Wang Lun, Zhang Jiaojiao, Xu Xiaoyong
College of Horticulture and Landscape Architecture, Yangzhou University, Yangzhou 225009, China.
Key Laboratory of Plant Functional Genomics of the Ministry of Education, Agricultural College of Yangzhou University, Yangzhou 225009, China.
Plants (Basel). 2024 Oct 15;13(20):2878. doi: 10.3390/plants13202878.
The epigenetic variation in protoplast regeneration is a topic that has attracted interest recently. To elucidate the role of DNA methylation in the regeneration of protoplasts from the ponkan (), this study employs the methylation-sensitive amplification polymorphism (MSAP) molecular marker technique to analyze changes in DNA methylation levels and patterns during the isolation and culture of protoplasts from ponkan and tobacco. Additionally, differential DNA methylation fragments are cloned, sequenced, and subjected to bioinformatics analysis. The results reveal that, for non-regenerable ponkan mesophyll protoplasts, DNA methylation levels increase by 3.98% after isolation and then show a trend of initial decrease followed by an increase during culture. In contrast, for regenerable ponkan callus protoplasts and tobacco mesophyll protoplasts, DNA methylation levels decrease by 1.75% and 2.33%, respectively, after isolation. During culture, the DNA methylation levels of ponkan callus protoplasts first increase and then decrease, while those of tobacco mesophyll protoplasts show an opposite trend of initial decrease followed by an increase. Regarding DNA methylation patterns, ponkan mesophyll protoplasts exhibit primarily hypermethylation changes accompanied by a small amount of gene demethylation, whereas ponkan callus protoplasts are dominated by demethylation changes with some genes undergoing hypermethylation. The methylation exhibits dynamic changes in protoplast isolation regeneration. By recovering, cloning, sequencing, and performing BLASTn alignment analysis on specific methylation modification sites in the ponkan, 18 DNA sequences with high homology are identified which are found to be involved in various biological functions, thereby establishing a foundational basis for genetic editing in protoplasts.
原生质体再生过程中的表观遗传变异是近年来备受关注的一个话题。为了阐明DNA甲基化在椪柑原生质体再生中的作用，本研究采用甲基化敏感扩增多态性（MSAP）分子标记技术，分析椪柑和烟草原生质体分离与培养过程中DNA甲基化水平和模式的变化。此外，对差异DNA甲基化片段进行克隆、测序，并进行生物信息学分析。结果表明，对于不可再生的椪柑叶肉原生质体，分离后DNA甲基化水平增加3.98%，随后在培养过程中呈现先下降后上升的趋势。相比之下，对于可再生的椪柑愈伤组织原生质体和烟草叶肉原生质体，分离后DNA甲基化水平分别下降1.75%和2.33%。在培养过程中，椪柑愈伤组织原生质体的DNA甲基化水平先升高后降低，而烟草叶肉原生质体则呈现相反的趋势，即先下降后上升。关于DNA甲基化模式，椪柑叶肉原生质体主要表现为超甲基化变化，伴有少量基因去甲基化，而椪柑愈伤组织原生质体则以去甲基化变化为主，部分基因发生超甲基化。甲基化在原生质体分离再生过程中呈现动态变化。通过对椪柑中特定甲基化修饰位点进行回收、克隆、测序及BLASTn比对分析，鉴定出18条具有高度同源性的DNA序列，发现它们参与了多种生物学功能，从而为原生质体的遗传编辑奠定了基础。
