【作者】 孙霞；

【导师】 王秀峰；

【作者基本信息】 山东农业大学 ， 观赏园艺， 2013， 博士

【摘要】 菊花（Chrysanthemum morifolium Ramat.）是菊科（Asteraceae）菊属（Chrysanthemum）植物，是世界四大鲜切花之一。栽培品种3000多个，用于切花菊的品种多为典型的短日照植物，光周期途径调控开花主要由光受体基因接受光信号，传递给节律钟基因，通过节律钟调控输出基因激活下游转录因子的表达，在叶中上调促进开花基因的表达，输送到茎尖与成花整合因子作用完成花芽分化。本研究以白色切花菊‘神马’为试材，因其典型的短日照特点，能较准确人工调控花芽分化进程，是研究菊花花芽分化分子机理的模式材料。通过大规模高通量测序，获得数据与NCBI核酸蛋白数据库进行比对，经生物信息分析，确定功能分类及涉及的代谢途径，获得菊花花芽分化期相关的基因表达信息，探索在菊花不同花芽分化阶段相关基因的差异表达富集模式及表达谱；对光周期途径的关键基因进行全长的克隆与表达分析，在分子水平上对菊花开花时间的网络途径有初步的了解，为后期进一步进行基因的功能验证及通过分子育种方法（反义抑制或转基因）培育目标花期新品种提供基础理论依据。本研究主要成果如下：1.本试验获得菊花花芽分化期转录组ESTs，对所有unigenes进行功能分类，收集到54,488条分属于分子功能、细胞组分和生物学过程的unigenes。其中参与分子功能的共12种，参与细胞组分的共11种，参与生物学过程的共18种。得到编码蛋白框（CDS）的核酸序列和氨基酸序列为54,644条。用ESTscan比对所有unigenes，得到其CDS的核酸序列和氨基酸序列均为8,506条。将所有unigenes与COG数据库BLAST比对显示，共获得22,871条与已知其他植物同源，属于23类直系同源簇群，未知功能的有780条；得到25,001条参与代谢通路的unigenes，从中收集到与植物节律代谢通路相关unigenes共269条，从中比对到参与光周期途径的光受体基因、生物节律钟基因和生物节律钟输入和输出基因等相似功能基因共20个。2.初步得出菊花花芽分化期涉及植物节律途径的数字基因表达谱：菊花光受体PHYB在花芽分化启动期表达量达最高值，是暗处理前的近五倍，随后稍有下降，到总苞鳞片分化中期仍维持暗处理前的近三倍，到分化完成一直还维持近两倍的表达量；节律钟组件APRS、 TOC1表达高峰值均出现在总苞鳞片分化中期；PIF3高峰值出现在花芽分化启动期，到总苞鳞片分化中期仍维持暗处理前的近三倍；FKF1在花芽分化启动期上调到高峰值，总苞鳞片分化中期下调表达，随后在小花原基分化中期和分化完成期又上调到高峰。节律钟输出基因GI在花芽分化启动期表达量达最高值，在总苞鳞片分化中期稍有下降，分化完成期又有回升。CO暗处理一天后反而表达量降低，在总苞鳞片分化中期表达量达最高值。CHS（查尔酮合成酶）在暗处理一天后表达量上调，启动期达近五倍的最高值，总苞鳞片分化中期表达量稍下降，随后在小花原基分化中期下调表达。3.初步得出菊花花芽分化期差异表达基因显著富集的最主要生化代谢途径和信号转导途径：集中在光合作用及核蛋白体途径中。4.初步得出菊花不同花芽分化阶段差异表达基因富集参与的生物功能：花芽未分化期，植物中蛋白质精氨酸甲基转移酶家族基因和蛋白参与基因转录调控、氧化还原酶活性基因转录加强；花芽分化启动期，脂肪酸合成酶、糖氢共转运蛋白活性基因差异表达富集，蛋白质精氨酸甲基转移酶的差异表达显著；总苞鳞片分化中期，信息显示富集在各种蛋白质、核苷酸糖的合成；小花原基分化中期，蛋白质精氨酸甲基转移酶活性加强，氧化还原酶活性基因转录加强；花冠分化末期即花芽分化完成期，基因差异表达富集仍在结构分子活动、砷酸盐还原酶活性和作为花和花粉发育的能量来源的脯氨酸的累积等方面。5.从菊花‘神马’中克隆得到节律钟输出基因GIGANTEA的cDNA全长序列，命名为CmGI基因，登录号为JQ043439。对其做序列信息分析；菊花CmGI的表达呈昼夜节律表达模式，高峰值出现在16:00；不同花芽分化阶段叶片中CmGI基因mRNA水平差异大，两个高峰值分别出现在花芽分化启动期和小花原基分化中期；盛花期表达量较高。6.从菊花‘神马’中克隆得到开花相关基因CmCOL的cDNA全长序列，登录号为KC589293，对其做序列信息分析；菊花CmCOL mRNA在叶中高表达，呈昼夜节律表达模式，表达高峰值出现在04:00；短日照处理能上调叶中CmCOL，对芽中的影响不明显；不同花芽分化阶段叶片中高峰值出现在小花原基分化中期；盛花期叶片中高表达。7.菊花CmFTL mRNA只在短日照条件下的叶片中呈现节律表达，00:00时为高峰值；芽中及长日照叶、芽中均检测不到昼夜起伏；不同花芽分化阶段叶片中CmFTLmRNA水平差异大，小花原基分化中期达最高峰值；花蕾期叶中高表达。8.研究显示菊花叶片感应光周期，短日照条件下CmGI首先于暗处理1d后启动上调，花芽分化启动期出现第一个峰值，而CmCOL则滞后CmGI，于花芽分化启动期轻微上调，暗示CmGI位于CmCOL上游，小花原基分化中期CmGI上调至高峰值时CmCOL上调到高峰。CmFTL在CmCOL微调时则无明显变化，在小花原基分化中期CmCOL上调至高峰值时CmFTL也上调到高峰值，暗示CmFTL滞后CmCOL，位于其下游。实验揭示菊花CmGI、CmCOL、CmFTL在SD条件下与拟南芥LD下及水稻SD下的GI-CO-FT调控模式相似。更多还原

【Abstract】 Chrysanthemum morifolium Ramat. is a chrysanthemum plant species in the Asteraceaefamily. This plant is one of four major cut flowers worldwide, and it has more than3000cultivated varieties. Cut flowers are typically obtained from short-day (SD) plant species, thephotoperiod regulation technique primarily controls flowering through light receptor geneproducts which receive a light signal and transmit the signal to circadian gene products. Thecircadian rhythm clock output gene activates the expression of the transcription factor andthen enhances the expression of the flowering-promoting gene in the leaves. Then transportedto the shoot-tip and functions together with flowering-integrating factors, regulation ofdownstream flowering-promoting factors in the photoperiod pathway.This study with white cut-flower chrysanthemum‘Jinba’as test materials, because of itstypical characteristics of short day, can be accurately controlled flower bud differentiationprocess, is the study of molecular mechanism of chrysanthemum flower bud differentiationmodel material. By high-throughput EST sequencing technique, the data compare with theNCBI nucleic acid protein database, through bioinformatics analysis, determine the functionclassification and metabolic pathways involved, into chrysanthemum flowering related geneexpression information, explore different in chrysanthemum flower bud differentiation