【作者】 韦朝领；

【导师】 陶汉之； 江昌俊；

【作者基本信息】 安徽农业大学 ， 茶学， 2003， 博士

【摘要】 茶树是我国重要的经济作物之一，茶叶为我国主要出口创汇农产品。我国茶区分布的气候范围南起热带季风气候北至暖温带季风气候；地形分布复杂，包括平原、丘陵、山地和高原。这些地区经常出现强光、季节性干旱、高（低）温等逆境，且茶树具有耐荫、喜湿的习性，因此加剧了光抑制程度，导致茶叶产量和品质降低。而有关茶树光抑制和光保护机制的研究非常缺乏。目前关于叶黄素循环在植物光保护中的作用及其关键酶—紫黄素脱环氧化酶（VDE）基因的结构及其调控是研究者关注的焦点。它对于阐明植物光抑制和光保护机理，以及提高光能转化利用效率具有重要意义。而且关于木本植物VDE基因的全长cDNA及其蛋白特征未见报道，特别是VDE中的Lipocalin蛋白特征区是否为影响催化活性的主要结构有待证实。为此本研究以茶树（品种为福鼎大白茶和龙井43）为试材，重点进行如下研究：一是关于茶树的光抑制及其恢复、叶黄素循环在茶树光保护中地位及其在自然强光和其它逆境中的表现特征，二是对茶树VDE进行全长cDNA克隆、序列的生物信息学分析和体外表达，并在此基础上对编码VDE蛋白中Lipocalin蛋白特征区的核酸序列进行体外定点突变、表达和活性鉴定。从而一方面进一步明确茶树光抑制机理，确立叶黄素循环在茶树光保护中的地位，并为茶树栽培和通过生物技术途径调控茶树体内的叶黄素循环、提高其抗逆能力和光合效率提供科学依据；另一方面将为其它木本植物的VDE研究奠定理论基础，同时进一步发展和丰富光合作用的光抑制理论。主要结果如下： 1．在没有其它逆境存在时，晴天自然光强下，连体叶片的光系统Ⅱ（PSⅡ）的光化学效率（F_v／F_m）自8：00时开始，随着光强的增加，由最高值逐渐降低，并于13：00时降到最低，福鼎大白茶和龙井43的最低值分别比最高值降低了7.9％和10.2％；福鼎大白茶和龙井43在14：00时的表观量子效率（AQY）比9：00时的降低了25.7％和28.9％，说明自然强光下茶树经常发生光抑制；而当光量子通量密度（PFD）超过1250μmol．m^-2．s^-1时，两个品种的F_v／F_m和PSⅡ潜在活性（F_v／F_o）就降低，证明茶树在晴天比较容易发生光抑制。发生光抑制时，它们的气孔导度和胞间CO₂浓度并未降低，表明在没有其它逆境存在时，自然强光是导致茶树光抑制现象发生的主要原因。自然光下，13：00时以后，F_v／F_m随着光强的减弱又逐渐升高，到18：00时，几乎接近上午最高值；一离体叶片的光抑制程度随着强光处理时间的延长而加重，160 min后，福鼎大白茶和龙井 43的 Fv”．比未处理前分别降低 25．0％和 26．2％，Fv”。分别降低65．l％和 67．5％，但是当光强减弱或在弱光下恢复2～4 h，又接近正常值，因此仅强光逆境引起的光抑制是可逆的。干旱胁迫不仅加重茶树光合作用的光抑制程度，而且降低其恢复能力。在同一条件下，龙井43所受的光抑制和弱光下恢复程度均比福鼎大白茶低，可见茶树所受光合作用的光抑制程度还因品种而异。 同等条件下，茶树龙井 43的倒 2、3、4叶的 Fv／F。值分别比各自的对照降低了35．5％、25．6％和6．2％，说明茶树幼叶比成熟度高的功能叶更易发生光抑制。 2．采取丙酮法提取茶树鲜叶中的叶黄素循环组分，分别在15C、22t和30oC室温下，用 Spherisorb Ca。（sum，250 nnnX4．6。）色谱柱，乙睛：甲醇（85：15）和甲醇：乙酸乙酯（68：32）为流动相，线性洗脱，在445 urn下检测。结果表明，不同温度条件下，高效液相色谱法都能够从茶树鲜叶提取物中检测到紫黄素（V人环氧玉米黄素u）和玉米黄素u人 而且分离效果好，重现性和精密度也很高，表明建立了稳定可靠的的茶树叶黄素循环组分测定方法。 3．经叶黄素循环抑制剂 一二硫苏糖醇（DTT）处理的茶树叶片，以 850u mol．丫’．s－‘的叮D照射120 min后，福鼎大白茶的叶黄素循环组分中的（A＋Z）含量降低了76．5儿结果导致非光化学碎灭门PQ入F丫F旷光化学碎灭系数…P入PS 11实际光化学量子效率（h PS 11R）和光合电子传递速率uTR)明显下降，而 F。显著上升，暗恢复后 Fv”。恢复程度小于未经 DTT处理的叶片，证明叶黄素循环是茶树光保护的主要途径。 叶黄素循环库w八亿)受季节影响较大，具体表现为夏季小，秋、冬季大，而一天中的日变化不明显。在季节变化中，V．A和Z的含量不受叶黄素循环库大小的影响。遮荫不仅使叶黄素循环库减小，而且大幅度降低了 A《的含量，在6、10月，福鼎大白茶分别降少了88．8％～96．3％和85．1％～96．1％，龙井43分别降少了 85．4％～98．8％和 74．7％～92．9％。 在晴天田间自然光照条件下，叶黄素循环的脱环氧化程度kA＋Z＊(V＋A＋Z门与 F／F．呈相反的日变化趋势，表明茶树叶片光抑制的发生，是依赖叶黄素循环的保护性反应。冬季叶黄素循环的脱环氧化程度比夏季大；干旱胁迫下叶黄 n一素循环的脱环氧化程度和NPQ都较对照大：遮荫茶树移到强光下，叶黄素循环脱环氧化程度比自然光下生长的叶片明显增大，尤更多还原

