【作者】 国静；

【导师】 张宪省；

【作者基本信息】 山东农业大学 ， 发育生物学， 2009， 博士

【摘要】 细胞分裂是生物体生长发育的基础。近年来,细胞周期的调控机制逐渐为人们所关注。在高等植物中,细胞周期关键调节因子在双子叶模式植物拟南芥中已经被系统分析,但在单子叶模式植物水稻(Oryza sativa)中研究则较少。水稻是我国重要的粮食作物,研究水稻细胞周期的调控机制对于有目的的改善水稻的农艺性状(如加速生长、增加株高、改善株型、提高千粒重等)进而提高水稻产量有着重要的意义。水稻基因组测序的完成及大量信息学数据的出现为查找水稻细胞周期关键调节因子提供了方便。为了获得水稻所有的细胞周期关键调节因子,本研究根据拟南芥基因组提供的信息,在水稻公共数据库中进行序列检索,将检索到的序列进行了信息挖掘,进而对水稻细胞周期关键调节基因的表达模式和功能进行了初步研究。本研究通过序列比对获得了90个水稻细胞周期关键调节因子的基因序列。这些调节因子包括44个细胞周期蛋白(Cyclin)、25个细胞周期蛋白依赖性激酶(CDK)、7个细胞周期蛋白依赖性激酶抑制剂(KRP)、9个E2F转录因子,另外还有2个成视网膜瘤蛋白(Rb),2个WEE激酶和1个细胞周期蛋白依赖性激酶亚单位(CKS)。对这些调节因子的蛋白序列进行了聚类分析并系统命名,其中有41个调控因子的名称是新命名的或对其原有的名称进行了更正。聚类分析以及蛋白质结构分析发现,水稻和拟南芥的细胞周期关键调节因子之间不仅在数目上是保守的,在蛋白序列和结构特征上同样比较保守。为了研究水稻细胞周期关键调节基因在水稻发育过程中的功能,首先利用半定量RT-PCR的方法初步研究了上述基因在水稻不同组织中的时空表达模式。根据表达模式,水稻细胞周期关键调节基因大致可分为五大类。这些基因中,同一基因家族成员之间具有相似的表达模式,而不同基因家族之间表达模式差异较大。暗示不同家族的细胞周期关键调节基因在水稻发育过程中可能起着不同的作用,而同一家族的基因成员之间可能存在功能冗余。为精确地了解水稻细胞周期关键调节基因所表达的细胞和组织,随机选取Orysa;CycA2;1、Orysa;CycB2;2、Orysa;CycU4;4与Orysa;CDKG;1四个基因进行了原位杂交分析。原位杂交结果显示水稻细胞周期关键调节基因具有时空表达特异性,特异地在细胞分裂旺盛的组织如根端分生组织、苗端分生组织以及发育的胚中表达。另外,原位杂交结果与半定量RT-PCR结果一致,证明通过半定量RT-PCR来研究基因表达模式的方法是比较可靠的。为了研究生长素和细胞分裂素对水稻细胞周期关键基因的调节作用,利用半定量RT-PCR的方法研究了这两种激素对上述基因表达的调控模式。结果显示,生长素调控表达的基因有29个,其中有25个基因的表达水平上调,4个基因的表达水平下调;细胞分裂素调控表达的基因有37个,其中,12个基因表达上调,25个基因表达下调;在这些表达变化的基因中,18个基因的表达同时受到生长素和细胞分裂素的调节。这表明生长素和细胞分裂素能够分别或着同时调节不同水稻细胞周期关键调节基因的表达。在种子中高水平表达的基因可能对种子的发育起着重要的作用。根据水稻90个细胞周期关键基因的表达模式,选取在种子中高水平表达的7个基因——Orysa;CycA2;1、Orysa;CycB1;1、Orysa;CycB2;2、Orysa;CDKE、Orysa;DEL1、Orysa;DP1以及Orysa;E2F4,通过遗传转化分析其在水稻种子发育过程中的功能。观察转基因水稻的表型发现Orysa;CycB1;1相关转基因水稻植株的生殖发育异常,因此,对Orysa;CycB1;1的功能进行了进一步的研究。对转基因植株进行表型分析发现,Pubi::CycB1;1RNAi转基因水稻生殖发育异常,表现为部分花粉和子房败育、结实率降低、种子发育异常、胚乳的发育受到抑制而胚的生长得到促进。组织学分析显示,授粉后3天(3 DAP)的转基因水稻种子胚乳发育出现异常,胚与对照相比没有显著差异,9 DAP的转基因种子中胚乳几乎消失,而胚与对照相比体积则增大三至四倍。在胚乳中特异敲除Orysa;CycB1;1发现,PGluA-1::CycB1;1RNAi转基因水稻能够产生与上述表型类似的种子,表明Orysa;CycB1;1直接调控水稻胚乳的发育,同时影响胚的发育。统计结果显示,Pubi::CycB1;1RNAi转基因水稻种子胚体积增大主要是由于细胞体积增加所致,与细胞数目无关。半定量RT-PCR、荧光实时定量PCR、原位杂交以及PCycB1;1::GUS转基因水稻种子的组织学染色结果显示,Orysa;CycB1;1在分裂旺盛的组织和细胞中表达,尤其在发育早期种子的胚和胚乳中均表达,暗示该基因对水稻种子胚和胚乳的发育起调控作用。对PGluA-1::CycB1;1RNAi转基因水稻胚乳的荧光实时定量PCR分析结果显示,种子发育相关基因FIE和GW2的表达量上调。芯片分析结果表明,Pubi::CycB1;1RNAi转基因水稻胚中,一些与发育进程、形态建成及细胞壁组织结构等相关的基因表达水平变化显著。暗示这些基因的表达可能受Orysa;CycB1;1调节。综上所述,得出以下结论:水稻与拟南芥的细胞周期关键调节基因在数目及结构上相当保守;不同家族的水稻细胞周期关键调节基因在水稻发育过程中起着不同的作用,而同一家族成员之间可能存在功能冗余现象;生长素和细胞分裂素能够分别或着同时调节不同水稻细胞周期关键调节基因的表达;Orysa;CycB1;1的正常表达为水稻生殖发育所必需,该基因直接调控水稻胚乳的发育,并影响胚的发育;Orysa;CycB1;1可能通过调节FIE和GW2基因调控胚乳的发育;Orysa;CycB1;1可能通过调节与发育进程、形态建成及细胞壁组织结构等相关的基因调控胚的发育。更多还原