phasedifferentially expressed enrichment pattern and expression of related genes，the photoperiodpathway related the key gene cloning and expression analysis, at the molecular level ofchrysanthemum flowering time of network way to have a preliminary understanding,So the information might be the base of system research the chrysanthemum into themolecular regulation mechanism and its biological function and sellecting flowering relatedgenes and using for transgenes.The main research results are as follows:1. Studies on transcriptomic express sequence tags (ESTs) of chrysanthemum duringfloral differentiation. sellected54,488unigenes which of moleculor functions, cell conpenentsand bioprocesses. All of the above unigenes involved12of molecular functions and11of cellcomponents, and18of bioprocesses. Sequences of nucleic acids and amino acids of54,644 encoding protein boxes (CDS) were sellected from all of the unigenes of chrysanthemumduring floral differentiation by BLAST software. Also8,506unigene sequences of nucleicacids and amino acids of CDS by ESTscan software. Comparison to the GenBanknon-redundant (nr) database in COG database revealed that22,871unigenes sequences weresimilar to other known plant homologous genes which classfied to23homologys and780unigenes sequences maybe new genes in chrysanthemum. Also25,001unigenes weresellected which of participating in metabolism pathway using KEGG database. From there269unigenes also sellected that related to participating circation rhythm metablism pathways,and also sellected20homologous genes which related to photoperiod pathways includingphotoreceptor genes, circadian rhthm clock genes, circadian rhythm import and export genes.2. Digital gene expression of chrysanthemum bud differentiation period involving plantrhythm pathways：Chrysanthemum light receptors PHYB orthologous genes expressed peaked at buddifferentiation initiation stage, is nearly five times more the pre-treatment level, then a slightdecline, until the involucre scales middle differentiation stage, nearly three times more thepre-treatment level，at bud differentiation completion stage，nearly two times the amount ofexpression.Circadian clock component APRS, TOC1expressed peaked at involucre scaledifferentiation mid-term stage.PIF3expressed peaked at bud differentiation initiation stage.FKF1express the change of the peak, dark treatment decreased expression a day after，the expression peaked at bud differentiation initiation stage and the floret primordiumdifferentiation and bud differentiation completion stages.Circadian clock output gene expression GI dark treatment after a day’s slight decline,expressed in bud differentiation initiation periods reaches peak, in the involucre scales middledifferentiation are down a bit, at bud differentiation completion stages have rebounded.Expression of CO dark treatment after a day’s slight decline, expressed in involucrescales differentiation medium quantity reaches peak, is nearly two times more thepre-treatment level; CHS on dark expression quantity rise, a day after start-up of the highest innearly five times more, involucre scales differentiation medium expression quantity slightlydecreased, then in the middle of the floret primordium differentiation decline.3. Preliminary concluded that chrysanthemum flower bud differentiation periodsignificant enrichment of differentially expressed genes of the main biochemical metabolic pathways and signal transduction pathways, concentrated in photosynthesis and ribosomepathways.4. Preliminary concluded that different chrysanthemum flower bud differentiation stageenrichment of differentially expressed genes involved in biological function：At the bud un-differentiated：single stranded RNA binding，poly-pyrimidine