紫花苜蓿不同发育时期次生壁合成调控的转录组分析

doi:10.3864/j.issn.0578-1752.2018.11.003

摘要/Abstract

摘要： 【目的】探讨紫花苜蓿次生壁合成的基因网络调控变化和表达模式，确定一些关键候选基因和转录因子，为紫花苜蓿次生壁加厚调控网络的分子机制研究奠定基础。【方法】对‘中苜1号’紫花苜蓿分枝期（S1）、现蕾期（S2）、初花期（S3）和盛花期（S4）的茎进行近红外光谱法测定次生壁主要物质含量和Illumina HiSeq^TM 2500高通量转录组测序。以紫花苜蓿的近缘物种蒺藜苜蓿（Medicago truncatula）基因组作为参考基因组进行序列比对并组装构建转录本。利用FPKM法计算基因表达量，以Fold change（差异表达倍数）≥2或≤0.5（表达上调或下调）、FDR（false discover rate）≤0.05为筛选条件，在3个相邻时期转录组比较组合中（S2vs S1，S3 vs S2，S4 vs S3）筛选差异表达基因。通过Gene Ontology数据库和KEGG Pathway数据库对紫花苜蓿不同发育时期差异表达基因的功能和参与的代谢途径进行注释。【结果】共获得41 734个基因在紫花苜蓿不同发育时期的转录表达情况，27个功能注释与紫花苜蓿纤维素、木质素合成密切相关的基因差异表达，其变化趋势与纤维素和木质素含量测定结果基本一致，即随着生长发育时期的变化，表达水平逐渐提高。研究表明，初花期是紫花苜蓿次生壁合成调控的转折期，纤维素和木质素含量与其合成基因表达量在初花期均显著提高。MTR_2g016630（Ces）和MTR_7g103590（Ces A1）等纤维素合成酶基因表达水平在初花期显著上升，木质素合成途径中，MTR_1g064090（PAL1）、MTR_1g111240（C4H）和MTR_2g104960（CCR）基因表达量在初花期或盛花期相比分枝期上调表达倍数达到10倍以上。本研究中27个与植物生长发育相关的转录因子在紫花苜蓿不同发育时期差异性表达，其中NAC家族和MYB家族转录因子有18个，也有少量WRKY、BHLH、ERF、C3H等转录因子。【结论】获得‘中苜1号’紫花苜蓿在4个生长发育时期茎的基因表达谱数据，共获得54个差异表达基因，其中稳定上调基因24个，稳定下调基因30个。这些基因很有可能参与紫花苜蓿次生壁的合成调控。

关键词: 紫花苜蓿, 发育时期, 次生壁, 转录组分析, 转录因子

Abstract: 【Objective】The aim of the study was to investigate the gene network regulation dynamics and expression pattern of secondary cell wall synthesis and to identify the key candidate genes and transcription factors in regulatory network for the understanding of molecular mechanism of the secondary cell wall thickening in alfalfa.【Method】The stem samples collected at vegetative stage (S1), flower bud stage (S2), early flower stage (S3) and late flower stage (S4) were used to determine the main substance contents in the secondary cell wall of the ‘Zhongmu No.1’ cultivar of alfalfa using a near-infrared-reflectance spectroscopy. The high-throughput transcriptome sequencing of these tissues were performed using an Illumina HiSeq^TM 2500 platform. The genome of Medicago truncatula was used as a reference to align the sequences and construct the Unigenes. The gene expression level was calculated in the term of Fragments Per Kilobase of transcript per Million fragments (FPKM). The thresholds to judge the significance of gene expression differences between three adjacent growth periods (S2 vs S1, S3 vs S2 and S4 vs S3) were set as fold change ≥2 or≤0.5 for up or down regulation with a false discovery rate (FDR) ≤ 0.001. The Gene Ontology database and KEGG Pathway database were used to annotate the functions and metabolic pathways of differentially expressed genes. 【Result】A total of 41 734 Unigenes were identified at different developmental stages of alfalfa in this study, of which 27 differentially expressed genes (DEGs) were annotated with functions closely related to cellulose and lignin biosynthesis. The expression levels of these DEGs gradually increased along with the accumulation of cellulose and lignin over the developmental stages. The early flower stage was found to be a transitional period at which the contents of cellulose and lignin in the secondary cell wall and expression of synthetic genes were all increased significantly. For instance, the expression of cellulose synthase genes of MTR_2g016630 (Ces) and MTR_7g103590 (Ces A1) were significantly increased at the early flower stage, the expression of lignin biosynthesis genes of MTR_1g064090 (PAL1), MTR_1g111240 (C4H) and MTR_2g104960 (CCR) were significantly up-regulated more than 10 times at the early or late flower stage compared to the vegetative stage. A total of 27 transcription factors were differentially expressed at different developmental stages of alfalfa which were related to plant growth and development, of which 18 belonged to NAC and MYB transcription factor family while the rest were of WRKY, BHLH, ERF and C3H transcription factor family.【Conclusion】We acquired the gene expression profiling data of the stem at four developmental stages of the ‘Zhongmu No. 1’ cultivar of alfalfa. A total of 54 DEGs were identified, of which 24 were up-regulated while 30 were downregulated. These genes were postulated to be involved in the synthesis and regulation of the secondary cell wall in alfalfa.

Key words: Medicago sativa;developmental stage, secondary cell wall, transcriptome analysis, transcription factor

刘希强，张涵，龚攀，宫文龙，王赞. 紫花苜蓿不同发育时期次生壁合成调控的转录组分析[J]. 中国农业科学, 2018, 51(11): 2049-2059.

LIU XiQiang, ZHANG Han, GONG Pan, GONG WenLong, WANG Zan. Transcriptome Analysis of Secondary Cell Wall Synthesis Regulation at Different Developmental Stages in Alfalfa (Medicago sativa L.)[J]. Scientia Agricultura Sinica, 2018, 51(11): 2049-2059.

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