甜玉米乳熟期果皮厚度相关基因的加权基因共表达网络分析

doi:10.7668/hbnxb.20193397

摘要/Abstract

摘要：

为深入探究甜玉米果皮厚度发育的分子机制,利用华南农业大学甜玉米课题组选育的果皮厚度差异较大的甜玉米自交系M03和M08为试验材料,对其授粉后15,19,23 d的果皮进行转录组测序。联合差异表达基因分析与加权基因共表达网络(WGCNA)分析,鉴定甜玉米乳熟期果皮厚度相关的共表达基因模块,根据模块内基因的连接度鉴定核心基因,利用qRT-PCR验证核心基因表达量的可靠性。比较M03和M08不同时期果皮转录组数据共筛选出4 748个差异表达基因(DEGs),DEGs主要富集到碳水化合物代谢过程、植物-病原体相互作用、植物MAPK信号通路。WGCNA分析共构建了18个共表达模块,通过筛选得到4个具有生物学意义且与果皮厚度显著相关(相关系数的绝对值>0.60)的特异性共表达模块,分别是Turquoise、Yellow、Magenta和Pink模块。GO和KEGG功能富集分析结果均表明,特异性模块富集到的天冬氨酸、丙氨酸和谷氨酸代谢、半胱氨酸和蛋氨酸代谢以及植物MAPK信号通路在甜玉米果皮厚度发育中具有重要的作用。选取连接度最高的前20个基因进行核心基因筛选,通过基因功能注释最终筛选到包括MYB转录因子、细胞周期蛋白(CYC15)、β-淀粉酶、细胞凋亡调节基因(BCL-2)在内的13个核心基因。以核心基因为节点构建的共表达网络中还包括生长素转录因子(ARF、AUX/IAA)、乙烯反应元件结合因子(ERF)、亮氨酸拉链(bZIP)等转录因子,功能预测表明,这些基因可能在甜玉米果皮厚度调控中发挥重要作用。

关键词: 甜玉米, 果皮厚度, WGCNA, 转录组测序

Abstract:

To further explore the molecular mechanism of pericarp thickness development in sweet corn, pericarp from sweet corn inbred lines M03 and M08 were used for transcriptome sequencing,these materials provided by the sweet corn breeding laboratory of South China Agricultural University.RNA-sequencing material were sampled at 15,19 and 23 days after pollination.Genes’ differentially expressed analysis combined with weighted gene co-expression network(WGCNA)analysis were used to identify the co-expressed gene modules related to pericarp thickness at the milk stage of sweet corn,and the hub genes were also identified.Quantitative real time-PCR(qRT-PCR)was used to validate the expression level of the hub genes.Here,a total of 4 748 differentially expressed genes(DEGs)were identified by comparing different development stages and different samples.Gene annotation analysis revealed that these DEGs were mainly enriched in carbohydrate metabolic processes,plant-pathogen interactions,and plant MAPK signaling pathways.Co-expression analysis identified 18 modules,in which four modules(Turquois,Yellow,Magenta and Pink)were significantly associated with pericarp thickness.GO and KEGG functional enrichment analysis were performed based on these four specific modules,which could be enriched in alanine aspartate and glutamate metabolism,MAPK signaling pathway-plant and cysteine and methionine metabolism.The top 20 hub genes were screed by calculating the gene connectivity in the corresponding net-works,13 hub genes including MYB transcription factor,cell cycle protein(CYC15),β-amylase,apoptosis regulatory gene(BCL-2),etc.were finally screened by functional annotation.Transcription factors such as auxin response factor(ARF,AUX/IAA),ethylene response element binding factor(ERF),and leucine zipper(bZIP)were also included in the co-expression network,and these genes may play a crucial role in pericarp development process.

Key words: Sweet corn, Pericarp thickness, WGCNA, RNA-sequencing

裴虎, 熊才运, 张亚辉, 任文闯, 李小琴, 黄君. 甜玉米乳熟期果皮厚度相关基因的加权基因共表达网络分析[J]. 华北农学报, 2023, 38(2): 31-42. doi: 10.7668/hbnxb.20193397.

PEI Hu, XIONG Caiyun, ZHANG Yahui, REN Wenchuang, LI Xiaoqin, HUANG Jun. Identification of Gene Co-expression Modules Related to Sweet Corn Pericarp by Weighted Gene Co-expression Network Analysis[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(2): 31-42. doi: 10.7668/hbnxb.20193397.

http://www.hbnxb.net/CN/Y2023/V38/I2/31

图/表 12

图1 基因共表达网络软阈值的确定 B纵坐标代表每一个软阈值对应的网络平均连接程度;C、D.在power值为14时拓扑网络符合程度的检测。

Fig.1 Soft threshold determination of gene co-expression network The vertical coordinate of Fig.B represents the mean connectivity corresponding to each soft threshold; C and D.The detection of topological network compliance at a power value of 14.

