本文關鍵詞： 凹葉厚樸 干旱脅迫 生理指標 蛋白組學 雙向電泳　出處：《福建農林大學》2017年碩士論文　論文類型：學位論文

【摘要】：凹葉厚樸(Magnolia officinalis subsp.bioba)是集藥用、材用和觀賞等功能為一體的珍貴樹種。水分是凹葉厚樸資源保護和人工栽培重要的限制性生態(tài)因子之一。研究凹葉厚樸對干旱脅迫的生理指標變化及響應機制,對提高凹葉厚樸抗旱性和指導抗旱品種選育具有重要意義。本文通過盆栽試驗,研究了持續(xù)干旱脅迫下(以6 d為一個間隔期,持續(xù)干旱30 d),3年生的凹葉厚樸幼苗的形態(tài)指標、土壤含水量(SWC)、葉片含水量(LWC)、葉片水勢(LWP)、光合特性等生理指標變化,運用2-DE技術與MALDI-TOF-MS手段相結合,對干旱脅迫下凹葉厚樸葉片差異表達蛋白進行分析。主要研究結果如下:1.凹葉厚樸幼苗隨著干旱程度的加深,葉片的萎蔫和葉卷程度逐漸增加。SWC為11.88%時出現(xiàn)發(fā)黃、斑點等傷害癥狀,SWC為7.37%時葉片邊緣卷曲、失水下垂,SWC為3.93%地上部分失水死亡。在干旱脅迫6～30d,SWC、LWC顯著降低,30d時SWC僅為對照組的13.86%;12 d后LWP急劇下降,30 d后LWP下降至-4.65 Mpa。凹葉厚樸葉綠素相對含量SPAD隨著SWC降低呈下降的趨勢。干旱脅迫下凹葉厚樸水分生理狀態(tài)變差,葉綠素含量顯著降低。2.干旱脅迫下凹葉厚樸幼苗葉片Pn、Tr、Gs顯著降低,Ci呈先下降后上升的趨勢;瞬時水分利用效率(PWUE)呈先升高后降低的趨勢;氣孔因素和非氣孔因素共同作用影響凹葉厚樸幼苗的光合作用,在干旱時間(0～18d)氣孔因素起主導作用,在干旱時間(18 d),非氣孔因素起主導作用。初始熒光(Fo)呈先升高后下降的趨勢,而最大光化學效率(Fv/Fm)、實際光化學效率(ΦPSⅡ)、表觀光合電子傳遞速率(ETR)隨著干旱脅迫程度的加劇逐漸降低;24 d后凹葉厚樸光合機構被破壞,Fv/Fm、ΦPSⅡ、ETR分別由0.79、0.65、21.84下降到0.42、0.41、3.93。光合特性指標與SWC、LWP極顯著相關(P0.01),氣孔是影響氣體交換參數(shù)變化的主導因素。3.干旱脅迫下脯氨酸(Pro)含量變化隨SWC下降呈先上升后下降的趨勢,在第18 d時達到峰值(29.96 μg·ml-1),與對照組相比增加了 250.1%,具有一定保水能力;干旱脅迫下0～18dLWC下降幅度較小,這與凹葉厚樸體內積累Pro進行滲透調節(jié)有關。在干旱脅迫0～30d,MDA含量顯著增加,24d時最高(32.34nmol·ml-1),與對照組相比增加了 121.4%。4.建立一套適合于凹葉厚樸葉片全蛋白2-DE體系,比較3種蛋白的提取方法,篩選最佳膠條承載量、等電聚焦條件。采用TCA/丙酮+Tris-酸法提取凹葉厚樸葉片蛋白效果最好,選用17cmpH5-8NL的IPG膠條,樣品上樣量為17μg,SDS-PAGE濃度為10%,按IEF程序Ⅱ進行2-DE分離和銀染,得到分辨率清晰、穩(wěn)定性高的2-DE圖譜。5.在該2-DE優(yōu)化體系基礎上,研究了對照組和干旱脅迫12 d后2-DE圖譜差異,共有45個顯著差異的蛋白點(Ration,p≤0.05)。其中豐度上調有10個,豐度下調有18個,新增蛋白點10個,消失蛋白點7個。通過MALDI-TOF-MS分析和NCBI、Uniport等數(shù)據(jù)庫搜索匹配,確定32個差異蛋白信息。6.對成功鑒定的32個蛋白進行了 COG功能分類、GO注釋分析及參與KEGG通路的分析。在凹葉厚樸幼葉中鑒定的差異蛋白,分別涉及到細胞結構(3.13%),氨基酸轉運與代謝(3.13%),光合作用(9.38%),翻譯后修飾、蛋白轉化、分子伴侶(12.50%),能量生產和轉換(15.63%),碳水化合物運輸和代謝(21.88%),未知功能蛋白分類(34.38%)。其中有20個酶類參與了 18個代謝通路,其中光合碳固定途徑有8個酶類參與,碳代謝有8個酶類參與,光合作用途徑7個酶類參與,乙醛酸鹽和二羧酸代謝有4個酶類參與,糖酵解和糖異生及氨基酸合成途徑各3個酶類參與。凹葉厚樸葉片在響應干旱脅迫過程中,相關蛋白表達量變化起到了重要作用,為研究凹葉厚樸響應干旱機制提供參考。
[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.

【學位授予單位】：福建農林大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S567.11

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