基于WGCNA鉴定全缘叶绿绒蒿类黄酮合成途径关键基因

doi:10.3864/j.issn.0578-1752.2024.15.011

摘要/Abstract

摘要：

【目的】黄酮类化合物具有抗炎、抗癌、抑菌等多种功效，是全缘叶绿绒蒿的主要药用成分之一。通过分析全缘叶绿绒蒿不同部位的空间代谢组与转录组信息，挖掘调控黄酮类化合物合成的关键基因，为研究全缘叶绿绒蒿类黄酮合成机制提供理论参考，并为提高类黄酮含量遗传育种奠定分子基础。【方法】以全缘叶绿绒蒿的根、茎、叶和花瓣为材料，对不同部位进行转录组测序，并通过空间代谢组数据分析黄酮类化合物在不同部位中的分布情况，利用加权基因共表达网络分析（WGCNA）鉴定出与黄酮类化合物合成密切相关的关键模块和关键基因。另外，挑选12个基因进行qRT-PCR，验证转录组数据的可靠性。【结果】全缘叶绿绒蒿中黄酮类化合物在不同部位呈现差异积累，花瓣是黄酮类化合物积累的主要部位，同时明确了8种主要黄酮类化合物。转录组测序共获得20 085个表达基因，仅在花中表达的基因有286个，是仅在其他部位中表达基因数量的3.6-4.2倍。利用WGCNA对过滤后的高表达差异基因进行划分，共获得14个共表达模块，确定了关键模块MEturquoise和MEgreen与8个主要黄酮类化合物显著相关（P<0.05）。KEGG分析发现这两个模块基因主要在代谢相关的通路中富集，在黄酮类化合物合成相关通路上也有基因富集，两个模块分别包含了18个和6个与黄酮类化合物合成相关的基因，并从模块中筛选到14个核心结构基因（5个CHS、2个HIDH、2个CCoAOMT以及FLS、CYP75B1、CHI、HCT和CYP73A）和1个转录因子HB2，这些基因多在花瓣或是茎中高表达。qRT-PCR所测基因表达变化趋势与转录组基本一致，表明利用该转录组数据所得出的分析结果可信。【结论】全缘叶绿绒蒿黄酮类化合物积累和基因表达在不同器官间具有显著差异，联合分析筛选到与黄酮类化合物积累密切相关的14个核心结构基因和1个转录因子，这些基因可能在调控全缘叶绿绒蒿不同器官黄酮类化合物的合成和差异积累过程中起到关键作用。

关键词: 全缘叶绿绒蒿, 类黄酮, 药用, 关键基因, 加权基因共表达网络分析（WGCNA）

Abstract:

【Objective】Flavonoids are known for their anti-inflammatory, anti-cancer, and antibacterial properties, and are one of the main medicinal components of Meconopsis integrifolia. By analyzing the spatial metabolome information and transcriptome data from various parts (roots, stems, leaves and petals) of M. integrifolia, the key genes regulating the flavonoid synthesis could be excavated. This research could provide valuable insights into the mechanism underlying flavonoids synthesis in M. integrifolia, paving the way for genetic breeding aimed at enhancing flavonoid content. 【Method】Transcriptome sequencing was conducted on the root, stem, leave and petal of M. integrifolia to analyze the distribution of flavonoids across different parts using spatial metabolomic data. The weighted gene co-expression network analysis (WGCNA) was employed to identify key modules and key genes closely related to flavonoid synthesis. To validate the reliability of the transcriptome data, 12 genes were selected for qRT-PCR analysis. 【Result】Flavonoids accumulation in M. integrifolia varied across different parts, with petals being the primary site of accumulation, where 8 main flavonoids were identified. Transcriptome sequencing revealed a total of 20 085 expressed genes, among which 286 genes expressed were exclusively expressed in flowers, showing 3.6-4.2 times more expression than that in other plant parts. Using WGCNA to categorize highly expressed differential genes, a total of 14 co-expression modules were identified, and the key modules, including MEturquoise and MEgreen, were significantly associated with 8 main flavonoids (P<0.05). KEGG analysis demonstrated that the genes within these two modules were primarily enriched in metabolism-related pathways, with some genes enriched in pathways related to flavonoid synthesis. MEturquoise and MEgreen comprised 18 and 6 genes related to flavonoid synthesis, respectively, and screened 14 core structural genes (5 CHSs, 2 HIDHs, 2 CCoAOMTs and FLS, CYP75B1, CHI, HCT, and CYP73A) and one transcription factor HB2, which were predominantly highly expressed in petals or stems. The consistent gene expression trends between qRT-PCR and transcriptome data were observed, which showed that the analysis results derived from the transcriptome data were reliable. 【Conclusion】The accumulation of flavonoids and gene expression patterns in different organs of M. integrifolia varied significantly, and 14 core structural genes and one transcription factor were screened to be closely related to the accumulation of flavonoids across different organs. These genes might play a key role in regulating the synthesis and differential accumulation of flavonoids in different organs of M. integrifolia.

