【摘要】 种子的萌发是植物新生命的开始,也是决定植物种群更新、物种延续的关键环节和影响粮食作物产量的重要因素.种子萌发事件是一个极其复杂的过程,其调控机制至今仍未完全知晓.本文基于拟南芥种子不同萌发过程的RNA-seq测序数据,分别从基因表达和pre-m RNA可变剪接层面系统地剖析了种子萌发过程中的组学特征和调控机制.首先,通过组学数据分析方法得到拟南芥的基因表达矩阵,系统地识别了种子不同萌发阶段间的差异表达基因,并解析了其生物学功能.结果显示,随着种子的萌发,一方面大量与糖酵解、DNA合成等代谢过程密切相关的基因被激活,保证了种子新陈代谢和基本的细胞活性的重新激活;另一方面,与氧化应激胁迫响应、有毒物质响应、热响应、水响应等相关的基因活性被抑制,保证了种子能够从休眠态顺利活化.其次,使用r MATS系统描绘了种子萌发过程中各阶段的pre-m RNA可变剪接图谱,鉴定了不同萌发阶段间的差异可变剪接事件,并分析了种子萌发过程中的可变剪接特征及差异可变剪接事件的生物学功能.该工作的开展将进一步促进种子萌发过程中的调控机制的阐明,并为相关实验工作的开展提供一定的帮助.更多还原

【Abstract】 Seed germination is the initial phase of life cycle for plants and plays a pivotal role in regeneration of plant population,continuation of plant species and crop yield. However, seed germination is an extremely complex biological process and its regulatory mechanisms remain unclear. It is well known that gene expression and pre-m RNA alternative splicing are vital to the central dogma. In Arabidopsis genome, ～60% multi-exon genes are alternatively spliced. Alternative splicing is involved in the response to environmental cues, including abiotic and biotic stresses, in the regulation of crucial developmental processes such as flowering, and in circadian timekeeping. The present paper studies the effect of gene expression and alternative splicing on seed germination in Arabidopsis.We constructed RNA-seq datasets from dry seeds, germinating seeds 1–3, Young seeds and Seeds in Arabidopsis and carried out quality control for the raw reads by using Fast QC and Trimmomatic. String Tie estimated transcript and gene expression from a mapping to the Arabidopsis genome obtained from Hisat2. We next created the gene expression matrix for Arabidopsis, which includes 37336 genes along the rows and 12 samples along the columns, and employed DESeq2 to systematically identify differentially expressed genes between different stages during seed germination. To elucidate the biological functions of differentially expressed genes, we performed a functional enrichment analysis of these genes using cluster Profiler tool. The results showed that many genes involved in metabolic processes, such as glycolysis and DNA synthesis, were activated during the seed germination. It ensures the reactivation of metabolism and basic cellular activities of seeds. Some genes involved in environmental responses, such as oxidative stress, toxic substance, heat, and water, were inhibited. This is essential for seeds to be successfully activated from a dormant state. Furthermore, r MATS was utilized to detect the pre-m RNA alternative splicing events and differential alternative splicing events under different conditions. We constructed the atlas of pre-m RNA alternative splicing at different stages of seed germination and analyzed the distribution characteristics of alternative splicing patterns across every stage. Besides, we detected the differential alternative splicing events between different germination stages and analyzed the biological functions of differential alternative splicing events during seed germination using cluster Profiler. A total of 18 genes, in which differential retained intron events were identified between dry seeds and germinating seeds 3, were enriched on splicing process. By visualization of alternative splicing events in AT4 G25500, which was done by using the rmats2 sashimiplot tool, the inclusion levels and functions of differential alternative splicing between different stages of seed germination were clearly illuminated.Overall, we investigated the dynamic changes of gene expressed levels and alternative splicing events during seed germination of Arabidopsis based on bioinformatics methods and dissected omics features and regulated mechanisms during seed germination from the perspective of gene expression and alternative splicing of pre-m RNA. This study is expected to be helpful for elucidating the regulated mechanisms of seed germination and to provide a guideline for relevant experimental works.更多还原