【作者】 刘芳；

【导师】 周蕴薇；

【作者基本信息】 东北林业大学 ， 园林植物与观赏园艺， 2014， 博士

【摘要】 百合是世界各国人们都喜爱的球根花卉,鳞茎秋季收获后便进入自然休眠的状态。需要经过一段时期的低温处理才会彻底解除休眠保证第二年正常开花生长。鳞茎休眠的解除是一个复杂的生物过程。激素、碳水化合物与内外环境因子及遗传基因等构成复杂的网络系统调控着休眠的整个过程。百合鳞茎休眠的研究目前主要集中在低温解除休眠过程中的形态变化和生理生化变化方面,而分子水平的研究甚少。本研究以细叶百合(Lilium pumilum)为试材,5℃低温解除休眠过程中,在对百合鳞茎休眠的细胞学及生理学研究的基础上,利用分子生物学技术,构建了百合鳞茎休眠解除过程中的抑制差减cDNA文库,获得大量与休眠调控相关的基因,并利用双向电泳技术手段来研究和分析细叶百合鳞茎通过低温解除休眠过程中差异表达的蛋白质及其调控功能,以期为进一步研究鳞茎休眠机制奠定基础。主要研究结果如下：1、通过石蜡切片和实体显微结构观察,低温冷藏期间,细叶百合花芽分化主要分为小花原基分化期,外轮花被原基分化期,内轮花被原基分化期,雄蕊和雌蕊原基分化期四个时期。鳞片及顶芽细胞内淀粉粒数量逐渐减少。冷藏36 d以后细胞分裂数量逐渐增加。透射电镜观察表明,低温处理0～12 d,鳞茎物质和能量代谢较弱,鳞茎不能萌发；12～48 d鳞茎萌发率较低,线粒体及高尔基体分泌囊泡增多,细胞通过质膜内吞及胞间连丝进行物质交流；48～72 d,核仁松散,内质网有序排列,在液泡膜与质膜边缘形成内质网桥,内质网桥一直延伸到核膜方向,胞内及胞间联系已经建立,鳞茎出现新生根；冷藏84 d时顶芽已伸长到鳞茎顶端。内质网桥延伸到胞间连丝,细胞器内部结构及数量增加,休眠完全打破。冷藏过程中百合鳞茎的顶芽随冷藏时间的推移渐渐伸长,呼吸速率呈现先下降后上升的趋势。冷藏时间越长,鳞茎萌发时间越短。2、在细叶百合鳞茎低温贮藏过程中,顶芽与鳞茎盘中的核酸含量发生了明显的变化,顶芽分生组织中DNA、RNA含量及RNA/DNA的比值在鳞茎冷藏36 d时变化最为明显,贮藏36 d是内外鳞片DNA及核酸总含量的转折点。顶芽和鳞茎盘冷藏0～36 d内可溶性蛋白质的含量升高,鳞茎不同部位可溶性蛋白质含量在冷藏48 d后迅速降低也是鳞茎开始解除休眠的重要体现。鳞茎休眠解除过程中伴随着旺盛的糖类代谢活动,淀粉含量下降,淀粉酶活性升高。贮藏前期,蔗糖含量,蔗糖代谢关键酶SS和SPS活性上升,SPS和SS协同控制蔗糖代谢及转运。冷藏条件下鳞片及顶芽内淀粉向可溶性糖方向代谢过程中促进了AGPase活性的增加。AGPase可能是休眠打破的一个重要的生理决定因素。鳞片、顶芽及鳞茎盘SOD、POD、CAT活性在冷藏前期下降,冷藏中后期,SOD、CAT、ASP活性升高,鳞片中POD的活性下降,顶芽及鳞茎盘POD、PPO在冷藏中期上升。各种代谢相关酶在不同器官中的作用并不完全相同。36 d是鳞茎解除休眠的起点,60 d时鳞茎基本解除休眠。3、利用抑制消减杂交技术构建了细叶百合鳞茎休眠及解除正、反向抑制消减文库。对正、反向文库随机挑选阳性克隆测序各获得100个表达序列标签(EST),在NCBI上进行BLASTx分析并用KOBAS系统将获得的unigene定位到Pathways中。分析结果表明,差异基因GO功能主要富集在蛋白质合成、细胞抗性及防御过程、信号转导、结合活性、转录活性、代谢过程等。休眠文库ESTs功能主要涉及胁迫响应方面,苯丙素生物合成及苯丙氨酸代谢途径,促分裂素原活化蛋白激酶/MAPK激酶及抗坏血酸代谢途径等与休眠有关。休眠解除文库在糖代谢、激素响应及信号转导方面表达量较高,淀粉及蔗糖代谢途径、α-亚麻酸代谢、亚油酸代谢途径、甲硫氨酸和半胱氨酸等氨基酸代谢途径、磷脂酰肌醇信号系统等参与了休眠解除过程。选取了8个差异表达基因用Real-time PCR检测分析,这些基因在百合鳞茎休眠解除过程中大部分上调表达。4、利用荧光差异凝胶电泳分离得到31个差异表达蛋白点。相对于休眠鳞茎,休眠解除鳞茎中上调表达的蛋白点有14个,占总数的45.16%；下调表达的蛋白点有17个,占总数的54.84%；应用MS质谱成功鉴定12个差异蛋白点,按功能划分为6类,分析表明,这些蛋白主要为胁迫类蛋白,可能涉及鳞茎内物质的代谢过程,进而调控鳞茎的休眠解除。更多还原

