【作者】 刘扬；

【导师】 赵洁；

【作者基本信息】 武汉大学 ， 发育生物学， 2012， 博士

【摘要】 复制因子C是一类在许多物种中相对保守的蛋白质复合体,它由5个蛋白质亚基组合而成,(?)PRFC1, RFC2, RFC3, RFC4和RFC5。在单细胞生物酵母的研究中发现,其它们参与了DNA复制、修复和细胞周期检验点的控制,但在植物中RFC的功能尚待进一步揭示,尤其是在植物生殖发育中其功能尚未报道。本研究以拟南芥为材料,利用分子生物学和细胞生物学技术,研究并揭示了AtRFC1参与拟南芥减数分裂过程中DNA修复的分子机理,并对RFC2, RFC3, RFC4和RFC5参与拟南芥胚胎发育的功能进行了初步的研究,获得的主要结果如下：1、对拟南芥5个RFC进行了生物信息学分析。首先,多重序列比对发现它们含有8个保守的结构域,这与其它物种的RFC是一致的。然而,AtRFC1蛋白的N端含有一个特有的结构域,即BRCT domain。序列比对和同源模建的结果都显示它与其它物种中的BRCT domain十分相似。从拟南芥突变体库中(http://www.arabidopsis.org/)获得了AtRFC1的T-DNA插入突变体,并命名为rfc1-2。经过种植鉴定后,发现rfc1-2纯合突变体植株的育性明显降低。进一步观察发现,rfc1-2雌、雄配子体的发育均呈现出了显著的缺陷。其中,rfc1-2突变体的单核小孢子难以极性化,导致后续的有丝分裂发生紊乱,最终大量花粉败育。作者还发现rfc1-2的胚囊大部分停滞在功能大孢子时期,难以继续分化成为具有功能的成熟胚囊。同时,对含有性细胞的花粉和体细胞花瓣的细胞核进行DAPI染色,发现rfc1-2花粉的有丝分裂是异常的,但其体细胞有丝分裂的形态是正常的。这些结果表明,rfc1-2突变体的雄性和雌性器官均发育异常。2、对野生型和突变体花粉母细胞的染色体进行DAPI染色,观察减数分裂时期的染色体形态特征,结果显示rfc1-2突变体的减数分裂异常紊乱,具体表现为：减数分裂前期Ⅰ中的细线期、偶线期和粗线期表现正常；双线期发生了染色体的双链断裂,这种断裂导致后续的染色质浓缩以及同源染色体和姐妹染色单体的分离发生异常。虽然,减数分裂的胞质分裂未受影响,仍然能形成四分体,但是每个小孢子细胞核的遗传物质是异常的。上述结果表明,rfc1-2突变体减数分裂时前期Ⅰ染色体表型异常,这可能是导致花粉败育的主要原因。3、通过rnRNA原位杂交技术,(?)(?)tRFC1在各个器官中的转录本水平进行了检测,结果发现,AtRFCl在花序分生组织和雌、雄器官中有着丰富的表达,尤其在花粉母细胞和大孢子母细胞中也检测到很高的信号分布。此外,利用免疫荧光技术检测到AtRFC1蛋白在减数分裂的染色体上也有明显的信号分布。这些结果表明,AtRFC1在有性生殖器官中具有丰富的表达水平,尤其是在花粉母细胞中的表达与其参与减数分裂的功能是一致的。4、通过rfc1-2分别与atspoll-1和atrad51突变体杂交,并对其双突变纯合体的后代进行表型分析,发现atspoll-1rfcl-2和atrad51rfcl-2双突变的表型均分别与atspoll-1和atrad51的单突变表型一致：atspoll-1rfcl-2和atspoll-1的减数分裂中期都表现为10个单价体；atradSl rfc1-2和atrad51在减数分裂后期和末期均表现出大量的染色体断裂。这表明AtRFCl是宅(?)AtSPOll-1的下游与AtRADS1一起发挥同源重组的功能。5、通过(?)(?)AtRFC2, AtRFC3, AtRFC4和AtRFC5基因突变体的鉴定后分析发现,这4个突变体均呈现出了胚胎纯合致死的表型。胚珠透明结果显示,败育的胚胎停滞在胚体为2-细胞时期。同时,突变体中胚乳细胞的分裂也受到了明显的抑制,其胚乳核体积增大,数目减少。通过转基因技术,重点对AtRFC4基因表达模式及其与其它亚基的相互作用进行了初步分析,GUS结果显示,AtRFC4基因在细胞分裂旺盛的组织中表达水平较高,如幼叶、保卫细胞、根尖和侧根原基。同时,mRNA原位杂交结果显示,早期胚胎中也存在EAtRFC4的转录。酵母双杂交结果表明,AtRFC4可以分别与(?)VtRFC1, AtRFC2, AtRFC3和AtRFC5相互作用。上述结果暗示,AtRFC4基因对拟南芥的生长发育是必需的。更多还原

