水稻OsBRI1基因启动子的克隆及功能分析
发布时间：2018-12-11 21:10
【摘要】：油菜素内酯(Brassinosteroids,BRs)是一种广泛存在于植物中的类固醇激素,已有的研究发现BRs有70多种,其中在被子植物中发现的最具活性的两类BRs为BL和CS。研究表明BRs是通过位于细胞膜上的跨膜类受体蛋白激酶BRI1感知和传递信号,进而激活下游基因的表达,调控植物生长发育的整个过程。通过GUS染色发现拟南芥AtBRI1在根部、叶柄、叶片、茎、花等组织中表达很高。RT-PCR检测结果也发现AtBRI1在拟南芥的整个植物体内均广泛表达,但RT-PCR检测水稻的OsBRI1发现其在根部表达不明显。因此BRI1在水稻和拟南芥中的表达存在差异,这种差异是基因本身造成的,还是由于启动子区的差异引起的呢?为了探究其原因,我们以十字花科和禾本科物种为研究对象,首先对十字花科和禾本科BRI1基因序列进行比对,发现BRI1的编码基因比较保守;随后对其启动子区进行了分析,发现其启动子序列存在多样性。因此推测,启动子区的多态性可能导致了BRI1蛋白在水稻和拟南芥中表达的差异。为此,我们克隆了水稻OsBRI1基因启动子并转化拟南芥,比较其与AtBRI1启动子的表达模式差异,探究BRI1蛋白在水稻和拟南芥中表达差异的原因。试验获得主要结果如下:1.本试验首先对十字花科、禾本科植物BRI1编码基因及其启动子序列进行生物信息学分析,得到BRI1的编码序列在这两科间保守,而启动子序列存在多样性。2.利用PlantCARE和PLACE网站上分析OsBRI1启动子含有的顺式作用元件,发现该序列含有多个启动子的基本元件TATA-box和CAAT-box,包含多种植物激素响应元件,如油菜素内酯响应元件、生长素响应元件、脱落酸响应元件及水杨酸响应元件;还包含多种植物逆境胁迫响应元件,如热激应答元件、干旱诱导相关的MYB结合位点及防卫和胁迫响应相关元件。同时还含有很多跟光响应相关的元件,如G-box、Sp1、GAG-motif等。3.使用PCR技术,根据已经公布的水稻日本晴基因序列,结合表达载体pCambia2300-GUS上的酶切位点,设计引物克隆得到水稻OsBRI1启动子,并成功将其与植物表达载体pCambia2300-GUS连接,转化,通过抗性筛选,得到转基因拟南芥。4.GUS染色表明,水稻OsBRI1启动子具有驱动GUS基因在拟南芥中广泛表达的活性。与已报道的拟南芥AtBRI1启动子在拟南芥上的表达模式没有明显差异,只是在根部没有AtBRI1启动子的表达量高,推测可能由于某些顺式作用元件来调控基因表达量的高低。
[Abstract]:Brassinolide (Brassinosteroids,BRs) is a kind of steroid hormone widely found in plants. Some studies have found that there are more than 70 kinds of BRs, of which the two most active BRs found in angiosperms are BL and CS.. It has been shown that BRs is a transmembrane receptor-like protein kinase (BRI1) which is located on the cell membrane to sense and transmit signals, which activates the expression of downstream genes and regulates the whole process of plant growth and development. By GUS staining, it was found that Arabidopsis AtBRI1 was highly expressed in root, petiole, leaf, stem, flower and so on. RT-PCR assay also showed that AtBRI1 was widely expressed in the whole plant of Arabidopsis thaliana. But the OsBRI1 of rice detected by RT-PCR was not significantly expressed in the root. Therefore, there are differences in the expression of BRI1 between rice and Arabidopsis. Is this difference caused by the gene itself or by the difference in promoter region? In order to find out the reason, we studied the species of Cruciferae and Gramineae. First, we compared the sequence of BRI1 gene between Cruciferae and Gramineae, and found that the coding genes of BRI1 were conserved. Then the promoter region was analyzed and the diversity of its promoter sequence was found. It is speculated that the polymorphism of promoter region may lead to the difference of BRI1 protein expression in rice and Arabidopsis thaliana. Therefore, we cloned rice OsBRI1 gene promoter and transformed Arabidopsis thaliana, compared its expression pattern with AtBRI1 promoter, and explored the reasons for the difference of BRI1 protein expression in rice and Arabidopsis thaliana. The main results are as follows: 1. In this experiment, the BRI1 coding genes and their promoter sequences of cruciferous and gramineous plants were analyzed by bioinformatics, and the coding sequences of BRI1 were conserved between the two families, and the promoter sequences were diverse. 2. Using PlantCARE and PLACE websites to analyze the cis-acting elements contained in the OsBRI1 promoter, it was found that the basic elements of the sequence containing multiple promoters, TATA-box and CAAT-box, contained a variety of plant hormone response elements, such as Brassinolide response elements. Auxin response element, abscisic acid response element and salicylic acid response element; There are also a variety of plant stress response elements, such as heat shock response elements, drought induced related MYB binding sites and defense and stress response related elements. It also contains many components related to light response, such as G-box Sp1, GAG-motif, etc. Using PCR technique, according to the published rice Nippon gene sequence and the restriction site of the expression vector pCambia2300-GUS, a primer was designed to clone the rice OsBRI1 promoter, and the promoter was successfully ligated and transformed into the plant expression vector pCambia2300-GUS. Transgenic Arabidopsis thaliana was obtained by resistance screening. 4.GUS staining showed that rice OsBRI1 promoter had the activity of driving the expression of GUS gene in Arabidopsis thaliana. The expression pattern of AtBRI1 promoter in Arabidopsis thaliana was not significantly different from that in Arabidopsis thaliana, but there was no high expression of AtBRI1 promoter in root, which suggested that some cis-acting elements might regulate the expression of gene in Arabidopsis thaliana.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
