干旱胁迫下凹叶厚朴的生理响应及差异蛋白表达分析

发布时间：2018-02-13 12:42

本文关键词： 凹叶厚朴干旱胁迫生理指标蛋白组学双向电泳　出处：《福建农林大学》2017年硕士论文　论文类型：学位论文

【摘要】：凹叶厚朴(Magnolia officinalis subsp.bioba)是集药用、材用和观赏等功能为一体的珍贵树种。水分是凹叶厚朴资源保护和人工栽培重要的限制性生态因子之一。研究凹叶厚朴对干旱胁迫的生理指标变化及响应机制,对提高凹叶厚朴抗旱性和指导抗旱品种选育具有重要意义。本文通过盆栽试验,研究了持续干旱胁迫下(以6 d为一个间隔期,持续干旱30 d),3年生的凹叶厚朴幼苗的形态指标、土壤含水量(SWC)、叶片含水量(LWC)、叶片水势(LWP)、光合特性等生理指标变化,运用2-DE技术与MALDI-TOF-MS手段相结合,对干旱胁迫下凹叶厚朴叶片差异表达蛋白进行分析。主要研究结果如下:1.凹叶厚朴幼苗随着干旱程度的加深,叶片的萎蔫和叶卷程度逐渐增加。SWC为11.88%时出现发黄、斑点等伤害症状,SWC为7.37%时叶片边缘卷曲、失水下垂,SWC为3.93%地上部分失水死亡。在干旱胁迫6～30d,SWC、LWC显著降低,30d时SWC仅为对照组的13.86%;12 d后LWP急剧下降,30 d后LWP下降至-4.65 Mpa。凹叶厚朴叶绿素相对含量SPAD随着SWC降低呈下降的趋势。干旱胁迫下凹叶厚朴水分生理状态变差,叶绿素含量显著降低。2.干旱胁迫下凹叶厚朴幼苗叶片Pn、Tr、Gs显著降低,Ci呈先下降后上升的趋势;瞬时水分利用效率(PWUE)呈先升高后降低的趋势;气孔因素和非气孔因素共同作用影响凹叶厚朴幼苗的光合作用,在干旱时间(0～18d)气孔因素起主导作用,在干旱时间(18 d),非气孔因素起主导作用。初始荧光(Fo)呈先升高后下降的趋势,而最大光化学效率(Fv/Fm)、实际光化学效率(ΦPSⅡ)、表观光合电子传递速率(ETR)随着干旱胁迫程度的加剧逐渐降低;24 d后凹叶厚朴光合机构被破坏,Fv/Fm、ΦPSⅡ、ETR分别由0.79、0.65、21.84下降到0.42、0.41、3.93。光合特性指标与SWC、LWP极显著相关(P0.01),气孔是影响气体交换参数变化的主导因素。3.干旱胁迫下脯氨酸(Pro)含量变化随SWC下降呈先上升后下降的趋势,在第18 d时达到峰值(29.96 μg·ml-1),与对照组相比增加了 250.1%,具有一定保水能力;干旱胁迫下0～18dLWC下降幅度较小,这与凹叶厚朴体内积累Pro进行渗透调节有关。在干旱胁迫0～30d,MDA含量显著增加,24d时最高(32.34nmol·ml-1),与对照组相比增加了 121.4%。4.建立一套适合于凹叶厚朴叶片全蛋白2-DE体系,比较3种蛋白的提取方法,筛选最佳胶条承载量、等电聚焦条件。采用TCA/丙酮+Tris-酸法提取凹叶厚朴叶片蛋白效果最好,选用17cmpH5-8NL的IPG胶条,样品上样量为17μg,SDS-PAGE浓度为10%,按IEF程序Ⅱ进行2-DE分离和银染,得到分辨率清晰、稳定性高的2-DE图谱。5.在该2-DE优化体系基础上,研究了对照组和干旱胁迫12 d后2-DE图谱差异,共有45个显著差异的蛋白点(Ration,p≤0.05)。其中丰度上调有10个,丰度下调有18个,新增蛋白点10个,消失蛋白点7个。通过MALDI-TOF-MS分析和NCBI、Uniport等数据库搜索匹配,确定32个差异蛋白信息。6.对成功鉴定的32个蛋白进行了 COG功能分类、GO注释分析及参与KEGG通路的分析。在凹叶厚朴幼叶中鉴定的差异蛋白,分别涉及到细胞结构(3.13%),氨基酸转运与代谢(3.13%),光合作用(9.38%),翻译后修饰、蛋白转化、分子伴侣(12.50%),能量生产和转换(15.63%),碳水化合物运输和代谢(21.88%),未知功能蛋白分类(34.38%)。其中有20个酶类参与了 18个代谢通路,其中光合碳固定途径有8个酶类参与,碳代谢有8个酶类参与,光合作用途径7个酶类参与,乙醛酸盐和二羧酸代谢有4个酶类参与,糖酵解和糖异生及氨基酸合成途径各3个酶类参与。凹叶厚朴叶片在响应干旱胁迫过程中,相关蛋白表达量变化起到了重要作用,为研究凹叶厚朴响应干旱机制提供参考。
[Abstract]:Ye Houpu (Magnolia officinalis subsp.bioba) concave is a set of medicinal and ornamental materials, rare species with other functions. Water is one of the important restrictive ecological factors of Magnolia officinalis resource protection and artificial cultivation of Magnolia officinalis on physiological changes of drought stress and response mechanism, to improve the drought resistance and magnoliabiloba breeding of drought resistant varieties are significant. In this paper, through pot experiment, studied under drought stress (up to 6 d for a period of continuous drought, 30 d), morphological index of 3 year old Magnolia officinalis seedlings, soil moisture (SWC), leaf water content, leaf water potential ((LWC) LWP), the changes of physiological indices of photosynthetic characteristics, the use of 2-DE technology and MALDI-TOF-MS technology combining with the analysis on the protein expression of Magnolia officinalis leaves under drought stress difference. The main results are as follows: 1. Magnolia officinalis seedlings with drought The deepening of leaf wilting and leaf volume increased when.SWC is 11.88%, appear yellow, spots and other symptoms, when SWC is 7.37%, the blade edge curl, water loss ptosis, SWC loss of part of the 3.93% on the ground of death. In drought stress of 6 ~ 30d, SWC, LWC decreased significantly, 30d SWC only as control a group of 13.86%; after 12 d LWP decreased sharply after 30 d LWP down to -4.65 Mpa. magnoliabiloba with SWC reduced the relative content of chlorophyll SPAD decreased. Variation of concave Ye Houpu moisture physiological state of drought stress, the chlorophyll content of.2. decreased significantly under drought stress in Magnolia officinalis seedling leaves Pn, Tr, Gs significantly decreased, Ci