水稻OsBRI1基因啟動子的克隆及功能分析
發(fā)布時間：2018-12-11 21:10
【摘要】：油菜素內(nèi)酯(Brassinosteroids,BRs)是一種廣泛存在于植物中的類固醇激素,已有的研究發(fā)現(xiàn)BRs有70多種,其中在被子植物中發(fā)現(xiàn)的最具活性的兩類BRs為BL和CS。研究表明BRs是通過位于細(xì)胞膜上的跨膜類受體蛋白激酶BRI1感知和傳遞信號,進(jìn)而激活下游基因的表達(dá),調(diào)控植物生長發(fā)育的整個過程。通過GUS染色發(fā)現(xiàn)擬南芥AtBRI1在根部、葉柄、葉片、莖、花等組織中表達(dá)很高。RT-PCR檢測結(jié)果也發(fā)現(xiàn)AtBRI1在擬南芥的整個植物體內(nèi)均廣泛表達(dá),但RT-PCR檢測水稻的OsBRI1發(fā)現(xiàn)其在根部表達(dá)不明顯。因此BRI1在水稻和擬南芥中的表達(dá)存在差異,這種差異是基因本身造成的,還是由于啟動子區(qū)的差異引起的呢?為了探究其原因,我們以十字花科和禾本科物種為研究對象,首先對十字花科和禾本科BRI1基因序列進(jìn)行比對,發(fā)現(xiàn)BRI1的編碼基因比較保守;隨后對其啟動子區(qū)進(jìn)行了分析,發(fā)現(xiàn)其啟動子序列存在多樣性。因此推測,啟動子區(qū)的多態(tài)性可能導(dǎo)致了BRI1蛋白在水稻和擬南芥中表達(dá)的差異。為此,我們克隆了水稻OsBRI1基因啟動子并轉(zhuǎn)化擬南芥,比較其與AtBRI1啟動子的表達(dá)模式差異,探究BRI1蛋白在水稻和擬南芥中表達(dá)差異的原因。試驗(yàn)獲得主要結(jié)果如下:1.本試驗(yàn)首先對十字花科、禾本科植物BRI1編碼基因及其啟動子序列進(jìn)行生物信息學(xué)分析,得到BRI1的編碼序列在這兩科間保守,而啟動子序列存在多樣性。2.利用PlantCARE和PLACE網(wǎng)站上分析OsBRI1啟動子含有的順式作用元件,發(fā)現(xiàn)該序列含有多個啟動子的基本元件TATA-box和CAAT-box,包含多種植物激素響應(yīng)元件,如油菜素內(nèi)酯響應(yīng)元件、生長素響應(yīng)元件、脫落酸響應(yīng)元件及水楊酸響應(yīng)元件;還包含多種植物逆境脅迫響應(yīng)元件,如熱激應(yīng)答元件、干旱誘導(dǎo)相關(guān)的MYB結(jié)合位點(diǎn)及防衛(wèi)和脅迫響應(yīng)相關(guān)元件。同時還含有很多跟光響應(yīng)相關(guān)的元件,如G-box、Sp1、GAG-motif等。3.使用PCR技術(shù),根據(jù)已經(jīng)公布的水稻日本晴基因序列,結(jié)合表達(dá)載體pCambia2300-GUS上的酶切位點(diǎn),設(shè)計(jì)引物克隆得到水稻OsBRI1啟動子,并成功將其與植物表達(dá)載體pCambia2300-GUS連接,轉(zhuǎn)化,通過抗性篩選,得到轉(zhuǎn)基因擬南芥。4.GUS染色表明,水稻OsBRI1啟動子具有驅(qū)動GUS基因在擬南芥中廣泛表達(dá)的活性。與已報(bào)道的擬南芥AtBRI1啟動子在擬南芥上的表達(dá)模式?jīng)]有明顯差異,只是在根部沒有AtBRI1啟動子的表達(dá)量高,推測可能由于某些順式作用元件來調(diào)控基因表達(dá)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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2
本文編號：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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2
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