發(fā)布時間：2018-03-26 03:38

  本文選題：花吊絲竹　切入點：PEG分級　出處：《福建農(nóng)林大學(xué)》2017年碩士論文

【摘要】：干旱的生長環(huán)境是植物生長發(fā)育中一個非常重要的限制因子,植物為適應(yīng)干旱的環(huán)境,會依據(jù)自身的習(xí)性啟動從植物外部形態(tài)、生理代謝、生化過程及分子水平等的一系列響應(yīng)�；ǖ踅z竹(Dendrocalamus minor var.amoenus)是重要的園林觀賞和經(jīng)濟筍用竹種。該竹子還在福建地區(qū)試行沿海沙地的引種,作為防風(fēng)固沙的新樹種,但是干旱、貧瘠的條件嚴(yán)重影響該竹種效益和特性。以2年生花吊絲竹母竹為供試材料,通過自然耗水進(jìn)行干旱處理,研究花吊絲竹生理參數(shù)的變化及蛋白的差異表達(dá)。主要研究結(jié)果如下:(1)2種蛋白提取方法(TCA/酚提結(jié)合法和PEG法)的SDS-PAGE分析表明:PEG法中5個組分的條帶差異明顯,全蛋白得到了有效分離,在16%PEG濃度的F3中富集到了較多高豐度蛋白,與NF中2條深色蛋白帶(15 kDa上箭頭和60 kDa下箭頭)位置基本相同;而F4及F5在相同區(qū)域并沒有高豐度蛋白,表明了花吊絲竹葉片全蛋白能通過PEG得到有效的分離。(2)2種蛋白提取方法(TCA/酚提結(jié)合法和PEG法)的2-DE分析表明:PEG分級法中F1-F5檢測蛋白總數(shù)超過1700個,但是相鄰組存在一定的重疊率;而TCA丙酮/酚提結(jié)合法只能檢測到570個左右的蛋白點,5個組與NF平均有439個蛋白點匹配,重疊率達(dá)到77.9%以上,顯著提高了 2-DE的分辨率,表明PEG法對于花吊絲竹低豐度蛋白分離及后續(xù)鑒定是切實可行且較為理想的,但由于繁瑣的操作步驟,會造成一定的蛋白損失。(3)干旱脅迫下花吊絲竹葉片光合作用參數(shù)變化為:凈光合速率(Pn)、氣孔導(dǎo)度(Gs)和蒸騰速率(Tr)都出現(xiàn)顯著下降,Pn對脅迫程度的響應(yīng)相較Gs和Tr更敏感;胞間CO2濃度(Ci)在0-15 d與對照組無顯著差異,而在15-26 d出現(xiàn)下降趨勢,在26 d達(dá)到最低,隨后急劇上升;氣孔限制值(Ls)在10-26 d呈現(xiàn)上升的趨勢,同樣在26 d出現(xiàn)峰值,26-30 d出現(xiàn)顯著的下降,Ci和Ls出現(xiàn)了相反的趨勢。表明在輕度干旱脅迫下,氣孔限制性因素是Pn下降的主要因素;而隨著脅迫程度加深,PSⅡ光合結(jié)構(gòu)損害和酶類失活,非氣孔限制因素是Pn下降的主導(dǎo)因素。(4)干旱脅迫下花吊絲竹葉片葉綠素?zé)晒鈪?shù)變化為:光系統(tǒng)Ⅱ(PSⅡ)的實際光化學(xué)效率(ΦPSⅡ)、表觀光合電子傳遞速率(ETR)和非光化學(xué)猝滅系數(shù)(qN)在脅迫后段(15-30 d)一直出現(xiàn)顯著下降,而葉片初始熒光(Fo)在后期呈現(xiàn)上升的趨勢。說明處理時間不長時,通過減少光捕獲、熱耗散和酶活的調(diào)節(jié)協(xié)同作用維持光合機構(gòu)功能,實現(xiàn)光保護(hù)。而隨著脅迫時間的延長,葉片的光合電子傳遞受阻,PSⅡ光合結(jié)構(gòu)受到嚴(yán)重?fù)p傷,正常的光合作用途徑和代謝途徑受阻。(5)干旱脅迫下花吊絲竹葉片相關(guān)物質(zhì)或保護(hù)酶類變化為:超氧化物歧化酶(SOD)和過氧化氫酶(CAT)的含量呈現(xiàn)先升高后降低的趨勢,而脯氨酸(PRO)和丙二醛(MDA)的含量則一直增加。表明在處理前期,PSⅡ光系統(tǒng)活性降低會引起光能過剩光抑制,形成過量的活性氧簇,造成膜脂過氧化,保護(hù)酶類活性受到干旱誘導(dǎo)出現(xiàn)上升,但是隨著脅迫程度加深,活性氧清除酶類的活性出現(xiàn)下降,嚴(yán)重?fù)p害細(xì)胞結(jié)構(gòu),各項生理代謝途徑受到損傷和阻遏。同時,CAT含量上升的幅度比SOD更大,花吊絲竹CAT對干旱脅迫反應(yīng)更加敏感。(6)干旱脅迫下花吊絲竹葉片差異表達(dá)蛋白的2-DE和MS實驗結(jié)果表明:干旱脅迫下出現(xiàn)41個顯著差異表達(dá)的蛋白點,表達(dá)上調(diào)的有23個,表達(dá)下調(diào)的有13個,新出現(xiàn)的有5個。對這些點進(jìn)行MALDI-TOF-MS質(zhì)譜鑒定分析,鑒定出33個蛋白點,這些差異表達(dá)蛋白質(zhì)與光合作用、能量代謝和逆境脅迫響應(yīng)有關(guān)。參與光合作用的蛋白質(zhì)為核酮糖1,5-二磷酸羧化酶/加氧酶(RuBisco)大亞基、RuBisc小鏈、RuBisco小亞基C、RuBisco活化酶、氧進(jìn)化增強蛋白(OEE)的不同構(gòu)型OEE1和OEE2和細(xì)胞色素b6-f復(fù)合體鐵硫蛋白。參與能量代謝的蛋白質(zhì)為ATP合成酶CF1β亞基。參與逆境脅迫響應(yīng)蛋白質(zhì)為萌發(fā)素類似蛋白、過氧化物還原蛋白BAS1、鐵蛋白-1。
[Abstract]:The growth environment arid plant growth is a very important factor in development, plants adapt to drought environment, will be based on their own habits starting from the plant morphology, physiological metabolism, a series of biochemical response process and molecular level. D. (Dendrocalamus minor var.amoenus) flower is an important landscape and ornamental bamboo economy use bamboo. The bamboo in Fujian area for the introduction of coastal sandy land, as a new species, windbreak and sand fixation but drought, barren conditions seriously affect the efficiency and the characteristics of bamboo. In 2 years of hanging flower Sizhu mother bamboo as experimental material, the water consumption of natural drought treatment, differential expression and protein research take D. physiological parameters. The main results are as follows: (1) 2 kinds of methods for extraction of protein (TCA/ thanphenol node method and PEG method) SDS-PAGE analysis showed that the PEG method of 5 components in the band differences Obviously, the proteins were separated effectively, at the concentration of 16%PEG in F3 enriched to more high abundance proteins, and 2 bands of protein NF in the dark (15 kDa and 60 kDa under the arrow arrow) position is basically the same; and F4 and F5 in the same area and no high abundance protein, that flower hanging silk leaf protein can effectively separated by PEG. (2) 2 kinds of methods for extraction of protein (TCA/ thanphenol node method and PEG method) 2-DE analysis showed that the total protein detection F1-F5 PEG classification method in more than 1700, but there is some overlap adjacent