基于联合测序的涡虫再生相关选择性剪接分析、验证及调控关系研究

Planarians are best known for their ability to regenerate complete animals from tiny fragments of their bodies. The type of regeneration undertaken by planarian is called epimorphic regeneration. The strong regenerative ability of planarians is empowered by a system of pluripotent stem cells, called neoblasts. Following injury, neoblasts migrate to the wound region, divide, and their progeny eventually differentiate to replace missing structures. Neoblasts also function in homeostasis by serving to replace cells lost during the normal cellular turnover. Neoblasts of planarian organisms are equivalent to stem cells; this makes these remarkable creatures an excellent model system for thestudy of stem cell biology. Although over the last years several efforts have been made to shed light on neoblast biology. However, full length mRNA isoforms are not captured.. In this study we will to combine second-generation sequencing and third-generation sequencing with a custom-designed method for isoform identification and quantification to generate a high-confidence isoform dataset for Planarian neoblasts.Based on the negative regulation relation between the miRNA and its target genes, the negative correlation analysis was carried out with signifieant miRNA expression pattem and construct the miRNA-Gene regulation network in post-transcriptional level. Finally, using quantitative real-time PCR and FISH to verify the key" node" miRNAs and genes. . In this way the results of the research will be helped to understanding the molecular principles governing the alternative splicing regulatory mechanisms of planarians regeneration, provides new ideas and useful information to the field of regenerative medicine base on stem cells.

涡虫因具有大量成体干细胞和强大的再生能力，成为理想的再生和干细胞研究模式生物，但其再生相关调控网络尚未阐明。选择性剪接作为真核细胞重要的mRNA转录后加工机制，在调控干细胞增殖、自我更新和分化方面也具有重要调控作用。本申请拟通过混合测序（Pacbio和Illumina），采用一种新型的分析方法，建立涡虫不同再生阶段的差异转录本库，研究mRNA不同转录本在涡虫再生过程中的动态表达模式，通过与miRNA组的负相关分析、靶基因预测、通路动态仿真等，构建miRNA和编码基因的综合调控关系网络，最后用定量PCR和原位杂交等表达差异显著的“节点”mRNA转录本进行实验验证，通过RNAi、过表达等方法对重要的“节点”基因进行功能研究，探寻涡虫可变剪切机制与再生和干细胞维持分化的关系，为再生生物学研究提供新的研究思路和和实验依据。

涡虫因具有大量成体干细胞和强大的再生能力，成为理想的再生和干细胞研究模式生物，但其再生相关调控网络尚未阐明。选择性剪接作为真核细胞重要的mRNA转录后加工机制，在调控干细胞增殖、自我更新和分化方面也具有重要调控作用。本项目通过混合测序（Pacbio和Illumina），建立涡虫不同再生阶段的差异转录本库，研究mRNA不同转录本在涡虫再生过程中的动态表达模式，构建miRNA和编码基因的综合调控关系网络，最后用定量PCR和原位杂交等表达差异显著的“节点”mRNA转录本进行实验验证，通过RNAi、过表达等方法对重要的“节点”基因进行功能研究，探寻涡虫可变剪切机制与再生和干细胞维持分化的关系。. 在本项目中，我们完成了涡虫全长转录组测序，发现了其中24个可变剪接转录本，完成了可变剪接基因的聚类分析、GO功能分析，Pathway功能分析蛋白互作分析，建立了再生相关转录本库与 miRNA 表达谱数据整合分析及调控网络。通过对上述调控网络分析，发现daf-12在再生过程中特异性高表达，而其调控基因对应的低表达，进一步研究发现敲低daf-12基因的表达，会导致尾部再生“头部”，产生两段同时具有眼点的“双头”表型缺陷，提示daf-12可能通过调控干细胞的增殖来影响涡虫再生的极性和神经再生。. 本研究初步探讨了涡虫可变剪切机制与再生和干细胞维持分化的关系，为再生生物学研究提供新的研究思路和和实验依据。