【作者】 黄鑫；

【导师】 郑国生； 戴思兰；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2008， 博士

【摘要】 牡丹是中国传统十大名花之一,正如许多多年生木本植物一样,每年冬季其花芽都将进入休眠状态,而且必须经过一定时期的低温处理才能彻底解除休眠,保证来年正常的开花展叶。牡丹花芽的休眠是典型的内休眠,迄今为止,对于内休眠调控的分子机理还知之甚少。以牡丹为材料,分离芽休眠调控的相关基因,研究其分子功能,对于揭示芽内休眠机理,人为调控花期,保证反季节催花的成功,具有重要的理论意义和实践价值。本研究以牡丹品种‘鲁荷红’为试材,通过观察自然低温条件下花芽的形态解剖变化,确定了花芽休眠解除的进程,认为冬季自然低温条件下(日平均气温为0-10℃)处理30-35d可彻底解除休眠。利用抑制性差减杂交(SSH)技术,以休眠解除芽的RNA为“tester”,休眠芽的RNA为“driver”,构建花芽休眠解除相关基因的差减cDNA文库,共筛选到31个与内休眠解除相关的差异基因,根据blast分析结果进行其分子功能预测,可将这31个基因分为9类:(1)结构功能:PsRPS7A,PsRPSL21,PsRPS,PsRPSL10,PsRPS7B,PsEDR,PsRPSL37,PsRPSL19,PsRPSL1,PsRPSL15,PsTUB;(2)激酶活性:PsSERK1;(3)蛋白质结合活性:PsXRCC1和PsPOB;(4)其他物质结合活性:PsMPT;(5)转运子活性:PsAEP;(6)其他酶活性:PsGA20和PsSAMT;(7)其他分子功能:PsPII;(8)未知功能基因:PsBDR1,PsBDR2,PsARP,PsFB2,PsCXE,PsDHN;(9)无blast匹配序列:PsBDR3-8。根据候选基因的功能推测和前人关于植物休眠调控的研究结果,选取了8个可能与内休眠解除相关的基因(PsPII,PsDHN,PsGA20,PsARP,PsMPT,PsCXE,PsSERK1和PsPOB),采用northern blot和RT-PCR技术分析了它们在花芽内休眠解除不同时期的表达模式变化,认为牡丹花芽内休眠解除的过程伴随着复杂物质代谢和能量消耗,差异基因主要参与植物生长发育及抗性胁迫等方面的调控,而且低温诱导的核糖体蛋白的表达可能也与花芽内休眠解除有关。为了深入研究生长素抑制蛋白基因(PsARP)在花芽内休眠解除过程中的分子作用,通过RACE-PCR技术扩增了其cDNA全长,对其编码的蛋白质氨基酸序列进行生物信息学分析,认为牡丹中该基因属于ARP/DRM基因家族,含有两个保守结构域,无信号肽及跨膜结构域。PsARP基因的亚细胞定位研究进一步证明该基因编码的蛋白为一细胞质蛋白。不同低温处理条件下,PsARP的表达模式变化的研究表明,该基因在低温诱导牡丹花芽内休眠解除过程中的表达量逐渐升高,高表达的PsARP mRNA可在正常生长条件下被恢复,表明该基因在花芽内休眠解除过程中可能起促进作用,但对正常生长条件下花芽发育的作用不大,推测PsARP的表达变化与花芽内束缚生长素和自由生长素之间的转变有关,自由生长素通过与生长素抑制蛋白(PsARP)基因相互作用,从而在转录后水平上调控花芽内休眠的进程,间接说明了生长素在调控花芽内休眠解除过程中也起一定的作用。PsARP在拟南芥中的超表达研究表明,转基因植株与野生型植株的表型差异不大,进一步说明该基因在植物生长发育过程中的作用不大。线粒体磷酸转移子基因(PsMPT)是从差减cDNA文库筛选到的另一在低温诱导花芽内休眠解除过程中表达量明显升高的克隆。通过RACE-PCR技术扩增PsMPT的全长cDNA序列,对其编码的蛋白质氨基酸序列进行生物信息学分析,发现PsMPT与其他物种中分离到的线粒体磷酸转移子(MPTs)的氨基酸序列相似性很高,均具有六个保守的跨膜结构域,而且其活性中心含有一个保守的半胱氨酸(Cys)残基。采用northern blot技术进一步分析PsMPT在不同低温处理条件下的表达模式变化,发现PsMPT的表达模式变化与花芽内休眠解除的进程及花芽内ATP含量的变化相吻合,推测PsMPT为一冷诱导表达基因,在花芽内休眠解除过程中,高表达的PsMPT促进ATP的合成,提高植物体内ATP/ADP的比值,从而使花芽内休眠解除所需的相关蛋白大量合成,彻底解除花芽内休眠。迄今为止,本研究首次证明了线粒体磷酸转移子参与花芽内休眠解除的过程,初步揭示了休眠解除过程伴随着ATP含量显著增加的分子机理。另外,PsMPT在拟南芥中的超表达研究表明,转基因拟南芥不同发育阶段的植株鲜重、叶片长度及叶片中ATP含量均比野生型植株的相应指标高,而且开花期提前4-5天,进一步说明了该基因的超表达增加了ATP的合成,促进了植株的生长发育。更多还原

