【作者】 董振营；

【导师】 刘宝；

【作者基本信息】 东北师范大学 ， 遗传学， 2009， 博士

【摘要】 实验室前期通过对预先选择的一些基因片段的研究表明,菰渐渗可以诱导受体水稻基因组产生一系列遗传和表观遗传变异。本实验首先通过采用甲基化敏感的扩增片断多态性(methylation-sensitive amplified polymorphism,MSAP)标记方法,在全基因组范围内估测了三个渐渗杂交系(RZ1, RZ2和RZ35)相对于亲本松前的甲基化变异程度和变异模式。共检测了2700个位点,每个位点代表一个同裂酶HpaII/MspI的识别位点,根据这对同裂酶的不同酶切方式,估测在亲本松前基因组有15.9%的5’-CCGG位点产生了内侧胞嘧啶的完全甲基化或者外侧胞嘧啶的半甲基化,与水稻亲本(松前)比较,这两种甲基化修饰程度在所研究渐渗系中显著提高,分别达到19.2%, 18.6%, 19.6%。以亲本松前的MSAP扩增图谱作为对照,在渐渗系MSAP扩增谱带可以归为四大类,每类又可以细分不同亚类。变化的甲基化模式包括5’-CCGG位点甲基化修饰程度的升高、甲基化的降低和内外侧甲基化修饰模式的互换。大多数发生在低拷贝位点的变化可以被southern杂交的方法所证实,采用相同方法发现在同一渐渗系不同单株间这种变化是一致的。根据MSAP扩增结果选择了31条甲基化修饰模式在渐渗系不同于亲本的扩增片段进行测序,发现这些序列分布于水稻不同12条染色体上,包括了编码蛋白的基因,转座子/反转座及基因的非同源序列。另外利用涵盖水稻12条染色体和两种细胞质基因组的微卫星(microsatellites,MS)序列对渐渗系进行了研究。微卫星也称作简单重复序列(simple sequence repeats,SSRs),是在真核生物基因组广泛存在的一种DNA序列,并且这种简单重复序列存在很高的突变几率。发现在所检测的三个渐渗系的微卫星位点均发生了大量相对于亲本的遗传变异,而且这种变异呈现一定规律,即在核基因组所有检测的基因编码区位点均表现出很高的保守性而在非编码区位点渐渗系表现出较高变异性(基因间序列位点变异率达100%,5’端调节区为66.7%,内含子区占83.3% )。而且更意外的是,虽然是母系遗传,在渐渗系细胞器基因组仍然检测到了变异,这种变异甚至发生在基因编码区。根据电泳图谱,共有五类变异模式被检测出来。通过对变异位点的序列分析,发现造成微卫星位点变异的主要原因是微卫星基序重复次数的改变,同时也有少量侧翼序列也产生了变异。通常解释微卫星变异主要机制有染色体重组或基因转换(gene conversion)及DNA复制滑移(DNA replication slippage)。但是无论涉及到哪一种机制,纠正DNA复制错误的错配修复(mismatch repair,MMR)基因均在这些过程中起着重要作用。Real-time PCR分析显示这些基因的表达均表现出相对于亲本的不同变化,说明在渐渗过程中或其后几个世代错配修复基因的表达发生了较大变化。同时发现错配修复基因启动子的DNA甲基化修饰也发生了一定改变,暗示DNA甲基化可能参与了错配修复基因表达改变。最后对渐渗诱导的甲基化改变可能机制及在作物育种的意义及渐渗系是研究微卫星功能的良好材料等问题进行了讨论。更多还原

【Abstract】 We have reported previously that introgression by Zizania latifolia resulted in extensive genetic and epigenetic changes in the recipient rice genome. In this study, using the methylation-sensitive amplified polymorphism (MSAP) method, we globally assessed the extent and pattern of cytosine methylation alterations in three typical introgression lines (RZ1, RZ2 and RZ35) relative to their rice parent at ~2,700 unbiased genomic loci each representing a recognition site cleaved by one or both of the isoschizomers, HpaII/MspI. Based on differential digestion by the isoschizomers, it is estimated that 15.9% of CCGG sites are either fully methylated at the internal Cs and/or hemi-methylated at the external Cs in the rice parental cultivar Matsumae. In comparison, a statistically significant increase in the overall level of both methylation types was detected in all three studied introgression lines (19.2, 18.6, 19.6%, respectively). Based on comparisons of MSAP profiles between the isoschizomers within the rice parent and between parent and the introgression lines, four major groups of MSAP banding patterns are recognized, which can be further divided into various subgroups as a result of inheritance of, or variation in, parental methylation patterns. The altered methylation patterns include hyper- and hypomethylation changes, as well as interconversion of hemi- to full-methylation, or vice versa, at the relevant CCGG site(s). Most alterations revealed by MSAP in low-copy loci can be validated by DNA gel blot analysis. The changed methylation patterns are uniform among randomly selected individuals for a given introgression line within or among selfed generations. Sequencing on 31 isolated fragments that showed different changing patterns in the introgression line(s) allowed their mapping onto variable regions on one or more of the 12 rice chromosomes. These segments include protein-coding genes, transposon/retrotransposons and sequences with no homology.At the same time, 98 microsatellites markers that mapped to all 12 rice chromosomes and the two cytoplasmic genomes were seleceted for detecting the variation in those specific sequences. Microsatellites, also called simple sequence repeats (SSRs), are widely dispersed in eukaryotes and have a high evolutional rate. In this study, we found that microsatellite variated extensively and rapidly in all three introgression lines. All of nuclear noncoding region located microsatellites extensively variated (100% in intergenic sequences, 66.7% in 5’ regulatory regions and 83.3% in introns), but no change was detected in coding regions. And more surprisingly, 50% chloroplast DNA loci and 14.3% mitochondrial DNA loci showed variation and the variation even happened in coding regions of cytoplamic DNA. According to electrophoresis in denaturing polyacrylamide sequencing gels, five classes of polymorphic bands were found, and sequence comparison of selected loci between introgression lines and its rice parent showed that the high degree of variability in the microsatellite containing sequences were mainly due to variation in the repeated regions, while variation in the flanking regions of the microsatellite also happened. As methylation variation, the changed microsatellite patterns are uniform among randomly selected individuals for a given introgression line within or among selfed generations. We also found expression of mismatch repair (mismatch repair,MMR) genens in introgression lines changed, considering the relation of MMR genes with genome stability, this may be one of the reason for microsatellite stability in introgression lines. Finally, issues about possible causes for the introgression-induced methylation changes and their implications for genome evolution and crop breeding as well as introgression lines can be very good materials for microsatellite function analysis are discussed.更多还原