水稻再生体系突变产生规律的研究

Plant tissue culture is an important way for rapid propagation, and is also a potent means to study plant genetics and molecular biology. Recent researchers have found that much more mutations could be introduced in the process of the tissue culture. However, no systematic research has been performed on this phenomenon. In our study, we constructed a rice tissue culture system and surveyed the mutations and methylation on callus, regenerated plants and their progeny at different stages. For the first time, the mitotic mutation rate during tissue culture was assessed by whole-genome sequencing. The mutation spectrum and distribution l of regenerated plants were compared with spontaneous mutations, in order to find the relationship among the number of mitotic divisions, the length of cell culture time, the dynamic change of methylation and the mutations. Figuring out these issues will lay the foundation for studying the molecular mechanisms of plant self-regulation on mutations.

植物组织培养是植株快速繁殖的重要途径，也是植物遗传、分子生物学研究中的重要技术手段。最近的研究发现，利用组织培养技术得到的再生植株存在较高的突变，但对其缺乏系统的研究。本研究拟利用水稻组织培养体系，对不同阶段的愈伤、再生植株以及其后代进行一系列的突变和甲基化追踪。利用全基因组测序的手段，精密评估组织培养过程中有丝分裂突变率，对再生植株的突变频谱及其分布规律与自发突变进行比较，系统探讨有丝分裂次数、细胞培养的时间长短和甲基化水平的动态变化与突变的关系。对这些科学问题的探讨，将会为解析植物调控自身突变分子机制的研究奠定基础。

为研究水稻组织培养过程中有丝分裂突变率和再生植株突变规律，本项目构建了水稻组织培养再生植株体系，并对其愈伤、再生植株及后代进行了全基因组测序和分析，对检测到的突变进行追踪和比较。结果表明，NIPB系列再生植株平均单核苷酸变异(SNV)为331.8，是亲本体细胞突变的44倍，说明组织培养过程会造成大量突变积累。通过突变追踪，估算水稻愈伤组织继代培养期间有丝分裂突变率约为1.1~4.0X10-8，分化培养阶段有丝分裂突变率约为0.3~10.1 X10-8。再生植株突变分布规律与突变频谱分析发现，突变主要发生在基因组非编码区域，编码区突变多为非同义突变；突变碱基存在明显偏好性，与亲本体细胞突变相比，C>T突变仍占主导，但C>A突变频率升高。与再生植株突变频谱不同，愈伤组织突变存在极强的C>A偏好，说明愈伤组织中大量突变被丢失。等位基因突变频率（VAF）分析显示再生植株在分化出苗过程中经历了单细胞瓶颈，其突变能够被后代稳定继承（＞93%），且不影响后代体细胞突变率。水稻再生体系突变规律的研究，为进一步解析组织培养期间突变的分子机制奠定了基础。

内容获取失败，请点击重试