【Abstract】 Tree peony (Paeonia suffruticosa Andr.) is one of ten famous traditional landscape flowers in China. Like many perennial woody plants, the floral buds will enter the“dormant”state every winter and must go through a period of chilling treatment to break dormancy before flowering well and developing normal leaves next year. The dormancy of tree peony floral buds is endo-dormancy. Until now, the molecular mechanism of endo-dormancy is not clear. It is helpful to explain the mechanism of endo-dormancy and further control the flowering time by isolating different expression genes from tree peony floral buds and studying their molecular functions. These kinds of studies have important academic significances and practical values.In our study, tree peony (Paeonia suffruticosa)‘Lu He Hong’was used as plant materials. By observing the floral buds anatomical changes under natural chilling treatment in winter to determine the progress of dormancy releasing, we regarded that about 30-35 d under the average temperature of about 0-10℃is required to break floral buds dormancy completely. A subtractive cDNA library was developed to study genes associated with the release of dormant floral buds in tree peony by using the RNA from burst floral buds as“tester”and that from dormant floral buds as“driver”. By differential screening, 31 genes which were associated with bud dormancy release were obtained. According to their blast results, the molecular functions of them were presumed as following: (1) structural molecular activity group which included PsRPS7A, PsRPSL21, PsRPS, PsRPSL10, PsRPS7B, PsEDR, PsRPSL37, PsRPSL19, PsRPSL1, PsRPSL15, PsTUB. (2) PsSERK1 was in kinase activity group. (3) PsXRCC1 and PsPOB in protein binding group. (4) PsMPT in other binding group. (5) PsAEP in transporter activity group. (6) PsGA20 and PsSAMT in other enzyme activity group. (7) PsPII in other molecular functions group. (8) PsBDR1, PsBDR2, PsARP, PsFB2, PsCXE, PsDHN in unknown molecular functions group. (9) The other six unmatched sequences (PsBDR 3-8) were classified into‘no matches’group.According to their putative function and literatures previously reporting an association with dormancy changes, eight cDNA clones, PsPII, PsDHN, PsGA20, PsARP, PsMPT, PsCXE, PsSERK1 and PsPOB were chosen from different molecular functional groups for further analysis by northern blot and semi-quantitative RT-PCR. The expression profiles of them during floral bud dormancy release indicated that bud breaking is a high energy and nutrition-consuming process. The transcriptional expressions of the isolated genes are related to growth regulation and stress response. Moreover, low-temperature-induced ribosomal proteins may be involved in floral bud breaking.In order to determine the molecular roles of auxin-repressed protein (PsARP) in bud dormancy release of tree peony, the full-length cDNA sequence was amplified by RACE-PCR and the deduced amino acid sequence were analyzed with the ExPASy Molecular Biology Sever (http://expasy.org/tools/). The results suggested that PsARP belongs to ARP/DRM gene family. And the putative protein contains two conserve domains without signal peptide and transmembrane domains. The subcellular localization of PsARP was further proved that putative PsARP is cytosolic protein. The expression pattern of PsARP under different chilling treatments showed up-regulated PsARP mRNA would accelerate bud dormancy release but not control bud development under normal conditions. So we presumed that the expression level of PsARP maybe relate to the interconversion between conjugated IAA and free IAA in floral buds. The free IAA probably affects floral bud dormancy process on post-transcriptal level by reacting with auxin-repressed protein (PsARP), implying indirectly that IAA also play a certain role in controlling floral bud dormancy release in tree peony. The characteristics of PsMPT-overexpression Arabidopsis plants by comparing to wild-type ones further indicated that PsARP maybe not control the normal growth.A putative mitochondrial phosphate transporter (PsMPT) gene is another clone from the subtractive cDNA library, which showed higher expression by chilling treatment during floral bud dormancy. By RACE-PCR, the full-length cDNA of PsMPT was obtained and the putative protein was also analyzed. The deduced amino acid sequence of PsMPT showed high identities with mitochondrial phosphate transporters in various organisms. The PsMPT protein also has the common six transmembrane domains while the conserved Cys residue exists in catalytic center. Northern bolt results and the characteristics of PsMPT-overexpression Arabidopsis plants indicated that the changes of the expression profile of PsMPT were correlated well with the process of floral bud dormancy release and the changes of the ATP content in floral buds under different chilling treatments, suggesting that PsMPT is a cold-responsive gene in tree peony and higher expressed PsMPT would promote the ATP synthesis and then directly increase the ATP/ADP ratio which regulates significant proteins synthesis to break floral bud dormancy release completely. To our knowledge, it is the first time to show the involvement of MPT in bud dormancy release and elucidate primarily the molecular mechanism of ATP content increasing in dormancy release.更多还原