【作者】 林燕；

【导师】 孙日飞； 张振贤；

【作者基本信息】 中国农业大学 ， 蔬菜学， 2014， 博士

【摘要】 植物游离小孢子培养技术在育种工作和基因工程研究中具有重要作用,目前小孢子胚胎发生机理尚未明确,严重限制了该技术的应用。大白菜小孢子培养体系比较成熟,且其基因组测序工作已经完成。因此,本研究以大白菜为试材,利用转录组测序技术(RNA-Seq)对小孢子胚胎发生早期相关基因的表达情况进行了研究,主要得到了以下结果：1.通过材料筛选,选定一份出胚率较高且能稳定出胚的材料"jihong82"为试材。研究了不同高温处理时间对小孢子出胚率的影响,确定了五份测序样品：TO(未处理的新鲜小孢子),T1-1(高温热激12h的小孢子),T1-2(常温培养12h的小孢子),T2-1(高温热激24h的小孢子)T2-2(常温培养24h的小孢子)。通过对三种RNA提取方法的比较发现超纯RNA提取试剂盒提取的总RNA质量较高,能够满足转录组测序的要求。2.测序一共得到了141.60M条reads,总碱基数为28.60G。每个样品的测序总核苷酸数均大于5Gb,Q30比例大于80%,五份样品的测序质量均较高。3.分析了六种可变剪接：ES, IR, AFE, ALE, Alt5’和Alt3’,其中内含子保留可变剪接(IR)占主要部分。通过将测序数据与参考基因组比对,进行了各样品的SNP分析。另外还搜索出了3102个已知SSRs并发现了478个新的SSRs。4.试验共发掘新基因的数量为2228个,将新基因序列与Nr, SwissProt, GO, COG, KEGG数据库进行比对,获得了1602个新基因的注释信息,为原有基因组注释进行了补充。5.将五份样品分组比对,统计了各组间差异表达基因的数量,并分析了差异基因在各处理中的表达情况,同时将差异表达基因与各大数据库进行了比对和功能注释。6.通过将差异基因进行GO注释和统计分析发现,在细胞组成分组中,差异表达基因在细胞组分、细胞和细胞器三个类型中所占比例最高；在分子功能分组中,结合、催化活性类型中差异表达基因所占比例最高；差异基因在生物学过程分组中的类型分布最多,其中在细胞过程、代谢过程等类型中所占比例较高。7.差异基因的COG直系同源分类结果显示,R(一般功能基因)功能类中所包含的基因数量最多,其次是T(信号转导机制)、K(转录)和L(复制、重组和修复)分类。8.差异基因的KEGG注释结果显示,差异基因显著富集的Pathway主要为植物激素信号转导、植物-病原菌互作、苯丙烷生物合成、淀粉和糖代谢以及苯丙氨酸代谢等。其中植物激素信号转导及淀粉和糖代谢途径中部分基因的表达情况与前人报道相符,苯丙烷生物合成和苯丙氨酸代谢途径中差异表达的相关蛋白与植物逆境防御机制相关。对以上途径中相关酶和基因的表达情况进行了深入分析,发现大部分基因在热激条件下显著上调表达,且热激处理24h条件下基因表达量比12h更高。更多还原

【Abstract】 The mechanism of microspore embryogenesis remains unclear till now, which limits the application of isolated microspore culture technology in plant breeding and genetic engineering research. The project of whole genome sequencing for Chinese cabbage has been completed, and the cultivation system of Chinese cabbage microspore was matured. In order to gain more insights into the mechanism of microspore embryogenesis, we performed transcriptome sequencing of microspores in the early stage of embryogenesis, the main conclusions are as follows:1. We chose "jihong82" as the donor plant and five samples as the sequencing material according to screening. The quality of RNA extracted by RNA-pure kit could meet the requirements of transcriptome sequencing.2. High-throughput Illumina RNA sequencing by paired-end sequencing were conducted, according to reference transcriptome and Data Analysis, and approximately141.60M high quality (HQ) reads,28.6Gb of sequence data were generated. For each of the samples, more than80percent of Q30were aligned on the reference genome.3. Six alternative splicing including ES, IR, AFE, ALE, Alt5’and Alt3’were studied, and the result showed that the number of IR was the most.In addition, SSRs were systematically tested, and478new SSRs were searched out.4.2228new transcripts were found by high throughput sequencing since present databases may be incomplete. The sequences of new genes were compared to the database of Nr, SwissProt, GO, COG and KEGG, and1602of them have annotation information.5. Based on RPKM method, we analysed differential expression genes between different samples. According to Hierarchical Cluster Analysis, a number of differentially expressed genes were gained in each group.6. Gene Ontology functional enrichment analyses revealed that the DEGs were primarily distributed in three cellular component functional classifications:cell part, cell and organelle, and two molecular function classifications:binding and catalytic activity, and the classifications in biological process were the most.7. Clusters of Orthologous Groups enrichment analyses showed that the DEGs were primarily associated with General function prediction only, Signal transduction, Transcription and Replication, recombination and repair.8. KEGG pathway enrichment analyses revealed that the DEGs were primarily associated with plant hormone signal transduction, starch and sucrose metabolism pathways, and so on. The expression of related enzymes and genes in the pathways rised significantly under heat shock condition, which suggesting that genes related with these pathways may play important roles in the early stage of microspore embryogenesis.更多还原