【作者】 胡昊；

【导师】 刘勇； 王有年；

【作者基本信息】 北京林业大学 ， 森林培育， 2012， 博士

【摘要】 桃是我国广泛栽培的重要核果类果树作物,同时也是国际公认的蔷薇科中的模式植物之一。同其它核果类果实类似,形成高度木质化的内果皮结构是桃果实发育过程中的一个重要特征。为了系统研究硬核期桃内果皮的发育形成机制,本研究以“久保”桃(Prunus persica L. cv. Okubo)为试材,通过测定硬核期果实生长参数,观察内果皮木质素的沉积动态确定果实硬核期；利用基于双向电泳和质谱的蛋白质组学技术识别鉴定了硬核期内果皮和中果皮的差异表达蛋白；克隆可能参与内果皮发育木质化的NAC和MYB类转录因子,并对这些基因在硬核期间的表达谱进行分析。研究结果如下：(1)果实生长参数和木质素沉积动态分析表明,内果皮发育木质化始于幼果快速生长期至果实硬核期的过渡期期间,即盛花期后35至45天。此期间木质素沉积由种子腔附近开始逐渐向外扩展,内果皮木质素含量也呈明显的指数型增长。(2)双向电泳蛋白质表达谱分析表明,硬核期内果皮和中果皮中的蛋白质点数均出现明显减少。通过差异双向电泳表达谱分析,共发现68个在硬核期内果皮和中果皮中都存在差异表达的蛋白质点。进一步质谱鉴定表明,这些差异表达的蛋白质点分别属于主要代谢、次生代谢、胁迫响应、蛋白质代谢、细胞结构形成和信号转导等功能途径。综合蛋白质表达谱和质谱鉴定结果,本试验发现硬核期内果皮的主要代谢途径受了到明显抑制,但是其中参与三羧酸循环的关键酶——丙酮酸脱氢酶的丰度却出现了异常增高(上调19.9倍),且其丰度变化与内果皮木质素变化呈高度正相关(R=0.940),因此推测它可能与内果皮的发育木质化过程有关；本研究发现硬核期桃内果皮的木质素生物合成途径与类黄酮生物合成途径相关酶类在蛋白质组学水平上存在明显的竞争关系,即内果皮在幼果快速生长期以类黄酮生物合成为主,在果实硬核期转为以木质素生物合成为主；本研究发现硬核期桃内果皮中存在组织特异的氧化胁迫现象,并推测它可能是激活内果皮发育木质化过程中细胞程序性死亡机制的重要因素之一。(3)本研究采用同源基因克隆法,从桃中克隆了2个NAC类转录因子和4个MYB类转录因子的全长编码区。氨基酸序列和系统进化分析表明,PpNST1/SND1和PpMYB46/63/83/85与毛果杨和拟南芥等参与调控次生细胞壁形成和木质化的NAC/MYB类转录因子具有较高同源性。荧光实时定量PCR分析表明,这些转录因子的基因表达水平随内果皮的木质化程度加强而不断升高,并于盛花期后52或59d达到最高表达量(上调10.0至325.2倍),且它们在内果皮中的表达水平都远高于中果皮。对PpNST1/SND1和PpMYB46/63/83/85下游可能的作用靶标进行进一步的表达分析表明,它们可能在内果皮发育木质化的转录调控网络中扮演重要角色。更多还原

【Abstract】 Peach(Prunus persica) is one of the economically important fruit crops in China, and also one of the model species in Rosaceae. Peach fruit is similar to other drupes, and featured with a hard lignified endocarp, which surrounding the seed.In order to investigate the development and formation of endocarp, peach cutivar ’Ohkubo’was used as material and several approaches were employed. The growth parameters and lignin deposition process were observed to determine hardening period; a proteomic approach based on two-dimensional electrophoresis together with mass spectrometry was used to explore the differentially expressed proteins; two NAC and four MYB transcription factor genes were cloned, and their expression patterns were examined. The main results and conclusions are as follows.(1) Analysis of growth parameters and lignin deposition revealed that the lignification of endocarp occurred in the transition period from the young fruit fast-growing stage to the fruit hardening period stage (between35and45days after flowering). During this period, the lignin deposition process featured a rapid expansion from seed cavity to the peripheral region of the endocarp, and the change of lignin content also fitted to an exponential growth curve.(2) Sixty-eight protein spots with different expression patterns were identified in both the endocarp and mesocarp during pit hardening, and the majorities were involved in primary or secondary metabolism. In contrast to most proteins associated with primary metabolism in the endocarp, whose expression is down-regulated, expression of pyruvate dehydrogenase (PDH) unexpectedly increased exponentially (up to19.9-fold). Moreover, its expression pattern was linearly positively correlated with the exponentially growing lignin content (R=0.940), which suggests that PDH may play a role in endocarp lignification. The proteome data also revealed different spatiotemporal expressions of enzymes involved in the lignin and flavonoid pathways that provided proteome-level evidence to support the hypothesis that these two pathways are competitive during endocarp development. In addition, the endocarp-specific oxidative stress was also observed and proposes that it may act as a stimulating factor in activating lignification and subsequent programmed cell death in the endocarp.(3) Two NAC domain and four MYB domain transcription factor genes were isolated from peach endocarp by homologous cloning method. The amino acid sequence alignment and phylogenetic analysis results indicated they were closely related to secondary cell wall formation-and lignification-related transcription factors in Populus trichocarpa and Arabidopsis thaliana. Quantitative real-time PCR analysis showed that the expression patterns of them were dramatically up-regulated during the lignification of peach endocarp (up to15.0to325.2-fold)), and their transcript expression levels were more abundant in endocarp than in mesocarp. Moreover, the expression analysis of the putative downstream targets of them suggested that they may play important role in endocarp developmental lignification.更多还原