【Abstract】 Replication factor C is a protein complex relatively conservative in many species, and it has a combination of five protein subunits, RFC1, RFC2, RFC3, RFC4, and RFC5. Studies in single-celled organism yeast demonstrate it involved in DNA replication, repair and cell cycle checkpoint control, but RFC function in plants has not to be revealed, in particular, its function in plant reproductive development. In this study, by using a series of molecular and cell biology techniques on Arabidopsis research material, we initially clarified the molecular mechanisms of DNA repair of the AtRFCl involved during meiosis. In addition, the roles of AtRFC2, AtRFC3, AtRFC4, and AtRFC5genes in Arabidopsis embryo development were investigated preliminarily. Our main results are as follows:1. We conducted a bioinformatics analysis for Arabidopsis five RFCs. Multiple sequence alignment shows that they contain eight conserved domains, which is consistent with other species of RFCs. However, the N-terminal of the AtRFC1protein contains a unique structural domain, the BRCT domain. Sequence alignment and homology-model reveal that it is very similar with other species BRCT domain. Therefore, we got AtRFCl T-DNA insertion mutant from the Arabidopsis mutant library (http://www.arabidopsis.org/) and named it rfc1-2. After mutant identification, we found that the fertility of the rfcl-2homozygous plants was significantly reduced. Further observation, we found that male and female gametophyte development has a significant defect. Among them, the mutant microspores are difficult to polarize, resulting in the subsequent mitosis disorder, and ultimately pollen sterility. As for the female gametophyte, we found that most of rfcl-2embryo sac arrested at the functional-megaspore stage, it was difficult to differentiate into functional mature embryo sac. DAPI staining of the pollen nucleus and the nucleus of somatic cell petals suggested that pollen mitosis was abnormal, whereas somatic cell mitotic morphology was normal. These results demonstrate both male and female gametophyte development were impaired.2. Meiosis chromosome morphological characteristics were observed by DAPI staining of chromosomes of pollen mother cells, the results showed that rfcl-2mutant meiosis was disrupted. Although chromosome is normal in leptotene, the early zygotene and pachytene, diplotene chromosome appeared some double-strand breaks, the rupture led to the subsequent defects of chromosome condensation and homologous chromosome separation. Although meiotic cytokinesis was not affected, still give rise to tetrads, but the genetic material of the nucleus of each microspore is impaired. The above results demonstrated that male meiosis during meiosis prophase I was affected and this defects should be the main reason or rfc1-2sterility.3. By using mRNA in situ hybridization technique, we detected AtRFC1transcription in various organs; the results showed that AtRFCl displayed abundant expression in the inflorescence meristem, and the male and female organs, especially in the microspore and megaspore mother cells. In addition, immunofluorescence technique was adopted to detect the AtRFCl signal distribution in the meiotic chromosomes. The results demonstrated that AtRFC1expressed mainly on reproductive tissues. Additionally, the high expression level in pollen mother cell was consistent with its function during meiosis.4. By hybridization, we obtained the atspoll-1rfc1-2and atrad51rfc1-2double mutants. Homozygous offspring analysis showed that the double mutant phenotypes were consistent with the single atspoll-1and atrad51mutants:both atspoll-1rfc1-2and atspoll-1displays ten univalents during metaphase I; many DNA fragmentations appeared in atrad51rfcl-2and atrad51during anaphase and telephase. The results indicated that AtRFCl should be function downstream of AtSPOll-1and work with AtRAD51during homologous recombination.5. In addition, we got the mutants of AtRFC2, AtRFC3, AtRFC4, and AtRFC5genes, the mutant identification and analysis indicated that these four mutants were the embryonic homozygous lethal phenotypes. Examination of ovules showed that mutant embryos arrested in the two-celled stage of embryo proper. Additionally, mutant endosperm cell division was severely suppressed, the size of endosperm nucleus increased, and their number decreased. By transgenic technique, we mainly analysed the AtRFC4expression pattern and its interaction with other subunits. The result showed that AtRFC4expressed highly in tissues undergoing active cell division, such as seedling, guard cell, root tip and lateral root tip. Meanwhile, in situ hybridization showed that the early embryo has its transcript. Yeast two hybrid showed that AtRFC4could interect with the other four subunits of RFC complex. The above results indicated that AtRFC4is necessary for Arabidopsis growth and development.更多还原