groups; while TCA provided with acetone / phenol method only detected about 570 protein spots, 439 protein spots, the average of 5 group and NF, the overlap rate reached more than 77.9%, significantly improved the resolution of 2-DE, show that the PEG method is feasible and ideal for the flower D. low abundance protein separation and subsequent identification, but due to the cumbersome The operation steps, will cause some loss of protein. (3) under drought stress changes of leaf photosynthesis of flower hanging wire parameters: net photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) were significantly decreased in Pn, Gs and Tr are more sensitive to the stress level in response; intercellular CO2 concentration (Ci) in 0-15 d had no significant difference with control group, while in the 15-26 d show a downward trend in the 26 D minimum, followed by a sharp rise; stomatal limiting value (Ls) at 10-26 D showed a rising trend in the same peak at 26 D, 26-30 D were significantly reduced. Ci and Ls showed the opposite trend. The results indicated that under mild drought stress, stomatal limitation factors were the main factors of Pn decline; with the deepening of water stress, PS II photosynthetic structure damage and enzyme deactivation, non stomatal limitation is the dominant factor of the decrease of Pn. (4) under drought stress flower hanging silk leaf chlorophyll fluorescence Parameter change: photosystem II (PS II) and the actual photochemical efficiency (PS II), apparent photosynthetic electron transport rate (ETR) and non photochemical quenching coefficient (qN) in the stress period (15-30 d) has declined significantly, while the leaf initial fluorescence (Fo) showed a rising trend in later period that processing time is not long, light harvesting by reducing the heat dissipation and enzyme activity regulation of synergy maintain photosyntheticapparatus, realize the light protection. And with the extension of stress time, the leaf photosynthetic electron transport was inhibited, PS II photosynthetic structure severely damaged, blocked the photosynthetic pathway and metabolic pathways of normal. (5) under drought stress, leaf flower hanging wire related substances or protective enzymes change: superoxide dismutase (SOD) and catalase (CAT) content increased firstly and then decreased, but the proline (PRO) and malondialdehyde (MDA) content has been increased. Ming in the early stage, PS decreased photosystem II activity can lead to excess light photoinhibition, excessive ROS formation, resulting in membrane lipid peroxidation and activities of protective enzymes under drought induced rise, but with the deepening of water stress, the activity of reactive oxygen scavenging enzymes declined, serious damage to cell structure, physiological metabolism the damage and repression. At the same time, the content of CAT increased more sharply than SOD, CAT spent D. to drought stress was more sensitive. (6) hanging wire from leaves of differentially expressed proteins between 2-DE and MS results indicate that the flowers under drought stress: 41 significantly differentially expressed protein spots up-regulated under drought stress. There are 23, 13 down regulated ones, the emergence of new 5. MALDI-TOF-MS mass spectrometry analysis of these points, identified 33 proteins, protein expression and Photosynthesis of these differences, energy generation Thanks and stress response. Proteins involved in photosynthesis for two 1,5- ribulose bisphosphate carboxylase / oxygenase (RuBisco) subunit, RuBisc chain, RuBisco small subunit C, RuBisco activating enzyme, oxygen evolution enhanced protein (OEE) of different configurations of OEE1 and OEE2 and cytochrome b6-f complex iron sulfur proteins involved in energy metabolism. The protein is ATP CF1 synthase beta subunit involved in stress response protein germin like proteins, peroxiredoxin BAS1, ferritin -1.

