花吊丝竹PEG蛋白分级及其在干旱胁迫下生理与蛋白的响应

发布时间：2018-03-26 03:38

本文选题：花吊丝竹　切入点：PEG分级　出处：《福建农林大学》2017年硕士论文

【摘要】：干旱的生长环境是植物生长发育中一个非常重要的限制因子,植物为适应干旱的环境,会依据自身的习性启动从植物外部形态、生理代谢、生化过程及分子水平等的一系列响应。花吊丝竹(Dendrocalamus minor var.amoenus)是重要的园林观赏和经济笋用竹种。该竹子还在福建地区试行沿海沙地的引种,作为防风固沙的新树种,但是干旱、贫瘠的条件严重影响该竹种效益和特性。以2年生花吊丝竹母竹为供试材料,通过自然耗水进行干旱处理,研究花吊丝竹生理参数的变化及蛋白的差异表达。主要研究结果如下:(1)2种蛋白提取方法(TCA/酚提结合法和PEG法)的SDS-PAGE分析表明:PEG法中5个组分的条带差异明显,全蛋白得到了有效分离,在16%PEG浓度的F3中富集到了较多高丰度蛋白,与NF中2条深色蛋白带(15 kDa上箭头和60 kDa下箭头)位置基本相同;而F4及F5在相同区域并没有高丰度蛋白,表明了花吊丝竹叶片全蛋白能通过PEG得到有效的分离。(2)2种蛋白提取方法(TCA/酚提结合法和PEG法)的2-DE分析表明:PEG分级法中F1-F5检测蛋白总数超过1700个,但是相邻组存在一定的重叠率;而TCA丙酮/酚提结合法只能检测到570个左右的蛋白点,5个组与NF平均有439个蛋白点匹配,重叠率达到77.9%以上,显著提高了 2-DE的分辨率,表明PEG法对于花吊丝竹低丰度蛋白分离及后续鉴定是切实可行且较为理想的,但由于繁琐的操作步骤,会造成一定的蛋白损失。(3)干旱胁迫下花吊丝竹叶片光合作用参数变化为:净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr)都出现显著下降,Pn对胁迫程度的响应相较Gs和Tr更敏感;胞间CO2浓度(Ci)在0-15 d与对照组无显著差异,而在15-26 d出现下降趋势,在26 d达到最低,随后急剧上升;气孔限制值(Ls)在10-26 d呈现上升的趋势,同样在26 d出现峰值,26-30 d出现显著的下降,Ci和Ls出现了相反的趋势。表明在轻度干旱胁迫下,气孔限制性因素是Pn下降的主要因素;而随着胁迫程度加深,PSⅡ光合结构损害和酶类失活,非气孔限制因素是Pn下降的主导因素。(4)干旱胁迫下花吊丝竹叶片叶绿素荧光参数变化为:光系统Ⅱ(PSⅡ)的实际光化学效率(ΦPSⅡ)、表观光合电子传递速率(ETR)和非光化学猝灭系数(qN)在胁迫后段(15-30 d)一直出现显著下降,而叶片初始荧光(Fo)在后期呈现上升的趋势。说明处理时间不长时,通过减少光捕获、热耗散和酶活的调节协同作用维持光合机构功能,实现光保护。而随着胁迫时间的延长,叶片的光合电子传递受阻,PSⅡ光合结构受到严重损伤,正常的光合作用途径和代谢途径受阻。(5)干旱胁迫下花吊丝竹叶片相关物质或保护酶类变化为:超氧化物歧化酶(SOD)和过氧化氢酶(CAT)的含量呈现先升高后降低的趋势,而脯氨酸(PRO)和丙二醛(MDA)的含量则一直增加。表明在处理前期,PSⅡ光系统活性降低会引起光能过剩光抑制,形成过量的活性氧簇,造成膜脂过氧化,保护酶类活性受到干旱诱导出现上升,但是随着胁迫程度加深,活性氧清除酶类的活性出现下降,严重损害细胞结构,各项生理代谢途径受到损伤和阻遏。同时,CAT含量上升的幅度比SOD更大,花吊丝竹CAT对干旱胁迫反应更加敏感。(6)干旱胁迫下花吊丝竹叶片差异表达蛋白的2-DE和MS实验结果表明:干旱胁迫下出现41个显著差异表达的蛋白点,表达上调的有23个,表达下调的有13个,新出现的有5个。对这些点进行MALDI-TOF-MS质谱鉴定分析,鉴定出33个蛋白点,这些差异表达蛋白质与光合作用、能量代谢和逆境胁迫响应有关。参与光合作用的蛋白质为核酮糖1,5-二磷酸羧化酶/加氧酶(RuBisco)大亚基、RuBisc小链、RuBisco小亚基C、RuBisco活化酶、氧进化增强蛋白(OEE)的不同构型OEE1和OEE2和细胞色素b6-f复合体铁硫蛋白。参与能量代谢的蛋白质为ATP合成酶CF1β亚基。参与逆境胁迫响应蛋白质为萌发素类似蛋白、过氧化物还原蛋白BAS1、铁蛋白-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.

【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】：S795

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