【Abstract】 Lily is one of the world famous bulb flowers, Natural dormancy is a characteristic of lily,Bulb dormancy can be completely broken after a period of low temperature treatment,and it will Normally flower and grow next year. Bulb dormancy-breaking is a complex biological process,Hormones,carbohydrates and the internal and external environmental factors and genetic constitute a complex network system to control the whole process of the dormancy.the research on dormancy mainly concentrated in morphology and physiological and biochemical changes during breaking dormancy under low temperature. Less in molecular level. We studied cytological and physiological changes of bulbs in Lilium pumilum during breaking dormancy under 5℃. On the basis of these research results, We constructed 2 SSH-cDNA libraries using dormant and dormancy breaking bulbs. a lot of dormancy regulation related genes were isolated, and we isolated differentially expressed proteins and analyzed their functions Using two-dimensional electrophoresis technology in the process of breaking dormancy under low temperature. In order to lay the foundation for further study bulb dormancy mechanism. The main results were as follows:1.Using paraffin section and stereoscopic microscopic observation, during cold storage, the lily flower bud differentiation is mainly divided into four periods:floret primordium differentiation stage, outer perianth primordium differentiation stage, inner perianth primordium differentiation stage, stamen and pistil primordium differentiation stage. the number of starch grains gradually reduced in Scales and apical bud cells. the number of fission cells increased gradually. after 36 d,Transmission Electron Microscope observation showed that the material and energy metabolism of bulbs were weak between 0 to 12 days and bulbs couldn’t sprout; the germination rate of bulbs was lower and mitochondria and golgi vesicles increased,Cells exchanged materials through the plasma membrane endocytosis and plasmodesmata between 12 to 48 days. Nucleoli were incompact and endoplasmic reticulums scattered orderly between 48 to 72 days, and endoplasm bridges formed at the edges of vacuole membrane and plasma membrane, with some of them extending to the nuclear envelope,Intracellular and intercellular connection had been established in this period. Bulbs appear new root;the apical bud has been extended to the top of the bulb after 84 d,Endoplasm bridges and the plasmodesmata connected in budding period, and the internal structures and quantity of organelles increased obviously,Dormancy is completely broken in this period.the apical bud elongated gradually during refrigerated condition. The respiratory rate showed a trend of decline after rising first. Cold storage time longer, shorter bulb germination time.2.The obvious changes of nucleic acid content in apical buds and basal plates of Lilium pumilum bulbs.The change of DNA, RNA content and RNA/DNA ratio in apical buds were most obvious when bulb refrigerated 36 d,36 d is a turning point of the total amount of nucleic acid, DNA in inner and outer scales.the content of soluble protein in apical buds and basal plates increased within 0～36 d during refrigerated condition. the content of soluble protein in different parts of the bulbs reduced rapidly after 48 d, which was the important symbol of bulb for breaking dormancy. the sugar metabolic activity was strong in the process of dormancy breaking, the starch content of bulbs decreased, the activity of amylase activity remains high.At the beginning of the storage, the sucrose content, sucrose metabolism key enzyme SS and SPS were on the rise, there was a synergy between SPS and SS in controling of sucrose metabolism and transport. During refrigerated conditions the metabolism direction of starch to soluble sugar promoted the increase of AGPase activity. AGPase is likely to be a important physiological deciding factor for dormancy breaking.SOD, POD, CAT activity in the early stage of the cold storage has a downward trend, during middle and later periods, SOD, CAT and ASP are elevated, POD activity of scales continued to decline in the process of cold storage, POD and PPO activity of apical buds and basal plates is also on the rise in the middle of the cold storage. The role of various metabolism related enzymes in different organs is not the same.,36 d is the starting point of bulb breaking dormancy, at 60 d bulb dormancy basicly broke.3.We constructed 2 SSH-cDNA libraries using dormant and dormancy breaking bulbs of Lilium pumilum. The forward library contained genes preferentially expressed in dormancy breaking bulbs and the reverse library contained genes preferentially expressed in dormant bulbs. 100 positive clones were selected respectively for sequencing from the two libraries. We analyzed ESTs in BLASTx of NCBI and unigenes positioning in the pathways using KOBAS system. Results indicated that in dormant bulb library, these functionally known genes were detected in stress response. Phenylalanine metabolism and MAPK signaling pathway were associated with dormancy. In bulb dormancy breaking library, GO function of different genes mainly enriched in protein synthesis, cell resistance and defense, signal transduction, binding activity, transcriptional activity and metabolic process, etc.the function of ESTs involved in sugar metabolism, hormone response and signal transduction,Linoleic acid metabolism and Starch and sucrose metabolic pathway was involved in dormancy breaking process. the establishment of both libraries laid a foundation for further understanding the molecular mechanism of lily during bulb dormancy.4. A total of 31 differentially expressed proteins were separated by using fluorescent difference gel electrophoresis, Among these proteins, there are 14 proteins up-regulated and 17 proteins down-regulated in the dormancy breaking bulbs. up-regulated proteins accounts for 45.16% of the total,and down-regulated proteins accounts for 54.84% of the total, These Proteins were classified by function into 6 categories. Comprehensive analysis shows that these proteins are mainly stress protein, may involve metabolic process of the bulbs, and regulating the bulb dormancy breaking.更多还原