【作者】 刘伟；

【导师】 张可伟； 程祝宽；

【作者基本信息】 浙江师范大学 ， 生物学， 2019， 硕士

【摘要】 减数分裂(Meiosis)是真核生物有性生殖过程中一种特殊的细胞分裂方式。减数分裂在维持物种间染色体数目稳定和促进物种遗传多样性等方面具有重要意义。在减数分裂前期I,同源染色体进行了配对、联会和重组等一系列重要的生物学事件,从而保证在后期I的准确分离。已有报道在芽殖酵母和哺乳动物中,性母细胞的同源重组和联会如果不能正常完成,负责监测减数分裂同源重组与细胞周期进程之间协同运行的监督机制将被激活,使减数细胞进程停滞在粗线期或进入细胞程序性死亡。这种监督机制被称为粗线期检验点(Pachytene checkpoint)。然而,目前在植物中有关粗线期检验点的运行机制尚不明确。本研究中,我们通过图位克隆的方法在水稻中克隆到一个调控减数分裂进程的基因MEL1。对mel1突变体扬花期花粉进行碘化钾染色,发现突变体为无花粉型。用野生型花粉对突变体授粉,仍不可结实,说明mel1突变体雌雄配子的育性都受到了影响。对野生型不同组织MEL1的表达量进行检测,发现其只在穗部表达。花药RNA原位杂交分析,表明MEL1在生殖细胞中特异表达。对mel1突变体花粉母细胞减数分裂时期的染色体进行花药树脂切片和DAPI染色观察,发现减数分裂进程停滞在粗线期,并且染色体异常凝集。根据细胞学观察结果,我们猜测在mel1突变体中可能发生了染色体降解和细胞凋亡。为了验证推测,我们在野生型和mel1突变体花粉母细胞中进行了TUNEL实验。结果显示,突变体停滞之后的花粉母细胞中检测出了强烈的细胞凋亡信号。进一步的q RT-PCR检测分析发现,细胞周期蛋白Os MCM2和CDC6表达量显著降低,而细胞周期依赖性激酶CDKB1和CDKB2表达量显著增加。为了研究MEL1影响减数分裂的分子机制,我们通过FISH和免疫荧光实验对突变体中减数分裂过程的重要事件进行了检测,发现其染色体不能正常配对和联会。以上结果表明了mel1突变体减数分裂进程停滞在了粗线期并由此引发了花粉母细胞的凋亡,最终导致了无法形成花粉。此外,MEL1编码一个AGO蛋白,已有报道表明AGO蛋白在染色体的表观修饰过程中发挥着重要作用。通过q RT-PCR检测发现,在mel1突变体中,参与组蛋白甲基化和DNA甲基化修饰的相关基因SDG704、SDG713和CMT3a、DRM2发生了显著变化。目前,在水稻中并未发现有减数分裂前期异常导致粗线期停滞的突变体。本研究中,发现mel1突变体停滞在减数分裂粗线期。我们推测这种停滞可能不同于哺乳动物中粗线期检验点的监督机制。鉴于目前对植物中细胞周期调控机制的研究较少,还需要进一步研究MEL1突变导致停滞的分子机制。但是,对植物减数分裂进程停滞相关基因的研究,将有助于理解植物减数分裂进程调控机制并对完善植物减数分裂调控网络有着非常重要的意义。更多还原

【Abstract】 Meiosis is a special type of cell division in the process of sexual reproduction in eukaryotes.The first meiotic division（meiosis I）is a process in which homologous chromosomes pair,recombine,synapse and segregate to enhance the close attachment of homologous chromosomes in prophase I.In budding yeast and mammals,there is a monitoring mechanism responsible for monitoring the synergy between meiotic homologous recombination and cell cycle progression,called the pachytene checkpoint.When homologous recombination and synapsis can not be completed properly,the pachytene checkpoint was activated,which makes the process of subtractive cells stagnate in pachytene or programmed cell death.However,the mechanism of the pachytene checkpoint in plants is still unclear.In this study,we report the cloning and characterization of MEIOSIS ARRESTED AT LEPTOTENE 1（MEL1）,which is specifically involved in meiosis in rice（Oryza sativa）anther development.I₂-KI staining showed that the mutant without pollen.Pollinating the mutant flowers with wild type pollen did not set any seeds,suggesting that the mutant is both male and female sterile.Real-time RT-PCR analysis revealed that the transcription of MEL1 was preferentially in young panicles and RNA in situ analysis indicated that the MEL1 was specifically expressed in germ cells.The meiotic chromosomes and transverse sections of rice anthers stained with DAPI were used for observation.These results indicate the meiosis process was arrested at pachytene.According to the cytological observation,we speculated that chromosome degradation and cell apoptosis might take place in mel1 mutants.To test this possibility,we performed TUNEL assay in wild type and mel1 PMCs.A strong TUNEL signals were detected in the arrested PMCs.To investigate the molecular mechanism by which MEL1 affects meiosis,we examined important events during meiosis in mutants by FISH and immunofluorescence.These results show that homologous chromosomes pairing and synapsis were incompleted.MEL1 encodes an AGO protein,which has been reported to play an important role in the epigenetic modification of chromosomes.Therefore,we analyzed the genes involved in histone methylation and DNA methylation by q RT-PCR in the mel1.The results showed that SDG704（histone lysine methyltransferase）and DRM2（DNA methyltransferase）have a significant increase in mel1 mutant.The mutants of meiotic arrested at pachytene were not be reported in rice.In this study,we found that the meiotic process was arrssted at pachytene in mel1 mutants.We believe that it different from the molecular mechanism of the pachytene checkpoint in mammals.Due to the mechanism of pachytene checkpoint was unclear in plants.However,studies on genes involved in plant meiosis processes will help to understand the regulatory mechanisms of meiosis processes in plant,and it is very important to improve the plant meiosis progression regulation network.更多还原