【作者】 王艳；

【导师】 张新忠； 韩振海； 王忆；

【作者基本信息】 中国农业大学 ， 果树学， 2014， 博士

【摘要】 由于童期较长,导致了苹果的育种周期长。高等植物在个体发育的过程中往往伴随着一系列基因的差异表达以及蛋白质的定性或是定量的变化,但是,多年生木本被子植物的个体发育过程中的生理和分子机制仍不清楚。在之前的研究中发现,一些蛋白酶在苹果实生树的个体发育过程中会发生翻译后修饰的现象,磷酸化修饰作为一个重要的翻译后修饰影响着蛋白的功能及代谢,但它在多年生木本被子植物不同发育阶段所发生的动态变化仍不十分清楚,所以研究磷酸化修饰在苹果实生树不同发育阶段的差异将有可能从蛋白质翻译后水平揭示阶段转变的分子机制,对进一步了解多年生木本被子植物的个体发育具有重要的理论意义。在本研究中,为了确认蛋白质磷酸化修饰在苹果实生树个体发育过程中存在差异以及在不同杂交组合的不同实生树中的一致性,以‘红玉’x‘金冠’和‘紫塞明珠’ב富士’两个杂交组合,分别为11年生和6年生的苹果实生树为试验材料,提取童期(J),成年营养生长期(V),成年生殖生长期(R)三个不同发育阶段的叶片蛋白,通过对Pro-Q Diamond染色效果进行对比以及蛋白去磷酸化方法进行优化,采用双向电泳与western blotting免疫印迹法结合对S6PDH的磷酸化修饰进行初步验证,确定试验体系。对蛋白进行去磷酸化处理并通过双向电泳对蛋白进行分离,同时将未进行去磷酸化处理的蛋白双向电泳分离之后采用Pro-Q Diamond进行染色,找出在在不同发育阶段差异表达的磷酸化蛋白点,并采用MALDI-TOF-TOF对其进行质谱鉴定。研究结果如下：1、通过与TCA/丙酮提蛋白法相结合优化了苹果叶片蛋白质CIP去磷酸化法,使用此方法可获得背景清晰、分辨率高的双向电泳图谱。利用蛋白质双向电泳和蛋白免疫印迹法Western blotting)对S6PDH在苹果实生树个体发育过程中发生的磷酸化修饰进行鉴定,证明采用CIP对蛋白进行去磷酸化的方法是可行的。2、在三株苹果杂种实生树中共检测出7组差异磷酸化蛋白质,经过MALDI-TOF-TOF质谱鉴定,5组共计107个蛋白质点获得鉴定结果：A6,A33,A48包括55个蛋白点,被鉴定为Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments,其蛋白表达量以及磷酸化程度在不同的发育阶段存在差异：A6组蛋白在三株实生树中均表现为生殖生长期蛋白表达量低于童期和成年营养生长期；A33组蛋白中A33-1在3棵实生树中均在童期表达较弱,A33-2在成年营养生长期的蛋白表达量高于童期和成年生殖生长期,A33-3在成年营养生长期的蛋白表达量高于童期和成年生殖生长期,且A33-3的蛋白表达量高于A33-1和A33-2；A48组蛋白中A48-1在3棵实生树中均在成年生殖期蛋白表达量弱,A48-2在成年营养生长期表达高于童期和成年生殖生长期,A48-3在成年营养生长期的表达量低于童期和成年生殖生长期。B38组27个蛋白点被鉴定为Ribulose bisphosphate carboxylase/oxygenase activase,这组蛋白点的表达量从童期到成年生殖生长期呈现下降的趋势。B38-2的蛋白表达量在不同实生树的不同发育阶段均高于B38-1和B38-3。C24组的25个蛋白点被鉴定为Tubulin beta chain,低等电点的磷酸化β-tubulin在童期和成年营养生长期的表达量较高,在三棵实生树的不同发育阶段等电点较高的蛋白点C24-3的蛋白表达量高于等电点较低的蛋白点。从这些结果中可以看出蛋白质磷酸化修饰在苹果实生树的个体发育过程中存在差异,且这些差异在不同杂交组合的不同实生树中是一致的。3、在三棵实生树中,叶片净光合速率分别在40节、80节、140节(07-07-115),30节、90节、120节(07-07-133)和30节、90节、140节(07-09-141)出现峰值。随着树体节位的升高,叶片狰光合速率发生变化,但这种变化没有随着节位升高而发生梯度的增加,所以在本试验中被大量鉴定出来与光合作用相关的Rubisco large subunit和Rubisco activase,它们的磷酸化作用与树体节位无关,而是与树体的个体发育相关。更多还原

