昼夜温差对铁皮石斛原球茎多糖含量的影响及机理研究

发布时间：2018-03-09 20:34

本文选题：铁皮石斛　切入点：多糖　出处：《福建农林大学》2015年硕士论文　论文类型：学位论文

【摘要】：铁皮石斛(Dendrobium officinale),是兰科石斛属药用植物,有着非常独特的价值。本研究以铁皮石斛原球茎为材料,探讨了有利于原球茎增殖、保存及多糖积累的最佳培养条件,并初步建立液体培养系,以测定其在不同昼夜温差下的多糖含量,在此基础上克隆了多糖合成途径中的2个关键的糖基转移酶基因—DoGAUT1和DoPGSIP6基因,并对该基因在不同昼夜温差下进行定量表达分析。主要研究结果如下：1铁皮石斛离体培养条件的优化选取铁皮石斛的原球茎为材料,优化铁皮石斛原球茎增殖与保存的离体培养条件。①采用单因素试验设计,比较了6-BA、椰乳、土豆配合使用对原球茎增殖的影响,试验数据得出1/2 MS+土豆泥50g/L较适合铁皮石斛原球茎增殖,添力(?)2.0 mg/L 6-BA后,原球茎鲜重增殖系数增高,但原球茎部分分化。②改变1/2 MS中KNO3、KH2PO4的比例,比较其对铁皮石斛原球茎增殖的影响,试验结果表明,当KNO3, KH2PO4分别为对照的0.5倍,1倍时,铁皮石斛原球茎增殖系数虽最高,但原球茎少量黄化,对照1/2 MS较适合铁皮石斛原球茎增殖。③对比不同的硝态氮和铵态氮配比对铁皮石斛原球茎增殖效果的影响,试验结果表明,当KNO3, NH4NO3均为对照的0.5倍且土豆泥为50g/L的培养基和1/2 MS+土豆泥50 g/L增殖系数相最高。④比较不同浓度的姜黄素和甜菜红苷对铁皮石斛原球茎离体保存的效果的影响,结果显示姜黄素为0.1-1.0g/L可以抑制原球茎生长,甜菜红苷浓度为0.1g/L保存效果最好。⑤以诱导出的铁皮石斛原球茎为材料,接种1/2 MS+土豆提取液50g/L的液体培养基中,比较了不同时间下铁皮石斛培养物生物量的变化,试验结果表明,在整个生育期内,原球茎的鲜重变化曲线为S型,30-40 d快速生长。2铁皮石斛离体培养物在不同昼夜温差处理条件下多糖含量变化以诱导出的铁皮石斛原球茎为材料,比较了不同时间下铁皮石斛培养物多糖的变化,在整个生长周期,原球茎多糖变化曲线呈S型。选取培育30 d的铁皮石斛原球茎为材料,对比了铁皮石斛多糖含量在不同昼夜温差下的差异。试验结果表明,处理温度为25℃/10℃,处理2d的多糖含量最高,且在一定的处理时间范围内,多糖含量先增加后下降的趋势；处理温度为25℃/15℃,该温度下多糖的含量随着处理时间的增加而增加,该温差是最适合促进次生代谢物多糖积累的温度；处理温度为25℃/20℃,多糖含量呈递增趋势,略低于相同时间下温度为25℃/15℃的处理。3铁皮石斛原球茎DoGAUT1和DoPGSIP6基因的克隆及生物信息学分析应用同源克隆与RACE技术结合,以铁皮石斛原球茎为材料,克隆了两个糖基转移酶基因DoGAUT1和DoPGSIP6,全长分别为2518、2010 bp,分别编码了705、549个氨基酸,登入号为1740985。生物信息学研究表明,DoGAUT1为亲水性跨膜蛋白,不含信号肽,形成卷曲螺旋结构,亚细胞定位于高尔基体。DoPGSIP6为疏水性跨膜蛋白,含信号肽,无卷曲螺旋结构,亚细胞定位于质膜。它们同属于GT8家族中的GT-A类的糖基转移酶,DoGAUT1蛋白在第394-692位含有典型的GT8_like_1未知功能的保守结构域,多个配体结合位点、2个金属离子结合位点。而DoPGSIP6蛋白在第29-276位含有GT8_Glycogenin保守结构域,多个底物结合位点、2个锰结合位点和一个保守基因序列DXD,该基序主要负责将不同的糖添加到糖链、碳酸盐、蛋白上。此外,DoPGSIP6蛋白在第448-522位含有CCC1_like保守结构域,它可能具有固氮的作用。进行进化树构建时发现DoGAUT1和DoPGSIP6基因推定的氨基酸序列分别与二穗短柄草,大豆进化关系最近,而与番茄、甜橙亲缘关系较远。4铁皮石斛DoGAUT1和DoPGSIP6基因在不同昼夜温差下的定量表达分析以18S rRNA为内参基因,对DoGAUT1基因和DoPGSIP6基因在不同昼夜温差下的表达水平进行相对定量表达分析。结果发现,昼夜温度为25℃/15℃时,DoGAUT1基因和DoPGSIP6基因表达水平与多糖含量呈正相关。DoPGSIP6在昼夜温差为15℃时,处理2d,DoPGSIP6基因相对表达量和多糖的含量均达到该温差处理条件下的最高水平。
[Abstract]:Dendrobium (Dendrobium officinale), is the orchid Dendrobium, has a very unique value. In this study, the tin Shi Huyuan bulbs as materials, discussed in favor of protocorm proliferation, preservation and accumulation of polysaccharides in the optimum culture conditions, and to establish a liquid culture system, to determine the content of polysaccharides in different the temperature difference between day and night, based on the cloning of 2 key glycosyl polysaccharide biosynthesis transferase gene DoGAUT1 and DoPGSIP6 gene, and the gene quantitative expression analysis in different temperature. The main results are as follows: 1 Dendrobium officinale from optimization of culture conditions of selected body of Dendrobium candidum protocorm material conditions in vitro optimization of protocorm proliferation and preservation. The single factor experiment, compared with 6-BA, coconut milk, potatoes with effect on the protocorm proliferation test. The data that 1/2 MS+ Mashed Potato 50g/L is suitable for protocorm proliferation, Tim (?) 2 mg/L 6-BA, increased fresh weight of protocorm proliferation coefficient, but part of protocorm differentiation. The change of KNO3 1/2 in MS, the ratio of KH2PO4, compare the protocorm proliferation effect of test results show that when KNO3, KH2PO4 were 0.5 times, 1 times, protocorm proliferation coefficient was the highest, but a small amount of protocorm chlorosis, control 1/2 MS is suitable for protocorm proliferation. The effect of the comparison of different nitrate nitrogen and ammonium nitrogen ratio on the iron skin effect of protocorm multiplication of Dendrobium the test results show that when KNO3, NH4NO3 are 0.5 times than the control and Mashed Potato 50g/L medium and 1/2 MS+ Mashed Potato 50 g/L. The highest proliferation coefficient of different concentrations of curcumin and red beet glycosides on protocorm in vitro preservation effect The results showed that curcumin, 