本文编号：2373227
[Abstract]:Brassinolide (Brassinosteroids,BRs) is a kind of steroid hormone widely found in plants. Some studies have found that there are more than 70 kinds of BRs, of which the two most active BRs found in angiosperms are BL and CS.. It has been shown that BRs is a transmembrane receptor-like protein kinase (BRI1) which is located on the cell membrane to sense and transmit signals, which activates the expression of downstream genes and regulates the whole process of plant growth and development. By GUS staining, it was found that Arabidopsis AtBRI1 was highly expressed in root, petiole, leaf, stem, flower and so on. RT-PCR assay also showed that AtBRI1 was widely expressed in the whole plant of Arabidopsis thaliana. But the OsBRI1 of rice detected by RT-PCR was not significantly expressed in the root. Therefore, there are differences in the expression of BRI1 between rice and Arabidopsis. Is this difference caused by the gene itself or by the difference in promoter region? In order to find out the reason, we studied the species of Cruciferae and Gramineae. First, we compared the sequence of BRI1 gene between Cruciferae and Gramineae, and found that the coding genes of BRI1 were conserved. Then the promoter region was analyzed and the diversity of its promoter sequence was found. It is speculated that the polymorphism of promoter region may lead to the difference of BRI1 protein expression in rice and Arabidopsis thaliana. Therefore, we cloned rice OsBRI1 gene promoter and transformed Arabidopsis thaliana, compared its expression pattern with AtBRI1 promoter, and explored the reasons for the difference of BRI1 protein expression in rice and Arabidopsis thaliana. The main results are as follows: 1. In this experiment, the BRI1 coding genes and their promoter sequences of cruciferous and gramineous plants were analyzed by bioinformatics, and the coding sequences of BRI1 were conserved between the two families, and the promoter sequences were diverse. 2. Using PlantCARE and PLACE websites to analyze the cis-acting elements contained in the OsBRI1 promoter, it was found that the basic elements of the sequence containing multiple promoters, TATA-box and CAAT-box, contained a variety of plant hormone response elements, such as Brassinolide response elements. Auxin response element, abscisic acid response element and salicylic acid response element; There are also a variety of plant stress response elements, such as heat shock response elements, drought induced related MYB binding sites and defense and stress response related elements. It also contains many components related to light response, such as G-box Sp1, GAG-motif, etc. Using PCR technique, according to the published rice Nippon gene sequence and the restriction site of the expression vector pCambia2300-GUS, a primer was designed to clone the rice OsBRI1 promoter, and the promoter was successfully ligated and transformed into the plant expression vector pCambia2300-GUS. Transgenic Arabidopsis thaliana was obtained by resistance screening. 4.GUS staining showed that rice OsBRI1 promoter had the activity of driving the expression of GUS gene in Arabidopsis thaliana. The expression pattern of AtBRI1 promoter in Arabidopsis thaliana was not significantly different from that in Arabidopsis thaliana, but there was no high expression of AtBRI1 promoter in root, which suggested that some cis-acting elements might regulate the expression of gene in Arabidopsis thaliana.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2
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