βGlcY对拟南芥根发育的影响及AGPs基因干扰载体的构建与油菜遗传转化

发布时间：2018-01-23 06:07

本文关键词： 甘蓝型油菜拟南芥 AGPs RNAi 遗传转化根　出处：《湖北大学》2016年硕士论文　论文类型：学位论文

【摘要】：阿拉伯半乳糖蛋白(arabinogalactan-proteins,AGPs)是一类高度糖基化的糖蛋白分子,其氨基酸序列中富含脯氨酸/羟脯氨酸。AGPs广泛分布于植物各种组织和细胞中,属于富含经脯氨酸的糖蛋白(Hyp-riehglyeoproteins,HRGPs)家族。阿拉伯半乳糖蛋白参与了植物细胞的增殖、分化、细胞程序性死亡、植物细胞胚胎发生等活动。1.本文利用甘蓝型油菜(BrassicanapusL.)作为实验的研究对象。选取了同样为十字花科的拟南芥中已经测定序列的两种AGP相关基因AtAGP3、AtAGP8,通过已有的文献及研究者已测出的序列合成基因,PCR产物先经SpeI/SacI双酶切,克隆至pTCK303载体,测序正确后再用BamHI/KpnI第二次双酶切克隆,以此来构建油菜的RNA干扰载体。同时以油菜种子萌发并准备工程感染菌株,将构建好的pTCK303-AtAGP3-1RNAi载体质粒、pTCK303-AtAGP3-2 RNAi 载体质粒、pTCK303-AtAGP3-3 RNAi 载体质粒、pTCK303-AtAGP8-1 RNAi 载体质粒、pTCK303-AtAGP8-2 RNAi 载体质粒、pTCK303-AtAGP8-3 RNAi载体质粒,分别转化农杆菌感受态EHA105后将其转化到油菜子叶柄中,并培养其愈伤组织至生根。2.本实验还研究了人工合成的βGlcY对拟南芥根生长发育的影响。将拟南芥种子分别于1/2 MS 培养基,1/2 MS+10 μM αG1cY培养基,1/2 MS+10μM βGlcY培养基,1/2 MS+10μM βGlcY1/2MS培养基萌发培养。发现:(1)用βGlcY试剂处理拟南芥种子,随着种子萌发,βGlcY与拟南芥根表皮细胞壁的AGPs结合使根部呈现红色,与对照相比,βGlcY显著抑制了根的生长。随着根重新转入1/2 MS培养基上继续培养,使抑制得以恢复。进一步细胞学观察表明,AGPs与βGlcY相互作用导致根伸长区的细胞体积变小,表皮细胞产生明显的膨胀现象。(2)对照组拟南芥根的维管细胞部位能检测到大量PIN1的表达,且PIN1定位于细胞的形态学基部。而用βGlcY试剂处理过的拟南芥根部只能检测到极少量分布的PIN1蛋白,说明βGlcY阻碍了 PIN1蛋白的分布,从而影响了生长素向根部的极性运输。用βGlcY试剂处理4天后再转移到1/2 MS培养基上,检测到拟南芥根部的荧光亮度回升,表明PIN1蛋白的含量有所增加。(3)对照组拟南芥根部在根冠分生组织细胞中能检测到DR5蛋白的表达。但βGlcY试剂处理过后,拟南芥根冠分生组织细胞的DR5蛋白表达增强。用βGlcY试剂处理4天后再转移到1/2 MS培养基上培养4天,发现DR5在根冠分生组织部位的表达恢复正常。这些结果说明βGlcY处理拟南芥根后,导致生长素响应的蛋白过度聚集在根冠分生组织部位,抑制了根的生长。(4)PIN2定位于根的表皮细胞和皮层细胞的形态学顶端质膜上,说明生长素在PIN2蛋白作用下从根的基部经过表皮细胞向根的上方运输,并维持表皮细胞的生长和发育。但用βGlcY试剂处理后,根表皮细胞的PIN2蛋白表达减少或不表达,说明βGlcY与AGPs相互作用改变了 PIN2蛋白在细胞质膜上的极性定位,阻碍了生长素的极性回流,从而影响表皮细胞的模式形成,引起表皮细胞的发生膨胀。但重新转移到1/2MS培养基上培养后,这种阻碍是可逆的。
[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.

【学位授予单位】：湖北大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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