荷花‘青玉’花瓣转录组测序以及花开放差异基因分析

发布时间：2019-05-20 10:00

【摘要】：荷花(Nelumbo nucifera Gaern)属于睡莲科莲属,是多年生草本植物花卉,具有极高的观赏和经济价值。荷花的花期时间较短,研究荷花花期的生物学基础相对滞后,所以提升荷花开花时间的研究尤为重要。转录组测序技术已成为研究分子功能的主流,极大地加快了生物相关功能的研究进展。本研究通过高通量Illumina HiSeq2500平台测序,研究‘青玉’花瓣开放的暄蕾期与盛花期的转录水平,最终获得50,154条unigene,总长度3,938,0851bp,平均长度是785.20bp,N50的长度是1,412bp。转录本中有23,753条unigene对比到GO数据库中,其中三大类中包含56组功能;有32,194条unigene对比分析到NR蛋白数据库中,同源蛋白质序列对比较高的是葡萄(Vitis vinifera)39.5%,可可树(Theobroma cacao)10.6%等。有10,374条unigene注释到COG数据库中,最多的是基因功能预测(General function prediction only)。有9,550条unigene注释到KEGG数据库中,其中RNA运输(RNA transport)有381条unigene,核糖体(Ribosome)有360条unigene。通过对样品进行差异表达分析,差异表达基因5,141条,其中上调基因3,832条,占差异表达基因的74.5%,下调基因1,309条,占差异表达基因的25.5%。2,728条差异基因注释在GO功能富集中,得到56组GO功能分类,代谢过程(Metabolic process)、细胞过程(Celluar process)、细胞(cell)、细胞部分(cell parts)、催化活性(catalytic activity)、结合性(binding)等功能的unigene较多。在三大类的topGO功能富集分析中细胞壁(cell wall)、亚铁红素结合(heme binding)、镉的响应(response to cadmium ion)节点最为显著。1,660条unigene注释在COG注释分类中,最显著的是基因功能预测(General function prediction only),这些基因在作用机制上有共同点而类聚在一起,从而推测基因功能。有1,500条差异基因注释到KEGG分类中,共有148条代谢通路,最高的代谢通路中是氧化磷酸化(Oxidative phosphorylation)与总unigene KEGG富集分析结果一致。按照基因功能找出差异性较大的调控荷花‘青玉’花开放的相关关键基因,促进乙烯合成的相关基因ACC和ERF;影响生长素合成的相关基因XTH和YUCCA;调控细胞分裂素合成的CKX基因;影响细胞壁生长的相关基因EXP。通过实时荧光定量Real-time PCR方法,以‘青玉’和‘重阳’两个品种花开放的6个时期,现蕾期、转色期、暄蕾期、初花期、盛花期、谢花期的花瓣为材料,对表达量较高的4个基因验证分析,在‘青玉’的暄蕾期与盛花期的表达量的变化与转录组测序基本一致,说明测序数据真实可靠。在‘青玉’与‘重阳’的花朵开放的6个时期中,不同基因在整个开花过程的表达量变化有所差异,说明基因在花开放中的不同作用,也推测不同的基因对花型也有调控作用。本研究揭示了控制花开放的关键基因,为延长花期和荷花育种提供有效分子数据信息。
[Abstract]:Lotus (Nelumbo nucifera Gaern) belongs to the genus Nymbiaceae. It is a perennial herbaceous flower and has high ornamental and economic value. The flowering time of lotus is short, and the biological basis of lotus flowering is relatively lagging behind, so it is particularly important to improve the flowering time of lotus. Transcriptional group sequencing technology has become the mainstream of molecular function research, which greatly accelerates the research progress of biological related functions. In this study, the transcriptional levels of bud stage and flowering stage of 'Qingyu' petals were studied by sequencing on a high throughput Illumina HiSeq2500 platform. Finally, the total length of 50154 unigene, was 3, 938, 0851 BP, the average length was 785.20 BP, and the length of N50 was 1412 BP. There are 23753 unigene in the transcript compared with the GO database, of which the three categories contain 56 groups of functions. 32194 unigene were compared and analyzed in NR protein database. The highest homologous protein sequence pairs were grape (Vitis vinifera) 39.5%, cocoa tree (Theobroma cacao) 10.6% and so on. There are 10374 unigene comments to the COG database, the most of which are gene function prediction (General function prediction only). There are 9550 unigene comments into the KEGG database, of which 381 unigene, ribosomal (Ribosome) have 381 unigene, ribosomal (Ribosome) and 360unigene. are transported by RNA. Through the differential expression analysis of the samples, there were 5141 differentially expressed genes, of which 3832 were up-regulated genes, accounting for 74.5% of the differentially expressed genes and 1309 down-regulated genes. It accounted for 25.5% of the differentially expressed genes. 2728 differentially expressed genes were annotated in GO. 56 groups of GO functional classification were obtained, and the partial (cell parts), of (cell), cells of (Celluar process), cells was obtained during the metabolic process of (Metabolic process), cells. 2. 2728 differentially expressed genes accounted for 25.5% of the differentially expressed genes. There are more unigene with catalytic activity (catalytic activity), binding (binding) and so on. Among the three types of topGO functional enrichment analysis, the response (response to cadmium ion) node of cell wall (cell wall), ferricopene combined with (heme binding), cadmium was the most significant. 1660 unigene comments were in the COG annotation classification. The most significant thing is that gene function predicts that (General function prediction only), genes have something in common and cluster together in the mechanism of action, thus speculating on gene function. There were 1500 differentially expressed genes in KEGG classification, and there were 148 metabolic pathways. The highest metabolic pathway was oxidative phosphorylation (Oxidative phosphorylation) and the results of total unigene KEGG enrichment analysis were consistent. According to the gene function, the key genes that regulate the flower opening of lotus' Qingyu 'were found out, and the genes related to ethylene synthesis, ACC and ERF;, which affected auxin synthesis, XTH and YUCCA;, were found. CKX gene regulating mitogen synthesis; EXP., a gene related to cell wall growth By real-time fluorescence quantitative Real-time PCR, the petals of 'Qingyu' and 'Chongyang' were used as materials in six periods of flower opening, bud stage, color conversion stage, bud stage, early flowering stage, full flowering stage and Xie flowering stage. For the four genes with high expression, the changes of expression at the bud stage and flowering stage of 'Qingyu' were basically consistent with those of the transcriptional group, which indicated that the sequencing data were true and reliable. In the six periods of flower blooming of 'Qingyu' and 'Chongyang', the expression of different genes was different during the whole flowering process, which indicated that different genes played different roles in flower opening, and it was speculated that different genes also played a regulatory role in flower type. This study revealed the key genes to control flower blooming and provided effective molecular data information for prolonged flowering and lotus breeding.
【学位授予单位】：河南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S682.32;Q943.2

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