【Abstract】 Cell division plays very important roles in many aspects of plant growth and development. Although extensive and systematic studies of core cell cycle genes such as cyclin-dependent kinases (CDK), cyclins, CDK inhibitors (CKIs), homologs of the retinoblastoma proteins (Rb) and the E2F transcription factors (E2F) have been reported in Arabidopsis, only a small number of core cell cycle genes were functionally examined in monocots. Rice, as a model plant species of monocots, is suitable for the identification of core cell cycle genes because of the availability of its whole genome sequences. In this study, we searched for the predicted core cell cycle genes from rice genome, and examined their expression patterns in various rice tissues and the regulation of their expression by cytokinin and auxin, and then determined the role of some candidate genes during rice development.In order to obtain the putative core cell cycle genes in rice, blast searches were conducted against the rice database using the Arabidopsis core cell cycle genes published as query sequences. Ninety putative core cell cycle proteins were identified and they belong to cyclin, CDK, E2F, CKI, CKS, Rb and Wee families, respectively. Phylogenetic and structural analysis indicated that both the number and the structure of the core cell cycle regulators are relatively conserved between the rice and Arabidopsis genomes.To understand the fuctions of rice core cell cycle genes, their expression patterns in rice were investigated by semi-quantitative RT-PCR. The tissues include roots, young leaves, spikelets, seeds (0, 1, 3, 6 days after pollination or DAP), endosperms (9, 12 and 15 DAP), and embryos (9 and 15 DAP). The results indicated that the expression of most of the core cell cycle genes are spatially regulated, and they can be divided into five classes based on their expression patterns. The expression patterns of some closely related core cell cycle genes are similar, suggesting functional redundancy of these core cell cycle genes.In situ hybridization were conducted to study the transcript localization of the core cell cycle genes in rice. Four genes including Orysa;CycA2;1, Orysa;CycB2;2, Orysa;CycU4;4 and Orysa;CDKG;1 were randomly selected and the results suggested that they are temporally and spatially regulated. Also, this data supports the expression profiles underlied by the RT-PCR analysis. Following auxin or cytokinin treatment, the expression of some core cell cycle genes was either upregulated or downregulated, indicating that auxin and/or cytokinin regulate the expression of these core cell cycle genes.Seven core cell cycle genes such as Orysa;CycA2;1, Orysa;CycB1;1, Orysa;CycB2;2, Orysa;CDKE, Orysa;DEL1, Orysa;DP1 and Orysa;E2F4 were chosen to study their functions in rice development by genetic transformation. The results showed that Pubi::CycB1;1RNAi transgenic rice plants displayed aberrant phenotypes, such as lower seed-setting rate and abnormal seed morphology. Histological analysis indicated that abortive endosperm while larger embryo with disturbed morphology and structures to some extent setted in the abnormal seeds compared with the control one. Further analysis showed that endosperm-specific knockdown of Orysa;CycB1;1 produced similar abnormal seeds, indicating that Orysa;CycB1;1 regulated the development of endosperm directly. Statistical analysis showed that the enlarged embryo size in Pubi::CycB1;1RNAi plants was due to the increased cell size.The expression patterns of Orysa;CycB1;1 was confirmed using semi-quantitative RT-PCR, qRT-PCR, in situ hybridization and histological staining of PCycB1;1::GUS transgenic rice seeds. The results indicated that Orysa;CycB1;1 was highly expressed in tissues or cells which divide rapidly, and its transcripts were identified in endosperm and embryo.Expression analysis of the seed development-related genes in PGluA-1::CycB1;1RNAi transgenic rice endosperm indicated that the transcript levels of FIE and GW2 were stronger than that of control. Microarray analysis revealed that many genes involved developmental processes, morphogenesis and cell wall organization were significantly upregulated or downregulated in transgenic embryos. It was suggested that these gene expression might be regulated by Orysa;CycB1;1 during seed deveopment.In conclusion, we have conducted genome-wide identification and expression analysis of rice core cell cycle genes, and provided the evidences that Orysa;CycB1;1 plays the important roles in endosperm and embryo development in rice. Our current results might provide basic information to understand the mechanism of cell cycle regulation and the functions of the core cell cycle genes in rice.更多还原