0.1-1.0g/L can inhibit the growth of protocorms, red beet glycoside concentration is 0.1g/L the best preservation effect. Based on the induced protocorm were inoculated with 1/2 MS+ potato extract 50g/L liquid medium, compared the change of biomass under different cultivation time of iron Dendrobium, test the results show that in the whole growth period, fresh weight change curve of protocorm of type S and 30-40 D.2 rapid growth of Dendrobium officinale in vitro culture in different dif conditions to induce changes of polysaccharide content of Dendrobium candidum protocorms as materials, studied under different time multi culture of Dendrobium candidum sugar, in the whole growth period, the curve of protocorm polysaccharide changes were S type. Select 30 d cultivation of Dendrobium protocorm as materials, compared the difference of Dendrobium officinale polysaccharide content in different temperature difference between day and night under test. The results show that the processing temperature is 25 DEG /10 DEG 2D, the polysaccharide content is the highest, and the processing time in a certain range, the polysaccharide content increased first and then decreased; with the temperature of 25 DEG /15 DEG C, the temperature of polysaccharide content increased with the increase of processing time, the temperature difference is the most suitable for the promotion of secondary metabolites accumulation of polysaccharide temperature; processing temperature is 25 DEG /20 DEG, presenting the polysaccharide content increased, slightly lower than the same time under the temperature of 25 DEG /15 DEG.3 protocorm DoGAUT1 and DoPGSIP6 gene cloning and bioinformatic analysis using homologous cloning and combined with RACE technology with protocorms as materials, cloned two glycosyltransferase genes DoGAUT1 and DoPGSIP6 in length were 25182010 BP, 705549 amino acids respectively encoding, lander on 1740985. bioinformatics showed that DoGAUT1 Pro Water transmembrane protein without signal peptide, forming a coiled coil structure, subcellular localization in Golgi.DoPGSIP6 was a hydrophobic transmembrane protein containing the signal peptide, no coiled coil structure, subcellular localization at the plasma membrane. They belong to the GT8 family of glycosyl transferase GT- in a conserved DoGAUT1 domain containing protein. Typical GT8_like_1 of unknown function in section 394-692 of the plurality of ligand binding sites, 2 metal binding sites. DoPGSIP6 protein contains a conserved GT8_Glycogenin domain in section 29-276, a plurality of substrate binding sites, 2 manganese binding sites and a conserved sequence of DXD gene, the motif is mainly responsible for the addition of different the sugar to the sugar chain, carbonate, protein. In addition, DoPGSIP6 protein contains a conserved CCC1_like domain in section 448-522, it may have the role of nitrogen fixation. Phylogenetic tree revealed DoGAUT1 and DoPGSIP6 gene The deduced amino acid sequence respectively with two distachyon, recent soybean evolution, and tomato, expression analysis with 18S rRNA as a reference gene quantitative orange distantly related.4 Dendrobium DoGAUT1 and DoPGSIP6 gene in different day and night temperature difference, the expression level of DoGAUT1 gene and DoPGSIP6 gene in the different temperature difference between day and night the relative quantitative expression analysis. The results showed that diurnal temperature of 25 DEG /15 DEG C, DoGAUT1 and DoPGSIP6 gene expression level was positively correlated with the content of polysaccharide in the.DoPGSIP6 diurnal temperature is 15 degrees centigrade, 2D, the relative expression of DoPGSIP6 gene and polysaccharide contents reached the highest level under the condition of temperature.

【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S567.239

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