镉胁迫下地钱转录组的性别特异性响应机制

doi:10.11983/CBB21189

摘要/Abstract

摘要： 地钱(Marchantia polymorpha)作为雌雄异株的苔纲植物, 具有对重金属胁迫敏感的特性, 且种群中雌雄比例差异较大, 是研究重金属环境下植物性二态的理想对象之一。为探究雌雄地钱转录组对重金属镉的响应机制差异, 利用高通量测序和加权基因共表达网络分析(WGCNA), 鉴定了在镉胁迫条件下与不同性别地钱有关的模块和基因。结果表明, 镉胁迫响应转录因子雌雄差异主要集中于bHLH、ERF和MYB家族。关键差异响应基因包括MARPO_0147s0038、MARPO_1326s0001和MARPO_0015s0058。差异响应通路雌性主要集中在信号转导通路, 雄性则集中于类黄酮生物合成。研究结果有助于揭示雌雄异株地钱对镉胁迫的反应过程和响应机制, 可为理解雌雄异株植物对重金属胁迫的性别特异性响应机制提供参考。

关键词: 地钱, 镉胁迫, 重金属, 转录组测序, 加权基因共表达网络分析

Abstract: Marchantia polymorpha, a dioecious bryophyte, is characterized by hypersensitivity to heavy metal stress. There is a great difference in proportion of female and male in population, which means M. polymorpha is an ideal object to study plant sexual dimorphism under heavy metal stress. We used high-throughput sequencing and weighted gene co-expression network analysis (WGCNA) to explore the differences in the transcriptomes of M. polymorpha between male and female in response to the cadmium (Cd). We identified modules and genes associated with different sexes under Cd stress. The results showed that transcription factors differentially responsive to Cd stress were mainly the members of bHLH, ERF and MYB family, and some hub genes such as MARPO_0147s0038, MARPO_1326s0001, and MARPO_0015s0058. The differentially responsive pathways were mainly focused on signaling pathways in females and flavonoid biosynthesis pathway in males. These findings will help to reveal the process and mechanism of dioecious M. polymorpha response to Cd stress and provide a reference for understanding the sex-specific response mechanism of dioecious plants to heavy metal stress.

Key words: Marchantia polymorpha, cadmium stress, heavy metal, RNA-seq, weighted gene co-expression network analysis (WGCNA)

徐海霞, 何静, 易航, 王丽. 镉胁迫下地钱转录组的性别特异性响应机制. 植物学报, 2022, 57(2): 182-196.

Haixia Xu, Jing He, Hang Yi, Li Wang. Sex Specific Response Mechanism of Transcriptome in Both Male and Female Marchantia polymorpha Under Cadmium Stress. Chinese Bulletin of Botany, 2022, 57(2): 182-196.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB21189

https://www.chinbullbotany.com/CN/Y2022/V57/I2/182

图/表 11

表1 qRT-PCR引物序列

Table 1 Primer sequences for qRT-PCR

Gene names	Primer sequences (5'-3')
MpPCS	F: GAGTCCTTTCGAGCGAATTCT
MpPCS	R: AATCTTCCAGCAGTAGCAGTAG
MpActin	F: AGGCATCTGGTATCCACGAG
MpActin	R: ACATGGTCGTTCCTCCAGAC
MARPO_0086s0070	F: ATTGTCACTTCAAACTGCTTCC R: CTTTGCCAGTATGCTTTCAACT
MARPO_0265s0002	F: TGGCGACTCTTAAAATAGCTCT R: GAAAGTATGGGATTCTTGCGAC
MARPO_0001s0061	F: GAGGAAAATCAACGATACAGGC R: CTTCTCTTGCAGTCAGTTGTTC
MARPO_0145s0031	F: CGTAGGGAGATCAGTGATTACC R: CCTTGTATAACTCACGATCGGA
MARPO_0003s0122	F: TGGACAGAAAACTCATTGGTCT R: ACTTCCTTCTCTCTCACCAATG
MARPO_0003s0268	F: GACTAGGGTTCCTTGGTTCTAC R: CATAATCGGGAAAACGTAACCC
MARPO_0011s0046	F: CGGCTCAGATGTTGATAAATCA R: ATGCACTTCGCTGATCTCATAA
MARPO_0032s0085	F: CCCCTCATCTCAAACTACTTGT R: TTGGAAAAGGAATTGCTGGATG

图1 镉处理后雌性(A)和雄性(B)地钱形态设置处理后0、24、48、96和168小时5个时间节点。Bars=2 cm

Figure 1 Morphology of female (A) and male (B) Marchantia polymorpha after cadmium (Cd) treatment Five time points after Cd treatment: 0, 24, 48, 96 and 168 h. Bars=2 cm

