本文關(guān)鍵詞： 甘藍(lán)型油菜 擬南芥 AGPs RNAi 遺傳轉(zhuǎn)化 根　出處：《湖北大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：阿拉伯半乳糖蛋白(arabinogalactan-proteins,AGPs)是一類高度糖基化的糖蛋白分子,其氨基酸序列中富含脯氨酸/羥脯氨酸。AGPs廣泛分布于植物各種組織和細(xì)胞中,屬于富含經(jīng)脯氨酸的糖蛋白(Hyp-riehglyeoproteins,HRGPs)家族。阿拉伯半乳糖蛋白參與了植物細(xì)胞的增殖、分化、細(xì)胞程序性死亡、植物細(xì)胞胚胎發(fā)生等活動(dòng)。1.本文利用甘藍(lán)型油菜(BrassicanapusL.)作為實(shí)驗(yàn)的研究對(duì)象。選取了同樣為十字花科的擬南芥中已經(jīng)測(cè)定序列的兩種AGP相關(guān)基因AtAGP3、AtAGP8,通過(guò)已有的文獻(xiàn)及研究者已測(cè)出的序列合成基因,PCR產(chǎn)物先經(jīng)SpeI/SacI雙酶切,克隆至pTCK303載體,測(cè)序正確后再用BamHI/KpnI第二次雙酶切克隆,以此來(lái)構(gòu)建油菜的RNA干擾載體。同時(shí)以油菜種子萌發(fā)并準(zhǔn)備工程感染菌株,將構(gòu)建好的pTCK303-AtAGP3-1RNAi載體質(zhì)粒、pTCK303-AtAGP3-2 RNAi 載體質(zhì)粒、pTCK303-AtAGP3-3 RNAi 載體質(zhì)粒、pTCK303-AtAGP8-1 RNAi 載體質(zhì)粒、pTCK303-AtAGP8-2 RNAi 載體質(zhì)粒、pTCK303-AtAGP8-3 RNAi載體質(zhì)粒,分別轉(zhuǎn)化農(nóng)桿菌感受態(tài)EHA105后將其轉(zhuǎn)化到油菜子葉柄中,并培養(yǎng)其愈傷組織至生根。2.本實(shí)驗(yàn)還研究了人工合成的βGlcY對(duì)擬南芥根生長(zhǎng)發(fā)育的影響。將擬南芥種子分別于1/2 MS 培養(yǎng)基,1/2 MS+10 μM αG1cY培養(yǎng)基,1/2 MS+10μM βGlcY培養(yǎng)基,1/2 MS+10μM βGlcY1/2MS培養(yǎng)基萌發(fā)培養(yǎng)。發(fā)現(xiàn):(1)用βGlcY試劑處理擬南芥種子,隨著種子萌發(fā),βGlcY與擬南芥根表皮細(xì)胞壁的AGPs結(jié)合使根部呈現(xiàn)紅色,與對(duì)照相比,βGlcY顯著抑制了根的生長(zhǎng)。隨著根重新轉(zhuǎn)入1/2 MS培養(yǎng)基上繼續(xù)培養(yǎng),使抑制得以恢復(fù)。進(jìn)一步細(xì)胞學(xué)觀察表明,AGPs與βGlcY相互作用導(dǎo)致根伸長(zhǎng)區(qū)的細(xì)胞體積變小,表皮細(xì)胞產(chǎn)生明顯的膨脹現(xiàn)象。(2)對(duì)照組擬南芥根的維管細(xì)胞部位能檢測(cè)到大量PIN1的表達(dá),且PIN1定位于細(xì)胞的形態(tài)學(xué)基部。而用βGlcY試劑處理過(guò)的擬南芥根部只能檢測(cè)到極少量分布的PIN1蛋白,說(shuō)明βGlcY阻礙了 PIN1蛋白的分布,從而影響了生長(zhǎng)素向根部的極性運(yùn)輸。用βGlcY試劑處理4天后再轉(zhuǎn)移到1/2 MS培養(yǎng)基上,檢測(cè)到擬南芥根部的熒光亮度回升,表明PIN1蛋白的含量有所增加。(3)對(duì)照組擬南芥根部在根冠分生組織細(xì)胞中能檢測(cè)到DR5蛋白的表達(dá)。但βGlcY試劑處理過(guò)后,擬南芥根冠分生組織細(xì)胞的DR5蛋白表達(dá)增強(qiáng)。用βGlcY試劑處理4天后再轉(zhuǎn)移到1/2 MS培養(yǎng)基上培養(yǎng)4天,發(fā)現(xiàn)DR5在根冠分生組織部位的表達(dá)恢復(fù)正常。這些結(jié)果說(shuō)明βGlcY處理擬南芥根后,導(dǎo)致生長(zhǎng)素響應(yīng)的蛋白過(guò)度聚集在根冠分生組織部位,抑制了根的生長(zhǎng)。(4)PIN2定位于根的表皮細(xì)胞和皮層細(xì)胞的形態(tài)學(xué)頂端質(zhì)膜上,說(shuō)明生長(zhǎng)素在PIN2蛋白作用下從根的基部經(jīng)過(guò)表皮細(xì)胞向根的上方運(yùn)輸,并維持表皮細(xì)胞的生長(zhǎng)和發(fā)育。但用βGlcY試劑處理后,根表皮細(xì)胞的PIN2蛋白表達(dá)減少或不表達(dá),說(shuō)明βGlcY與AGPs相互作用改變了 PIN2蛋白在細(xì)胞質(zhì)膜上的極性定位,阻礙了生長(zhǎng)素的極性回流,從而影響表皮細(xì)胞的模式形成,引起表皮細(xì)胞的發(fā)生膨脹。但重新轉(zhuǎn)移到1/2MS培養(yǎng)基上培養(yǎng)后,這種阻礙是可逆的。
[Abstract]:Arabia arabinogalactan proteins (arabinogalactan-proteins, AGPs) is a kind of glycoprotein highly glycosylated, its amino acid sequence is rich in proline / hydroxyproline.AGPs is widely distributed in various tissues and plant cells, the proline rich glycoprotein belonging to the family (Hyp-riehglyeoproteins, HRGPs). Arabia arabinogalactan proteins involved in plant cell proliferation and differentiation, programmed cell death, plant somatic embryogenesis and other activities.1. using Brassica napus (BrassicanapusL.) as the research object. The experiment selected two AGP AtAGP3 gene sequence has been determined, the same as the cruciferous Arabidopsis AtAGP8 gene sequence synthesis through literature and previous studies have been measured. The PCR products by SpeI/SacI and double enzyme digestion, cloned into pTCK303 vector. After sequencing and BamHI/KpnI second double enzyme digestion to clone. Construction of RNA interference vector. At the same time to rape rape seeds and prepare engineering