棉花体胚发生及植株再生的DNA甲基化与基因表达特征分析

Somatic embryogenesis (SE) is a notable illustration of cell totipotency and has been viewed as a potential model system for the theory study of development biology among the higher organisms. And SE is the key step and technique limitation on genetic engineering in cotton. In this study, implementing new generation technique of MeDIP-seq, investigation of genome-wide methylation modification will be performed on two genotypic cotton with different embryogenic and plant regeneration ability during all staged SE and plant regeneration developmental process. In addition, differential expression profiling analysis will be conducted as well using RNA-seq assay. Furthermore, we will perform comparative analysis on methylation modification and expression profiling of two different genotypes to identify valued target genes associated with cotton SE and plant regeneration. Finally, based on our previous study on SE, to better understand the concerted regulatory mechanism involving gene expression and related methylation during this important reprogramming process, all research data from different levels will be integrated and associated systemically. We then use the systems biology strategy and bioinformatics approach to construct a updated SE association interaction network. A complex molecular system impacted by gene expression and methylation will be unraveled by SE association network, which provide the gene resource and theory basis for SE and plant regeneration in vitro during genetic engineering in cotton. This study is to dissect molecular basis of SE and plant regeneration in cotton from the perspective of systems biology, and will be of great importance in both uncovering the mechanism of the key scientific question of cell totipotency and cotton biotechnology breeding.

体细胞胚胎发生是细胞全能性的经典示例，是生物学理论研究的良好模型，也是棉花转基因遗传工程的重要环节和技术瓶颈。本项目针对棉花体胚发生和植株再生的不同基因型，应用MeDIP-seq技术分别探明早期体胚发生起始、中期体胚发育及后期植株再生过程的基因组DNA甲基化模式和位点，同时应用RNA-seq技术进行基因表达谱分析。通过比较分析不同基因型的DNA甲基化及基因表达差异，筛选并鉴定不同发育时期参入体胚发生和植株再生的特异甲基化基因及表达基因。进而整合关联不同基因型、各个发育时期的DNA甲基化与基因表达数据，分析受甲基化调控的基因，在我们前期研究基础上，通过系统生物学策略构建更完整的基因甲基化修饰和表达互作下的调控网络。本项目从系统生物学视角解析体胚发生和植株再生的分子基础，为体外调控这一过程并拓展棉花遗传工程提供理论依据和基因资源，对揭示细胞全能性分子机理和促进生物技术育种发展都具有重要意义。

该项目围绕不同体细胞胚胎发生能力的棉花基因型，应用单碱基分辨率甲基化测序BS-seq，分别解析早期体胚发生起始、中期体胚发育及后期植株再生过程中的基因组DNA甲基化模式和位点，同时应用RNA-seq技术进行基因转录组分析。通过本项目研究，获取了以下三个重要的研究成果。第一，研究分析表明DNA甲基化主要发生在基因的上下游区域，解析了高体胚基因型YZ和低体胚基因型LM在体胚发生各个发育时期的DNA甲基化修饰模式。并且不同类型的甲基化C碱基（CG, CHG与CHH）在体胚发生过程中存在着不同的甲基化修饰特征。其中，CG类型甲基化修饰在胚性分化过程中具有基因型特异性；CHH类型甲基化修饰在胚性分化过程中具有发育时期特异性。第二，挖掘出两个基因型在体胚发生各个发育时期的差异甲基化基因和差异表达基因。通过GO和KEGG分析注释了差异基因在两个基因型不同发育时期参与的生物过程和代谢通路。第三，整合关联以上两方面的数据，鉴定了体胚各个发育时期中受甲基化修饰的差异表达基因和互作调控模式，并通过RT-PCR和Methylation-sensitive HRM（甲基化特异性高分辨率溶解曲线）方法验证了全基因组甲基化与转录组分析结果。目前已经接受或发表基金署名的英文论文4篇，另有2篇第一标注的相关论文已投稿。培养研究生4名（2名硕士研究生已毕业，1名博士研究生和1名硕士研究生在读）。本项目研究为解析体外调控体细胞胚胎发生的分子机制和拓展棉花遗传工程提供了理论依据和基因资源。无论是对细胞全能性理论研究还是对转基因生物技术应用，都具有非常重要的意义。