【作者】 宁德利；

【导师】 王柏臣；

【作者基本信息】 东北林业大学 ， 林木基因组学， 2013， 博士

【摘要】 对于生长在温带和寒带地区的树种来说,生长与休眠周期交替是它们生存和生长的一个重要的自适应特征。植物芽休眠的诱导到解除是个复杂的生物学过程,它涉及到细胞信号转导、碳水化合物代谢、激素调控、氨基酸的转运、营养物质合成及能量的产生、细胞分裂及分生组织分化及等多个方面,是多种基因协同作用的结果。本研究综合运用无标记定量分析技术和Solexa测序方法对生长在中国哈尔滨(E126°41’,N45°45’)分别代表进入休眠、保持休眠和解除休眠三个阶段的小黑杨的芽进行蛋白质组学和转录组学的研究,系统地分析了杨树顶芽从休眠诱导到解除的分子调控机制,初步阐释调控杨树芽休眠诱导到解除的调控网络,发掘关键基因。进一步从整体水平上了解林木芽休眠诱导和解除的分子机理。通过蛋白质组学的研究,我们鉴定到的74个差异蛋白一共被分为14类,涵盖了大范围的生物功能与活动路径。本研究所获得的差异蛋白主要参与了碳水化合物代谢、氧化还原调控、氨基酸运输与代谢和胁迫响应等生物途径,所占比例分别为22%,19%,10%,8%。在表达差异显著的蛋白中,31个蛋白在芽休眠的三个阶段中呈上调表达,5个蛋白下调表达,而其余的38个蛋白在三个时期休眠芽中特异表达。根据亚细胞定位分析,发现这74个蛋白质主要位于细胞质(64%)、线粒体(10%)、叶绿体(4%),还有18%的蛋白质不清楚在细胞中的具体位置和分布。在杨树芽休眠过程中,参与糖酵解及磷酸戊糖途径的相关酶在芽2中表现为上调表达,主要为芽休眠提供必要的能量储备,其主要形式就是碳水化合物。同时植物产生的渗透调节物质,包括蔗糖、葡萄糖、果糖、半乳糖等,通过调节代谢途径来减少或消除非生物逆境所带来的伤害。此外,消除活性氧等有害物质的过氧化物酶类蛋白和防御蛋白在芽2休眠保持时期上调表达,为杨树芽顺利过冬提供了保证。在转录组学研究方面,我们将变化倍数(log2值)>2和<-2、统计学有效值(P-value值)<0.001、错误发生率(FDR)<0.01作为筛选基因的标准,共鉴定到了562个表达显著差异的基因,其中154个上调和408个下调的基因。它们涉及转录调控、产物代谢、蛋白激酶调控、逆境胁迫应答、激素信号转导等生物功能,从中可以看出差异表达的基因与植物的抗逆反应及生长发育调控有着密切关系。在所鉴定到的表达差异显著的基因中与转录相关的基因多在芽1阶段大量表达,特别是与核糖体相关基因的表达,说明杨树芽休眠过程首先发生在转录水平；而与细胞周期相关基因多在芽1和芽3阶段上调表达,标志着芽休眠的进入和解除。在代谢和能量转换的通路中的差异基因,mRNA与蛋白质表达变化趋势基本相同。那些在mRNA与蛋白质表达变化差异显著的基因,将为我们今后对抗逆基因转录后调控机制的研究提供参考信息。更多还原

【Abstract】 Induction and break of bud dormancy are important features for perennial plants surviving extreme seasonal variations in climate. However, the molecular mechanism of the dormancy regulation, still remain poorly understood. To better understand the molecular basis of poplar bud dormancy, in this study, we employed label-free quantitative proteomics and Solexa sequencing transcriptomics for investigation of complex protein expression in Populus simonii×P. nigra dormant buds during dormancy induction, dormancy, and dormancy break in Harbin, China,(E126°37’, N45°42’), with the aim of providing an overview of the biology of bud dormancy and elucidating the mechanisms responsible for differences in metabolic pathways. We used a label-free quantitative proteomics method for investigation of differential protein expression in apical buds of poplar. Among these identified over300proteins during poplar bud dormancy, there are74significantly altered proteins, most of which involved in carbohydrate metabolism (22%), redox regulation (19%), amino acid transport and metabolism (10%), and stress response (8%). Thirty-one of these proteins were up-regulated, five were down-regulated during three phases, and thirty-eight were expressed specifically under different conditions. Pathway analysis suggests that there are still the presence of various physiological activities and a particular influence on photosynthesis and energy metabolism during poplar bud dormancy. Differential expression patterns were identified for key enzymes involved in major metabolic pathways such as glycolysis and the pentose phosphate pathway, thus manifesting the interplay of intricate molecular events in energy generation for new protein synthesis in the dormant buds. Furthermore, there are significant changes present in redox regulation and defense response proteins, for instance in peroxidase and ascorbate peroxidase.In the transcriptomics study,we analyzed the most differentially regulated genes with a log2ratio>2or <-2using a greater statistically significant value (P<0.001) as well as false discovery rates (FDR<0.01), representing154up-and408down-regulated transcripts. The differential genes are involved in transcription regulation, energy metabolism, protein kinase, stress response, hormone signal transduction and other biological function, and are association with stress resistance of plant growth. Among them, some genes related transcription are over express in bud1, especially the ribosome gene expression, suggest that poplar bud dormancy process firstly occurs in transcription level; The cell cycle related genes in bud1and bud3are up expression that imply the bud dormancy into and break. Analysis finds that mRNA and protein involve in the metabolism and energy conversion expression trend is similar. While mRNA and Protein expression abundant of the genes that are related to metabolism and energy conversion is significantly different. The inconsistency of the transcription and protein expression profiles may provide important clues to the research of post-transcriptional regulation of stress resistance genes.更多还原