表1 qRT-PCR 所用引物

Tab.1 Sequences of the primers used in qRT-PCR

模块 Module	基因名 Gene ID	正向引物(5’-3’) Forward primer(5’-3’)	反向引物(5’-3’) Reverse primer (5’-3’)
松石绿	Zm00001d005884	TGGTGGATCTGGCCCTGATA	AAAAAGTTTGCGGCTCCGTC
Turquoise	Zm00001d012892	CAGCTATCTCCCGCTTCCAG	CATGCCAGCTCGTCCATTTG
	Zm00001d023282	CTCCAGCACGGACAACATCT	TGTCAATGGCCGAGGAAGTC
黄色	Zm00001d014291	AGTCTCTTGTCCCGTCGAGA	AACTGCTGAGTCATCCTCGC
Yellow	Zm00001d012460	GTCCCTTGTGGAGACCTTGG	TCTCAAACGTCTCCCACTGC
	Zm00001d022227	GCCTAGCGTCTGTAGCCTTT	TTGCGGATTGCTCGTGTACT
	Zm00001d021396	GCTAGGTCCATTCCTTGGCA	ATGATGGGAGGGGCAAACAG
洋红色	Zm00001d005199	AAGGTTCCGCTGTCCCAAAT	TCGCAGTGCTTCTTCTCCTG
Magenta	Zm00001d041214	TGGCCATTTCCAGAAGGTCC	GAAGGACGAGGAGGTTGTCG
	Zm00001d048143	TCACCTCAGCGACAGTAGGA	AAATCACTCTACGGCCACCC
	Zm00001d027619	GGGTGACTGGGGTACGGTA	GCAGCAGAACATCAAACCCA
粉色	Zm00001d013572	CAGGCTCAGGGACGAGATTC	CTACCGGGCTGCTGTCATAG
Pink	Zm00001d042717	GGCAACACGAGTGGTTCCTT	CCCCGAAGCAGAGGAAGTAG
	ubiquitin	GCAGTGCTGCAGTTCTACAAGG	GCAGTAGTGGCGGTCGAAGTGG

图2 M08和M03在3个不同时期的籽粒果皮扫描电镜图片以及果皮厚度变化 A-C.M03授粉后15,19,23 d的果皮扫描电镜结果;D-F.M08授粉后15,19,23 d的扫描电镜结果;G.M03和M08的果皮厚度变化,柱中字母为 Duncan’s多重比较结果,不同字母表示材料间在P<0.05水平差异显著。

Fig.2 SEM of seed pericarp and changes in pericarp thickness of M08 and M03 at three different periods A-C.SEM results of 15,19 and 23 days after M03 pollination;D-F.SEM results of 15,19 and 23 days after M08 pollination;G.The change of pericarp thickness of M03 and M08.The letters in the column are Duncan’s multiple comparison results,different letters indicate significant difference between materials at the level of P<0.05.

表2 参试样品转录组数据质量

Tab.2 Quality of the transcriptome data of each sample

样品编号 Sample-ID	总数据 Total reads	待分析数据 Clean reads	GC含量/% GC content	≥Q30/%	比对率/% Mapped Reads	外显子/% Exon	内含子/% Intron	基因间区/% Intergenic
M03-DAP15-1	54 442 418	27 221 209	54.81	93.39	81.92	91.27	2.25	6.48
M03-DAP15-2	52 524 708	26 262 354	54.29	93.20	83.83	90.93	2.44	6.63
M03-DAP15-3	57 364 228	28 682 114	55.26	93.71	84.50	91.97	1.98	6.05
M03-DAP19-1	52 218 286	26 109 143	55.43	93.69	82.08	91.79	2.02	6.19
M03-DAP19-2	47 828 436	23 914 218	54.29	93.72	81.74	91.68	2.07	6.25
M03-DAP19-3	49 153 314	24 576 657	55.15	93.55	83.17	91.80	2.01	6.19
M03-DAP23-1	54 265 260	27 132 630	55.51	93.42	82.95	90.90	2.35	6.75
M03-DAP23-2	52 659 674	26 329 837	55.80	93.61	83.61	90.85	2.64	6.51
M03-DAP23-3	52 420 156	26 210 078	55.05	93.49	83.15	90.90	2.35	6.75
M08-DAP15-1	52 932 600	26 466 300	54.90	93.56	83.07	90.32	2.54	7.14
M08-DAP15-2	54 342 162	27 171 081	53.42	93.46	78.82	87.38	3.37	9.25
M08-DAP15-3	52 076 478	26 038 239	53.57	93.11	80.06	88.01	3.42	8.57
M08-DAP19-1	51 359 308	25 679 654	55.93	94.02	83.75	91.34	2.31	6.35
M08-DAP19-2	51 528 224	25 764 112	55.21	94.24	83.68	90.58	2.63	6.79
M08-DAP19-3	54 932 698	27 466 349	55.04	94.25	83.78	90.88	2.52	6.60
M08-DAP23-1	57 996 594	28 998 297	54.98	93.76	82.94	90.69	2.47	6.84
M08-DAP23-2	57 301 120	28 650 560	55.10	94.39	83.10	90.80	2.40	6.80
M08-DAP23-3	55 910 494	27 955 247	54.61	94.00	83.59	90.71	2.44	6.85

