真空渗透转化技术转化模式植物拟南芥,得到了批量的转基因植株。ARF3转基因植株经抗性筛选,得到了12株T1代转基因植株;ARF10转基因植株经抗性筛选,得到11株T1代转基因植株。PCR检测证明,目的基因已成功导入模式植物拟南芥中;RT-PCR检测目的基因在转基因植株中得到表达。对转基因植株表型的观察,在得到转基因植株纯合子之后进行。
Cotton is an important global economic crop. The cotton fiber is the main product, which has become more and more important for its advantages. Compared to other fibers, it has better traping body heat,better keeping moisture and permeable. In view of these nature advantages, the world has paid more and more attention to the quality of the cotton fiber. As the development of molecular biology,the use of genetic engineering to improve the quality of the cotton fiber has a good prospect of application and extension. For example, through seperating the aim sequence from the cotton genome and conducting it into the cotton, we find the fiber of the transgenic cottons is influenced. Though there have been many successful examples of cotton fiber improvement using genetic engineering technology, the report of ARFs influence on cotton fiber is still not reported recently.
The auxin plays an important role in the process of plant upgrowth. It is reported auxin can promote the elongation of cotton fiber cell three days or two days before blossoming, the fiber cell can get elonggation ability throungh IAA-stimulation. Adding to IAA before blooming previously increases the cotton fiber quantity. ARF is the key factor in auxin signal transduction pathway, which can receive the signals caused by auxin and activate or repress the expression of the backward genes.
In this research,through electronic cloning technology and bioinformation knowledge, we have seperated a new ARF gene by RACE technology, named ARF3. The GenBank accession number is EF467605. We have also got the whole cDNA sequence of ARF10 by RT-PCR method. We connect the two cDNA sequences to plant expression vector pCAMBIA1301, named pCAMBIA1301-ARF3 and pCAMBIA1301-ARF10. By vacuum infiltration technology, the two plant expression vectors have been transmitted into Arabidoposis thaliana. We have got many transgenic plants. The PCR detection proves the sequnece has successfully recombined with the genome of Arabidoposis thaliana. We have also proved its expression by the method of RT-PCR.We will take note of the phenotype of the transgenic plants after we get homozygotes.
引文
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