贵紫麦1号籽粒色素形成相关基因的差异表达  

Differential Expression of Grain Pigment Related Genes of Guizimai No.1

作　　者：徐熙 任明见[1] 李鲁华 杨喜翠 徐如宏[1] XU Xi;REN MingJian;LI LuHua;YANG XiCui;XU RuHong(College of Agriculture, Guizhou University/Guizhou Sub-Center of National Wheat Improvement Center, Guiyang 55002)

机构地区：[1]贵州大学农学院/国家小麦改良中心贵州分中心,贵阳550025

出　　处：《中国农业科学》2018年第2期203-216,共14页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31660390);贵州省农业成果转化计划(黔科合成果(2016)4022号);国家"七大农作物育种"重点专项(2017YFD0100900);贵州省作物学省级重点学科建设计划(黔学位合字ZDXK[2014]8号);贵州省普通高等学校粮油作物遗传改良与生理生态特色重点实验室项目(黔教合KY字[2015]333)

摘　　要：【目的】探明贵紫麦1号小麦灌浆期变紫后和变紫前2个时期籽粒的转录组差异,发掘影响贵紫麦1号花青素合成的关键基因和关键酶,丰富小麦籽粒色素转录组数据信息,为转录因子的克隆及表达提供参考。【方法】利用Illumina Hiseq 2000TM高通量测序技术对贵紫麦1号籽粒变紫前和变紫后2个时期进行转录组测序、文库构建及建库质量评估,对测序结果进行信息学分析。采用TTM对read count数据进行标准化处理,随后用DEGseq进行差异分析,设定q-value<0.005且|log2(fold change)|>1为阈值。通过筛选分析,获得两者间差异表达基因,按照无参转录组分析方法,对差异表达基因进行BLAST搜索,Nr数据库比对,GO功能富集及KEGG pathway分析,找出与花青素相关的关键基因和关键酶,并结合q RT-PCR验证所找到的关键基因及关键酶在不同时期的表达水平,掌握这些关键基因的信息。【结果】测序结果表明,贵紫麦1号变紫后和变紫前分别获得13.36 G和12.69 G的clean bases,clean reads为106 906 108条和101 547 534条,占原始序列的93.73%和94.90%。通过Trinity软件对所得clean reads进行拼接,共获得170 396条转录本,长度为119 020 625。拼接clean reads后获得119 572条Unigenes。在BLAST搜索中,119 572个高质量独特序列中有86 004条(71.92%)Unigenes与现有基因模型具有至少1个显著匹配。在Nr数据库比对结果鉴定了至少5种具有与来自节节麦、乌拉尔图小麦、二穗短柄草、大麦、小麦等已知基因同一性且序列相似性高的Unigenes。KOG数据库比对结果显示,注释成功的基因按KOG的26个group进行分类,注释在一般功能基因,蛋白质翻译后修饰与转运、分子伴侣及翻译、核糖体结构与生物合成等类别基因所占比重较大,分别为15.79%、14.51%和10.54%。643个差异基因中,236个呈上调趋势,407个呈下调趋势。GO注释表明,按照基因参与的生物过程、所处的细胞组分、具【Objective】 The objective of this study is to investigate the transcriptome differences after and before purplechanging periods of grain at filling stage of Guizimai No.1, explore the key genes and enzymes that contribute to the biogenesis of anthocyanin, and then enrich the transcriptome data of grain pigment in wheat, provide references for the cloning and expression of the transcription factor. 【Method】 RNA-seq, construction library and quality assessment were carried out for two periods before and after purple-changing of Guizimai No.1 by using the Illumina Hiseq 2000 TM sequencing platform, and the sequencing results was analyzed by bioinformatics. TTM was used to standardize the read count data, then DEGseq was used to analyze the difference, and the q-value0.005 and ｜ log2（fold change） ｜1 were set as the threshold. The differential expression genes（DEGs） were obtained through selecting, in accordance with the transcriptome sequencing, then these DEGs were analyzed by BLAST search, NR annotated, GO functional enrichment and KEGG pathway method to find out the key genes and enzymes associated with anthocyanins, and combined q RT-PCR to verify the expression level of the key genes and key enzymes in different periods, finally the information of these key genes was mastered. 【Result】 The RNA-seq results showed that 13.36 G and 12.69 G clean bases were obtained, 106 906 108 and 101 547 534 clean reads accounted for 93.73% and 94.90% of the raw reads after and before purple-changing of Guizimai No.1, respectively. Clean reads were spliced by Trinity, totally 170 396 transcripts were obtained with a length of 119 020 625. There were 119 572 Unigenes after splicing clean reads. In the BLAST search, 86 004（71.92%） Unigenes out of 119 572 high quality unique sequences had at least one significant match to existing gene models. According to Unigenes＇ Nr database alignment, at least 5 Unigenes with similar gene identities and known sequence homologies to Aegilops tauschii, Triticum urartu

关 键 词：贵紫麦1号 籽粒 灌浆期 花青素 转录组 高通量测序 

分 类 号：S512.1[农业科学—作物学]