strains, the pTCK303-AtAGP3-1RNAi plasmid was constructed. PTCK303-AtAGP3-2 RNAi vector plasmid pTCK303-AtAGP3-3 RNAi plasmid pTCK303-AtAGP8-1 RNAi plasmid, pTCK303-AtAGP8-2 RNAi plasmid, pTCK303-AtAGP8-3 RNAi plasmid was transformed into Agrobacterium competent EHA105 transformed into rape sub petiole, and develop callus to rooting of.2. this paper has also studied the effects of beta synthetic GlcY on the growth and development of Arabidopsis thaliana roots. The Arabidopsis seeds in 1/2 MS medium, 1/2 MS+10 M alpha G1cY medium, 1/2 MS+10 M beta GlcY medium, 1/2 MS+10 M beta GlcY1/2MS medium germination culture. Found that: (1) treated with beta GlcY reagent with Arabidopsis seed, seed germination, beta GlcY and Arabidopsis root epidermal cell wall The AGPs binding to the root red, compared with the control group, P GlcY significantly inhibited root growth. With the root 1/2 re transferred to MS medium to culture, to suppress the recovery. Further cytological observations showed that AGPs and GlcY interact to beta cell body and root elongation zone is the product of smaller, epidermal cells have obvious the phenomenon of expansion. (2) the control expression of a large number of PIN1 cell fractions of Arabidopsis root vascular morphology, base and localization of PIN1 in cells. In Arabidopsis roots can only detect beta GlcY reagent treated to a very small amount of the distribution of the PIN1 protein, indicating that P GlcY hinders the distribution of PIN1 protein. Which affect the auxin polar transport to the roots. Beta GlcY reagent for 4 days and then transferred to 1/2 MS medium, detected the fluorescence brightness of Arabidopsis roots rise, showed that the content of PIN1 protein increased in the control group (3). Arabidopsis roots can detect the expression of DR5 protein in root meristematic cells. But the beta GlcY reagent after processing, enhance the expression of Arabidopsis meristem cell DR5 protein. After 4 days and then transferred to the 1/2 MS medium for 4 days using beta GlcY reagent treatment, the DR5 expression in the root cap meristem. These results returned to normal position TGF GlcY treated Arabidopsis root, resulting in excessive accumulation of auxin response protein in root meristem region, inhibit root growth. (4) PIN2 localized in the root epidermal and cortical cells of the apical membrane morphology, indicating that auxin in PIN2 protein function from the base of the root epidermal cells to transport through the above root, and maintain the epidermal cell growth and development. But with the beta GlcY reagent treatment, the expression of PIN2 protein in root epidermal cells reduced or no expression of TGF GlcY and AGPs interaction The polar location of PIN2 protein on the cytoplasmic membrane changed, which blocked the polar regressions of ghrelin, thereby affecting the formation of epidermal cells and causing the expansion of epidermal cells. However, when retransferred to 1/2MS medium, the hindrance was reversible.

【學(xué)位授予單位】：湖北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q943.2

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1 胡欣;βGlcY對(duì)擬南芥根發(fā)育的影響及AGPs基因干擾載體的構(gòu)建與油菜遺傳轉(zhuǎn)化[D];湖北大學(xué);2016年

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本文編號(hào)：1456904