tractbinding，protein-arginine N-methyltransferase，oxidoreductase activity，acting on paireddonors，with incorporation or reduction of molecular oxygen. At the bud differentiationinitiation：protein-arginine N-methyl transferase activity，fatty acid synthase activity，sugar-hydrogen symporter activity. At the involucre scale differentiation mid-term stages：protein transmembrane transporter activity，pyrimidine nucleotide sugar transmembranetransporter activity. At the floret primordia differentiated mid-term stage：oxidoreductaseactivity，oxidizing metal ions，NAD-NADP as acceptor，protein-arginine N-methyltransferaseactivity. At the corolla differentiation mid-term and bud differentiation completion stages：structural molecule activity，guanyl ribonucleotide binding，arsenate reductase activity.5. The cDNA sequence of GIGANTEA was cloned from chrysanthemum‘Jniba’，The sequence was submitted to GenBank, and the registration number is JQ043439. Sequenceinformation is done in the analysis. Fluorescent relative quantitative analysis shows that theexpression patterns of chrysanthemum CmGI are circadian rhythms expression, with its peakexpression occurring at16:00. At different flower bud differentiation stage, the CmGI gene inthe leaf blade mRNA level is different, two peak values were appeared in the flower buddifferentiation start-up and floret primordia middle differentiation periods. CmGI mRNA ishighly expressed in the leaves at the full-blossom stage.6. We cloned the flowering gene CmCOL (GenBank accession:KC589293) fromChrysanthemum morifolium‘Jinba’. Sequence information is done in the analysis. Relativefluorescence quantitative analysis showed that CmCOL mRNA is most highly expressed inthe leaves and demonstrates a circadian expression pattern, with its peak expression occurringat04:00. A short-day treatment elevated CmCOL mRNA expression in the leaves but did notsignificantly affect expression in the buds. The CmCOL mRNA level in the leaves changedsignificantly at various flower bud differentiation stages, and it peaked at the floret primordiadifferentiation mid-term stage. CmCOL mRNA is highly expressed in the leaves at thefull-blossom stage.7. Relative fluorescence quantitative analysis showed that CmFTL mRNA is most highly expressed in the leaves and demonstrates a circadian expression pattern, with its peakexpression occurring at00:00. A short-day treatment elevated CmFTL mRNA expression inthe leaves but did not significantly affect expression in the buds. The CmFTL mRNA level inthe leaves changed significantly at various flower bud differentiation stages, and it peaked atthe floret primordia differentiation mid-term stage. CmFTL mRNA is highly expressed in theleaves at the bud stage.8. The present study showed that Chrysanthemum morifolium leaves could sense thephotoperiod. Under SD conditions, CmGI mRNA expression began to increase one day afterthe SD treatment and reached the first peak at the bud differentiation initiation stage. CmCOLmRNA expression lagged behind that of CmGI; it increaseds lightly at the bud differentiationinitiation stage, suggesting that CmGI operates upstream of CmCOL. CmCOL expressionreached its peak at the floret primordia differentiated mid-term stage, which occurred afterCmGI expression peaked. The expression of CmFTL and CmCOL did not change significantlyunder micro-regulation. CmFTL mRNA expression peaked at the floret primordiadifferentiated mid-term stage, which occurred after CmCOL mRNA expression peaked,suggesting that CmFTL operates downstream of CmCOL. These results suggest that theGI-CO-FT regulation pattern is similar in Chrysanthemum morifolium under SD conditions,arabidopsis thaliana under LD conditions and Oryza sativa under SD conditions.更多还原