图3 不同材料不同时期DEGs数量统计及韦恩图

Fig.3 Number of DEGs in different datasets and venn diagram

图4 DEGs的GO富集和KEGG富集分析 A.3个不同颜色柱状图分别代表GO富集的三大类;B.纵轴代表富集的 KEGG 通路,横轴代表富集基因的比率,点大小代表路径中富集的基因数,颜色代表多重测试校正的 P 值。图7同。

Fig.4 GO and KEGG enrichment analysis of DEGs A.Three different color histograms respectively represent the three categories of GO enrichment;B.The vertical axis represents the enriched KEGG pathway,and the horizontal axis represents the ratio of enriched genes,the point size represents the number of genes enriched in the path,and the color represents the P value corrected by multiple tests.The same as Fig.7.

图5 基因共表达模块以及各模块所包含的基因数 A.基因聚类树,每个聚类树对应一个基因模块,聚类等级相同的模块用同样的颜色表示; B.聚类出的18个模块以及每个模块所包含基因个数。

Fig.5 Gene co-expression module and the number of genes contained in each module A. A gene cluster tree,each cluster tree corresponds to a gene module,and with the same clustering level are represented by the same color;B.The 18 modules clustered and the number of genes contained in each module.

图6 表型-模块相关性热图和模块-DEGs共有基因数 A.各模块与果皮厚度之间的相关系数热图,红色框为特异性模块;B.特异性模块与DEGs共有基因数。

Fig.6 Phenotype-module correlation heatmap and number of genes shared by significant modules and DEGs A.The heat map of correlation coefficient between each module and peel thickness,and the red box is the specific module; B.The number of genes shared by specific modules and DEGs.

图7 特异性模块KEGG富集分析

Fig.7 KEGG enrichment analysis of significant module

表3 模块中核心基因在拟南芥中的同源基因功能注释

Tab.3 Functional notes of homologous genes of hub genes in module in Arabidopsis

模块 Module	核心基因 Hub gene	拟南芥中的同源基因 Homologous in A. thaliana	基因功能 Gene function
黄色	Zm00001d014291	AT4G33500	蛋白磷酸酶2C家族蛋白
Yellow	Zm00001d012460	AT3G51780	拟南芥BAG家族蛋白,是哺乳动物细胞凋亡调节器的植物同源物
	Zm00001d022227	AT3G47600	Myb结构域蛋白94
	Zm00001d021396	AT5G20380	编码一种无机磷酸盐转运体(PHT4;5)
松石绿	Zm00001d005884	AT1G49720.1	脱落酸反应元件结合因子1
Turquoise	Zm00001d012892	AT3G05420.2	酰基辅酶A结合蛋白4
	Zm00001d023282	AT3G28910.1	Myb结构域蛋白30
洋红色	Zm00001d005199	AT2G42960.1	蛋白激酶超家族蛋白
Magenta	Zm00001d041214	AT3G47950	质膜H+ATP酶
	Zm00001d048143	AT1G70210.1	细胞周期蛋白D1;1
	Zm00001d027619	AT3G23920.1	编码叶绿体β-淀粉酶
粉色	Zm00001d013572	AT4G23140.2	富含半胱氨酸的RLK(受体样蛋白激酶)6
Pink	Zm00001d042717	AT5G61890.1	编码ERF/AP2转录因子家族的ERF(乙烯反应因子)亚家族B-4的一个基因

图8 特异性模块的基因共表达网络及其核心基因 A~D分别为Turquoise模块、Yellow模块、Pink模块、Magenta模块的基因网络;红色大点为核心基因,灰色矩形为模块内与表型相关基因的标注。

Fig.8 Gene co-expression network and hub genes of significant modules A-D are the gene networks of Turquoise module,Yellow module,Pink module and Magenta module respectively; the large red dot is the hub gene,and the gray rectangle is the annotation of phenotype related genes in the module.

图9 核心基因表达量的qRT-PCR验证柱状图表示qRT-PCR结果,折线图表示RNA-seq结果;横坐标中3和8分别代表M03和M08,a,b,c分别代表授粉后15,19,23 d;柱中字母为 Duncan’s多重比较结果,不同字母表示材料间在P<0.05水平差异显著。

Fig.9 qRT-PCR validation of hub genes The bar graph represents the qRT-PCR results,and the broken line graph represents the RNA-seq results;in the abscissa,3 and 8 represent M03 and M08 respectively,and a,b and c represent 15,19 and 23 day after pollination respectively.The letters in the column are Duncan’s multiple comparison results,different letters indicate that there is a significant difference between materials at the level of P<0.05.

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