【Abstract】 The breeding period for apple is comparatively long mainly because of its long juvenile phase. The process of individual development in higher plants was necessarily accompanied by a series of gene differential expression and protein qualitative or quantitative changes, but the physiological and molecualr mechanisms during the developmental process of individuals in perennial woody angiosperm plants are still unclear. Previous studies showed that post-translational modifications happened for some proteases in individual apple seedling plant during its developmental processes. Protein phosphorylation is an important post-translational modification affecting protein function and metabolism; therefore it is posssible to uncover the molecular mechanism of phase change at protein post-translational level by studying the various phosphorylations in apple seedling plant during different developmental stages, which is important to further understand the individual development in woody perennial angiosperm plants.In this study, to confirm the accordance of protein phosphorylation among different apple seedlings derived from different hybrid crosses, proteins from three different developmental stages’ leaves of three apple seedlings derived from two hybrid crosses (’Jonathan’ x ’Golden Delicious’ and ’Jonathan’ x’Zisai Pearl’ x ’Red Fuji’)were extracted, then optimize the Pro-Q Diamond staining and protein dephosphorylation methods. Combine2-DE and western blotting to preliminary identified the phosphorylation of S6PDH and then confirmed the testing system. Proteins were extracted and subjected to alkaline phosphatase pre-treatment, following separated by two-dimensional gel electrophoresis, meanwhile used Pro-Q Diamond to stain the protein that have not dephosphorylation, then find out the differentially expressed phosphoproteins and identified by MALDI-TOF-TOF mass spectrometry. The results were shown as follows:1. The modified dephosphorylation method combined with the TCA/acetone protocol can easyily get clear background and phosphoprotein spots could be visualized on2-DE map. Combined2-DE with western blotting to identified phosphorylation of S6PDH during ontogeny in the Malus seedlings, we found it is feasible to identified the phosphorylation of protein by using alkaline phosphatase to dephosphorylation of proteins2. Five groups of seven groups’ total of107phosphorylated protein spots in three apple seedlings were identified by MALDI-TOF-TOF:Group A6, A33, A48contains55spots and they were identified as ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large-chain fragments, they are varied significantly in protein abundance and degree of phosphorylation among ontogenetic phases:the global abundance of A6in the reproductive phase of the three seedlings was significantly lower than in juvenile and adult vegetative phases; A33-1expression was relatively weak during the juvenile phase of the three seedlings, the expression abundance of A33-2was higher during the adult vegetative phase than in juvenile and adult reproductive phases, the protein in spot A33-3was expressed more abundantly during the adult vegetative phase than in juvenile and adult reproductive phases and the expression abundance of A33-3 was greater than that of A33-1and A33-2; The expression of protein in A48-1was relatively lower in the reproductive phase, A48-2was expressed more abundantly in the adult vegetative phase than in juvenile and adult reproductive phases. The expression abundance of A48-3was likewise lower in the adult vegetative phase than in juvenile and reproductive phases.Group B38contains27spots and they were identified as Rubisco activase, the abundance of Rubisco activase was characterized by a declining gradient from juvenile to the reproductive phases; The expression of spot B38-2was more abundant than that of B38-1and B38-3during all ontogenetic phases of the three seedlings.Group C24contains25spots and they were identified as Tubulin beta chain. More extensively phosphorylated tubulin beta chain spots with lower isoelectric points were most abundant during juvenile and adult vegetative phases. Spot C24-3, with a higher isoelectric point, was expressed more abundantly than spots with lower isoelectric points during all three phases in the three seedlings.From the results we can see that protein phosphorylation varied significantly during vegetative phase change in apple seedlings. Most of the observed changes were consistent among seedlings and between hybrid populations.3. For the three apple seedlings, net photosynthesis rate of node40,80,140(07-07-115), node30,90,120(07-07-133) and node30,90,140(07-09-141) were peak. With the increase of leaf s node, net photosynthetic rate also changes, but the net photosynthesis rate did not gradient increasing with the rise of the node. Thus we thought the phosphorylation events on Rubisco large subunit and Rubisco activase were affected by the different ontogenetic stages but not the leaf’s level.更多还原