表2 转录组质控数据

Table 2 Transcriptome quality control data

Sample_ID	Raw reads	Clean reads	Error (%)	Q20 (%)	Q30 (%)	GC (%)	Total mapped (%)
CK_F1	50740902	50191272	0.0241	98.38	95.02	50.92	94.74
CK_F2	58049938	57506790	0.0242	98.36	94.95	50.98	89.75
CK_F3	53602814	53058498	0.0245	98.23	94.63	51.23	95.59
CK_M1	49826850	49313610	0.0243	98.31	94.84	51.14	94.22
CK_M2	52605578	51996578	0.0245	98.25	94.67	51.13	91.95
CK_M3	50064978	49523028	0.0242	98.36	94.96	50.94	90.51
Cd_F1	53604084	53084914	0.0241	98.40	95.03	51.02	94.65
Cd_F2	48343374	47896356	0.0255	97.87	93.60	50.53	95.16
Cd_F3	48556392	48058072	0.0247	98.15	94.43	50.90	96.26
Cd_M1	51066022	50567610	0.0244	98.28	94.75	50.66	87.88
Cd_M2	51079094	50542790	0.0244	98.28	94.75	50.48	90.25
Cd_M3	54825492	54241196	0.0249	98.06	94.19	50.61	93.78

图2 雌雄地钱样本间相关性分析 (A) 雌性和雄性样本间平均基因表达水平与总体连接度的相关性散点图(雌性作为x轴, 雄性作为y轴); (B) 样本间相关性的聚类热图

Figure 2 Correlation analysis among female and male Marchantia polymorpha samples (A) Scatter plot of correlation about average level of gene expression and overall connectivity between female and male samples (females as x-axis and males as y-axis); (B) Clustered heatmap of correlation among samples

图3 雌雄地钱响应镉胁迫差异表达基因统计 (A) 差异表达基因(DEGs)数目柱状图; (B) 不同变化趋势的差异基因数目韦恩图

Figure 3 Statistics of differentially expressed genes between female and male Marchantia polymorpha in response to cadmium stress (A) Histograms of differentially expressed genes (DEGs); (B) Venn diagram of number of differential genes with different changing trends

图4 雌雄地钱响应镉胁迫差异表达转录因子

Figure 4 Differentially expressed transcription factors between female and male Marchantia polymorpha in response to cadmium stress

图5 雌雄地钱响应镉胁迫的加权基因共表达网络分析 (A) 基因与模块的聚类树状图; (B) 模块与表型之间的相关性热图。红色表示正相关, 蓝色表示负相关。

Figure 5 Weighted gene co-expression network analysis of female and male Marchantia polymorpha response to cadmium stress (A) Cluster dendrogram of genes and modules; (B) Heatmap of correlation between modules and phenotypes. Red indicates positive correlation and blue indicates negative correlation.

图6 目标模块的富集分析 (A) salmon模块前10条GO条目富集结果; (B) black模块前10条GO条目富集结果; (C) salmon模块前10条KEGG条目富集结果; (D) black模块前10条KEGG条目富集结果

Figure 6 Enrichment analysis of target modules (A) Top ten GO enrichment of salmon module; (B) Top ten GO enrichment of black module; (C) Top ten KEGG enrichment of salmon module; (D) Top ten KEGG enrichment of black module

图7 salmon模块和black模块的共表达网络图和核心基因圆点直径从小到大、从浅色到深色代表degree逐渐增大。

Figure 7 Co-expression network diagram and hub genes of the salmon module and black module The diameter of dot from small to large, with light to dark colors represents the increasement in degree.

表3 salmon模块和black模块中核心基因功能注释

Table 3 Functional annotation of hub genes in salmon module and black module

Module	Gene names	Protein names
black	MARPO_0147s0038	Phytocyanin domain-containing protein
black	MARPO_1326s0001	ANK_REP_REGION domain-containing protein
black	MARPO_0015s0058	NAC domain-containing protein
black	MARPO_0068s0011	Hydrolase_4 domain-containing protein
black	MARPO_0034s0124	Protein kinase domain-containing protein
salmon	MARPO_4159s0001	Protein kinase domain-containing protein (fragment)
salmon	MARPO_0097s0006	SCP domain-containing protein
salmon	MARPO_0047s0057	FAD-binding FR-type domain-containing protein
salmon	MARPO_0009s0238	S-protein homolog
salmon	MARPO_0047s0055	FAD-binding FR-type domain-containing protein

图8 基因表达半定量分析及表达模式验证 (A) 半定量RT-PCR检测4个样本中marker基因(MpPCS)的转录, MpPCS为33个PCR周期, MpAct (作为内参基因)为27个PCR周期; (B) 通过qRT-PCR验证表达数据

Figure 8 Semi-quantitative analysis of gene expression and expression pattern validation (A) Semi-quantitative RT-PCR was used to detect transcription of marker genes (MpPCS) in four samples, 33 PCR cycles for MpPCS, 27 PCR cycles for MpAct (as an internal reference gene); (B) The expression data were verified by qRT-PCR

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