【Abstract】 Tea plant(CameIlia Sinensis (L.) O. Kuntzes) is one of the important industrial crops and export agricultural products in China. The scope of climate which tea areas distributes is between the tropical monsoon climate zone and warm temperate zone in China, and the topography is complex , with plains, hills, high mountains and plateaus. The stress conditions, including strong light intensity, seasonal drought, high or low temperature, often happen in tea areas, additionally, tea plant has habits and characteristics of resisting shade and tending to humid , which leads to photoinhibition of tea plant more easily, and decreases the yield and quality of tea. However, no report of photoinhibition and photoprotection in tea plant is found. At present, the role of xanthophyll cycle in plant photoprtect and the regulation of its key enzyme, violaxanthin de-epoxidase (VDE), are hot spot of study focused on. People mainly utilize methods of molecular biology to study xanthophyll cycle, despite of involving in many other methods. But, up to now, the cDNA of VDE gene and the features of VDE protein in xylophyta yet have not been found. In addition, it is unknown that whether lipocalin protein domain in VDE has functional signification with respect to catalytic activity.Here we place emphasis on studying two aspects in tea plant (cultivars: Fuding-dabaicha and longjing 43): one is of the photoinhibition and its recovery, the role of xanthophyll cycle in photoprotection and the characteristics of it contents change in nature light and in stress conditions, another is of the cloning, analysis with bioinformatics, site-mutated, expression and identification of activity in vitro of the cDNA of VDE gene. All of these further expend the theory of tea plant photosynthesis, and open new opportunities for possible enhancing stress conditions tolerance and photosynthetic efficiency in cultivation and breeding of tea plant. At the same time, these supply the references for the studies on VDEs in other xylophyta, further perfect the theory of plant photoinhibition and give more conclusive evidences for the important position of xanthophyll cycle in plant photoprotection. The significant results are showed as following:, 1. With increase of photon flux density(PFD) since 8:00, photochemical efficiency of PS II(Fv/Fm) of intact conjoined leaves of tea plant decreased gradually, andreached the lowest at 13:00, with Fv/Fm s of Fuding-dabaicha(FD) and Longjing 43(LJ) at 13:00 being lower by 7.9% and 10.2% than these at 8:00 respectively, at the same time, apparent quantum yield(AQY) of tea plant decreased notably after exposed to midday high irradiance, with AQYs of FD and LJ at 14:00 being lower by 25.7% and 28.9% than these at 9:00 respectively, under natural light in sunny day without other stress conditions. it indicates that photoinhibition of photosynthesis often occurs in tea plant under natural high light intensity. Fv/Fm s and potential activities of PS II (Fv/Fo)of FD and LJ decreasing when PFD at 1250 u mol.m-2.s-1 indicate photoinhibition of photosynthesis occurs in tea plant easily. The occurrence of photoinhibition with no decreases of stomatal conductance(Gs) and intercellular CO2 concentration(Ci) shows that natural high light intensity is the essential cause of photoinhibition in tea plant. The extent of photoinhibition of leaf disks of tea plant became more serious with the time of strong light treatment extending, with Fv/Fm s of FD and LJ decreased by 25.0% and 26.2% respectively, and FV/Fo s of FD and LJ decreased by 65.1% and 67.5% Respectively, after exposed to high artificial light intensity for 160 minutes. It indicates that the photoinhibition only caused by strong light is reversible , with the facts that Fv/Fm went up gradually after 13:00,and was close to the maximum at 18:00 in nature, and that Fv/Fm nearly reached to the untreated(control) after recovery of 2~4h in weak light in artificial light.Drought not only made the extent of photoinhibition of tea plant serious but also made the ability更多还原