水稻颖花开放前浆片转录组变化

doi:10.3864/j.issn.0578-1752.2016.06.001

摘要/Abstract

摘要： 【目的】水稻颖花开放是由其基部的一对浆片吸水膨大所启动。测序技术的发展为从细胞整体水平研究浆片对颖花开放的分子响应，从而对掌握浆片调控颖花开放的内在分子机制提供更为快速、有效的方法。【方法】以常规籼稻品种中早25为材料，应用Illumina测序技术对水稻颖花开放前12 h和临开放前1 h 2个时间点的浆片转录组进行测序，将所得高质量的序列（clean reads）与籼稻9311参考序列比对，获得唯一比对上某一参考基因匹配的reads（unique reads），采用RPKM法计算基因表达量，并在此基础上以FDR≤0.001和|log₂Ratio|≥1为条件筛选出两样本间差异表达的基因，通过与Gene Ontology（GO）数据库、KEGG pathway数据库以及联合蛋白质数据库中的UniProtKB等数据库比对注释差异表达基因的功能和可能参与的分子调控途径。【结果】从水稻颖花开放前12 h和临开放前1 h 2个时间点的浆片转录组中分别检测到有26 369和26 157个表达基因，2个时间点的转录组之间存在3 924个差异表达基因，其中2 623个基因呈现下调表达和1 301个基因呈现上调表达，有105个基因表达差异倍数高达100倍以上（即|log₂Ratio|≥6.7）。GO分析结果表明被注释分子功能的差异基因有1 624个，其中21.7%基因具有离子结合活性、10.3%具有氧化还原酶活性、5.4%具有转运活性。富集于生物学过程的差异基因有1 313个，其中15.0%的差异基因参与定位、12.4%参与运输、4.7%参与碳水化合物代谢、4.5%参与脂类代谢。Pathway显著性富集分析发现有2 229个差异基因参与了123条代谢途径，其中，富集基因数目较多的途径主要包括次生物质生物合成（399个）、植物激素信号转导（152个）、核苷酸代谢（146个）以及淀粉和糖代谢（64个）等路径。花前1 h浆片中特异表达且唯一比对上基因的reads数（Uniq-reads_ num）超过30的基因的功能主要涉及细胞壁重塑、质膜的稳定性、能量代谢、基因转录调节以及信号转导等生命活动。茉莉酸及其类似物对水稻颖花开放有强烈的诱导效应，差异表达基因中有16个基因参与了茉莉酸生物合成途径以及11个基因参与了茉莉酸的信号转导过程，且随着颖花开放时间的临近，多数基因表达水平显著提高。【结论】获得颖花开放前12 h和临开放前1 h浆片中差异表达基因的表达变化模式及其功能信息，发现参与调控碳水化合物代谢与运输、细胞能量代谢、细胞壁结构修饰以及茉莉酸等激素代谢与信号转导等生理过程的基因在颖花临开前被强烈诱导或抑制表达，提示这些基因与浆片细胞吸水膨大调控水稻颖花开放密切相关。

关键词: 水稻, 浆片, 颖花开放时间, 转录组, Illumina测序

Abstract: 【Objective】The rice floret opening will be driven by that a pair of lodicules lying between the lemma and the stamen base in the floret will expand rapidly when absorbing abundant water and lever away the rigid lemma. However, little is known about the molecular mechanism of the lodicules regulating the time of floret opening. With the development of sequencing technology, a faster and more effective way is provided to study the mechanism of the responses of lodicules to floret opening time at the whole level of cell.【Method】In this study, conventional indica rice germplasm Zhongzao 25 was used as the material and the transcriptomes of lodicules at 12 h and 1 h before floret opening were sequenced using Illumina sequencing technology. First, clean reads were mapped to the reference sequences of indica 9311 and the unique matched reads were obtained; second, the gene expression level was calculated by using RPKM method; Then, false discovery rate≤ 0.001 and the absolute value of Log₂Ratio ≥ 1 were used as the threshold to judge the significance of gene expression differences. Finally, the functions and pathways of differentially expressed genes (DEGs) were annotated by comparing with the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) and Universal Protein Knowledgebase (UniProtKB).【Result】The expressed genes from the two transcriptomes of lodicules at 12 h and 1 h before floret opening were 26 369 and 26 157 respectively, and 3 924 differentially expressed genes (DEGs) were screened between the two transcriptomes. 2 623 DEGs were down-regulated and 1 301 DEGs were up-regulated genes. Moreover, change folds of 105 DEGs were more than 100 folds (|log₂Ratio|≥6.7) of lodicules during the two stages before floret opening. Gene ontology enrichment analysis showed that 1 624 DEGs could be mapped as functional molecule, 21.7% DEGs of which had ion-binding activity, 7.5% DEGs of which had oxidoreductase activity and 5.7% DEGs of which had transporter activity. Meanwhile, 1 313 DEGs were involved in biological processes, 15.0% DEGs participated in localization processes, 12.4% DEGs participated in transport process, 4.7% DEGs participated in carbohydrate metabolic process and 4.5% DEGs participated in lipid metabolic process. KEGG pathway enrichment analysis revealed that 2 229 DEGs were involved in 123 different metabolic pathways，which enriched larger numbers of DEGs including biosynthesis of secondary metabolites (339 genes), plant hormone signal transduction (152 genes), nucleotide metabolism (146 genes), and starch and sucrose metabolism (64 genes). The functions of those genes, which were specifically expressed at 1 hour before floret opening and the unique match reads number of which exceeded 30, were mainly concerned in the regulation of physiological processes of cytoskeleton remodeling, membrane stability, energy metabolism, transcription regulation, and signal transduction. Jasmonic acid and analogs have a strong inductive effect on floret opening in rice, 16 DEGs were involved in jasmonic acid biosynthesis pathway and 11 DEGs were involved in jasmonic acid signal transduction pathway, whose expression levels increased significantly as the floret opening time approached.【Conclusion】The transcriptome data of rice lodicules was obtained using RNA sequencing technology. The expression profiling data of DEGs and molecular function of DEGs were also collected. The differential activation or suppression of key DEGs in energy metabolism, carbohydrates metabolism and translocation, cell walls structure modification and hormones metabolism and signaling pathway in lodicules suggested that these DEGs were closely related to water absorption and expansion of lodicules towards the regulating of the floret opening time.

Key words: rice (Oryza sativa L.), lodicule, floret opening time, transcriptome, Illumina sequencing

付永琦，向妙莲，蒋海燕，何永明，曾晓春. 水稻颖花开放前浆片转录组变化[J]. 中国农业科学, 2016, 49(6): 1017-1033.

FU Yong-qi, XIANG Miao-lian, JIANG Hai-yan, HE Yong-ming, ZENG Xiao-chun. Transcriptome Profiling of Lodicules Before Floret Opening in Oryza sativa L.[J]. Scientia Agricultura Sinica, 2016, 49(6): 1017-1033.

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