increased and then decreased; instantaneous water use efficiency (PWUE) increased and then decreased; interaction of stomatal and non stomatal factors of Magnolia officinalis seedlings photosynthesis, drought in time (0 ~ 18D) stomatal factors The leading role in the drought time (18 d), non stomatal factors play a leading role. The initial fluorescence (Fo) was first increased and then decreased, and the maximal photochemical efficiency (Fv/Fm), the actual photochemical efficiency (PS II), apparent photosynthetic electron transport rate (ETR) with the aggravation of drought stress gradually decreased; after 24 D magnoliabiloba photosynthetic apparatus was damaged, with Fv/Fm, PS II, ETR were decreased from 0.79,0.65,21.84 to 0.42,0.41,3.93. photosynthetic characteristics and SWC, LWP were significantly correlated (P0.01), stomatal is the dominant factor affecting gas exchange parameters of.3. drought stress proline (Pro) content was first increased and then decreased with decrease of SWC, reached the peak at eighteenth D (29.96 g ML-1), compared with the control group increased by 250.1%, with a certain ability of keeping water; under drought stress of 0 ~ 18dLWC decreased slightly, and the accumulation of Magnolia officinalis Pro penetration Regulation. Under drought stress of 0 ~ 30d, MDA content increased significantly, the highest 24D (32.34nmol - ML-1), compared with the control group increased 121.4%.4. to set up a suit of Magnolia biloba leaf protein 2-DE system, comparison of 3 protein extraction method, screening the best adhesive capacity, isoelectric focusing conditions. The extraction of Magnolia officinalis leaf protein had the best effect using TCA/ +Tris- acid acetone method, IPG strip with 17cmpH5-8NL, sample volume is 17 g, the concentration of SDS-PAGE was 10%, according to the IEF program II separation by 2-DE and silver staining, get the resolution clear, high stability of 2-DE.5. in the 2-DE based optimization system on 12 d after 2-DE of control group and the difference of drought stress, there were 45 different protein spots (Ration, P < 0.05). Among them, 10 genes were up-regulated and 18 were downregulated, 10 new protein spots, 7 protein spots disappeared by MALDI-TOF-MS. Analysis and NCBI, Uniport and other database searching and matching of COG function to determine the classification of 32 proteins.6. 32 protein identified successfully, GO annotation analysis and is involved in the KEGG pathway. These proteins in Magnolia officinalis leaves, respectively related to cell structure (3.13%), amino acid transport and metabolism (3.13%), photosynthesis (9.38%), posttranslational modification, protein conversion, molecular chaperone (12.50%), energy production and conversion (15.63%), carbohydrate transport and metabolism (21.88%), unknown function protein classification (34.38%). There are 20 enzymes involved in 18 metabolic pathways, including photosynthetic carbon fixation there are 8 ways to participate in carbon metabolism enzymes, involved 8 enzymes involved in photosynthesis pathway enzymes 7, glyoxylate and two carboxylic acid metabolism in 4 enzymes involved in glycolysis and gluconeogenesis and amino acid synthesis pathway of the 3 enzymes. Magnoliabiloba In response to drought stress, the changes of the expression of related proteins play an important role in the response to drought stress. It provides a reference for the study of the response of Magnolia officinalis to the drought mechanism.

【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S567.11

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