【作者】 王蓉；

【导师】 郭文武； 陈春丽；

【作者基本信息】 华中农业大学 ， 果树学， 2016， 硕士

【摘要】 核仁显性是一种普遍存在于植物和动物杂种中的表观遗传现象,与rRNA基因表达有关。在柑橘体细胞杂种中发现的核仁共显性现象为研究多倍体基因组遗传规律、基因表达调控提供了新思路。本实验通过荧光原位杂交(FISH)、蛋白免疫荧光等技术,分析柑橘体细胞杂种及其亲本的45S rDNA、DNA甲基化和H3K9ac在细胞核和染色体上的分布,并对DNA甲基化和H3K9ac与rRNA基因表达之间的关系进行了探讨,从分子细胞学角度研究核仁共显性产生原因。1.45S rDNA在亲本和体细胞杂种中的分布通过荧光原位杂交,观察发现哈姆林甜橙(Citrus sinensis?Hamlin‘)有3个45S rDNA位点,2个为活性NORs;枸头橙(Citrus aurantium)中有4个45S rDNA位点,均具有转录活性,但其中一个活性较低;体细胞杂种BG(朋娜脐橙+枸头橙,Citrus sinensis?Bonanza‘+Citrus aurantium)有7个45S rDNA位点,只有1个不具有转录活性。这进一步验证了核仁共显性现象的存在,而且表明枸头橙中转录活性较低的NOR有可能在体细胞杂种中被激活。2.DNA甲基化和H3K9ac在亲本和体细胞杂种中的分布通过蛋白免疫荧光定位,分析发现在亲本和杂种中DNA甲基化和H3K9ac分布规律相同。在有丝分裂间期细胞核中,DNA甲基化在DAPI+异染色质区分布较多,而在常染色质区和核仁区分布较少;H3K9ac在常染色质区分布较多,异染色质区和核仁区分布较少。在中期染色体上,DNA甲基化分布均匀,而H3K9ac一般在染色体近中部和末端分布较多。3.DNA甲基化和H3K9ac与45S rRNA基因表达的关系结合荧光原位杂交和蛋白免疫荧光技术,发现亲本和体细胞杂种中活性NORs解聚部分DNA甲基化程度低,不具有活性的45S rDNA位点甲基化程度高,且枸头橙中活性较低的NOR在杂种中被激活后,甲基化程度明显降低,表明核仁共显性中45S rRNA基因表达与DNA甲基化有关。所有45S rDNA位点在亲本和体细胞杂种间期细胞核中H3K9ac乙酰化程度低,而在染色体上,活性NORs解聚部分乙酰化程度高,浓缩的45S rDNA位点乙酰化程度低,说明H3K9ac与核仁共显性45S rRNA基因表达也有一定的关系。更多还原

【Abstract】 Nucleolar dominance is an epigenetic phenomenon which exists in plant and animal hybrids generally and relates to the expression of r RNA genes. The discovery of nucleolar codominance in Citrus somatic hybrids provides a new way to research the genetic law of genome and regulation of gene expression in polyploid. Using fluorescence in situ hybridization(FISH) and immunolocalization, we analyzed the distribution of 45 S r DNA, DNA methylation, H3K9 ac in Citrus somatic hybrid and its parents and discussed the relationship of methylation, acetylation and r RNA gene expression. From theperspective ofmolecular cytology, the mechanism of nucleolar codominance was researched.1. The distribution of 45 S r DNA in somatic hybrid and its parentsWith FISH, we found that there were three 45 S r DNA sites in Citrus sinensis ?Hamlin‘, two of them were active NORs; four 45 S r DNA sites in Citrus aurantium were all active, but one of them had low activity; there were seven 45 S r DNA sites in BG(Citrus sinensis ? Bonanza ‘+Citrus aurantium), only one was inactive. This results demonstrated the existence of nucleolar codominance and inferred that the low active NOR in Citrus aurantium may be activated in hybrid.2. The distribution of DNA methylation and H3K9 acin somatic hybrid and its parentsWe inferred that the distribution patterns of DNA methylation and H3K9 ac were the same in parents and somatic hybrid according to immunolocalization. In interphase nuclei, DAPI+ heterochromatic regions were hypermethylated, euchromatin and nucleolus were less methylated; however, H3K9 ac was enriched in euchromatic regions, heterochromatin and nucleolus showed a low level of acetylation. DNA methylation signals were homogenously distributed and most of H3K9 ac signals were localized at proximal and terminal regions in metaphase chromosome.3. The relationship between DNA methylation, H3K9 ac and 45 S r RNA gene expressionCombining the two methods, we found that the dispersed regions of active NORs were weakly methylated and inactive 45 S r DNA sites showed a high level of methylation, what is more, the methylation level of the low active NOR in C. aurantium was significantly decreased in somatic hybrid, all of this showed that 45 S r RNA gene expression of nucleolar codominance had a relationship with DNA methylation. All 45 S r DNA sites were less acetylated in interphase nuclei, but in metaphase chromosome, the dispersed regions of active NORs were found strongly H3K9-acelyted and condensed 45 S r DNA sites showed a low level of it, these results also illustrated that H3K9 ac was related to the expression of 45 S r RNA gene.更多还原