【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S795

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 謝惠玲;劉杰;陳珊;王經(jīng)源;傅偉;李圓萍;王微;肖清鐵;鄭新宇;黃錦文;林瑞余;林文雄;;紫蘇葉片響應(yīng)鎘脅迫的蛋白質(zhì)差異表達(dá)分析[J];中國生態(tài)農(nóng)業(yè)學(xué)報;2014年10期

2 馬成梅;梁永增;鐘俐;;甜瓜葉片蛋白質(zhì)2-DE中IPG膠條及染色方法的選擇[J];西北農(nóng)業(yè)學(xué)報;2013年11期

3 李紅麗;劉迪秋;何華;張南南;葛鋒;陳朝銀;;類萌發(fā)素蛋白在植物防衛(wèi)反應(yīng)中的作用[J];植物生理學(xué)報;2013年04期

4 李娟;彭鎮(zhèn)華;高健;陳媛文;;干旱脅迫下黃條金剛竹的光合和葉綠素?zé)晒馓匦訹J];應(yīng)用生態(tài)學(xué)報;2011年06期

5 張仁和;鄭友軍;馬國勝;張興華;路海東;史俊通;薛吉全;;干旱脅迫對玉米苗期葉片光合作用和保護(hù)酶的影響[J];生態(tài)學(xué)報;2011年05期

6 程雷;王蕾蕾;程備久;范軍;;玉米過氧化物還原蛋白BAS1的原核表達(dá)及其功能研究[J];中國生物工程雜志;2010年11期

7 卜令鐸;張仁和;常宇;薛吉全;韓苗苗;;苗期玉米葉片光合特性對水分脅迫的響應(yīng)[J];生態(tài)學(xué)報;2010年05期

8 張淑勇;周澤福;張光燦;夏江寶;;半干旱黃土丘陵區(qū)天然次生灌木山桃(Prunus davidiana)與山杏(Prunus sibirica L.)葉片氣體交換參數(shù)日動態(tài)差異[J];生態(tài)學(xué)報;2009年01期

9 畢建杰;劉建棟;葉寶興;謝連杰;;干旱脅迫對夏玉米葉片光合及葉綠素?zé)晒獾挠绊慬J];氣象與環(huán)境科學(xué);2008年01期

10 孟林;毛培春;張國芳;史曉霞;;17個苜蓿品種苗期抗旱性鑒定[J];草業(yè)科學(xué);2008年01期

相關(guān)碩士學(xué)位論文 前4條

1 張國君;水稻鈣調(diào)蛋白對鎘脅迫的分子響應(yīng)[D];福建農(nóng)林大學(xué);2013年

2 郭偉志;干旱脅迫下不同杉木無性系的若干生理響應(yīng)和差異蛋白質(zhì)組分析[D];福建農(nóng)林大學(xué);2012年

3 翁兆霞;鎘脅迫下秋茄（Kandelia candel）根系蛋白質(zhì)組變化及差異表達(dá)蛋白功能研究[D];福建農(nóng)林大學(xué);2011年

4 王旭;PEG分級沉淀方法及其在擬南芥低溫脅迫蛋白質(zhì)組學(xué)研究中的應(yīng)用[D];吉林大學(xué);2007年

，

本文編號：1666170