Key words: Meconopsis integrifolia, flavonoids, medicinal, key genes, WGCNA

陈晓涓, 王海菊, 王富敏, 雍清青, 黄顺满, 屈燕. 基于WGCNA鉴定全缘叶绿绒蒿类黄酮合成途径关键基因[J]. 中国农业科学, 2024, 57(15): 3053-3070.

CHEN XiaoJuan, WANG HaiJu, WANG FuMin, YONG QingQing, HUANG ShunMan, QU Yan. Identification of Key Genes in the Flavonoid Synthesis Pathway of Meconopsis integrifolia Based on WGCNA[J]. Scientia Agricultura Sinica, 2024, 57(15): 3053-3070.

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https://www.chinaagrisci.com/CN/Y2024/V57/I15/3053

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非类黄酮合成通路内的核心基因功能注释"

核心基因 Hub gene	核心基因在拟南芥的同源基因 Hub gene in A. thaliana	基因功能 Gene function
QYY_transcript_8026	AT2G34410.2	编码已知参与新型隐球菌多糖O-乙酰化的蛋白质Cas1p的同源物。该蛋白质在高尔基体中表达，并参与次生壁生物合成过程中木聚糖的乙酰化 Encodes a homolog of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. The protein is expressed in the Golgi and is involved in the acetylation of xylan during secondary wall biosynthesis
QYY_transcript_7230	AT5G03760.1	编码农杆菌介导的植物遗传转化所需的β-甘露聚糖合酶涉及细菌和宿主植物之间的复杂相互作用。3' UTR参与转录调控，该基因在根的伸长区表达 Encodes a beta-mannan synthase that is required for agrobacterium-mediated plant genetic transformation involves a complex interaction between the bacterium and the host plant. 3' UTR is involved in transcriptional regulation and the gene is expressed in the elongation zone of the root
QYY_transcript_22662	AT4G05530.1	编码短链脱氢酶/还原酶（SDR）酶家族的过氧化物酶体成员。IBR1功能的丧失导致对吲哚-3-丁酸的抗性增加，而不影响植物对IAA、NAA和2,4-D的反应。该酶可能负责催化IBA向IAA的β氧化样转化中的脱氢步骤 Encodes a peroxisomal member of the short-chain dehydrogenase/reductase (SDR) family of enzymes. Loss of IBR1 function causes increased resistance to indole-3-butyric acid without affecting plant responses to IAA, NAA, and 2,4-D. This enzyme may be responsible for catalyzing a dehydrogenation step in the beta- oxidation-like conversion of IBA to IAA
QYY_transcript_20137	AT4G24330.1	假设性蛋白质（DUF1682） Hypothetical protein (DUF1682); (source:Araport11)
QYY_transcript_21126	AT5G17770.1	编码NADH：细胞色素（Cyt）b5还原酶，该酶对NADH对重组Cyt b5（AtB5-A）的还原具有严格的特异性，而当NADPH用作电子供体时，未观察到Cyt b5还原 Encodes NADH:cytochrome (Cyt) b5 reductase that displayed strict specificity to NADH for the reduction of a recombinant Cyt b5 (AtB5-A), whereas no Cyt b5 reduction was observed when NADPH was used as the electron donor
QYY_transcript_7030	-	-

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表6

两个关键模块内的关键转录因子"

模块 Module	基因 Gene	基因ID Gene ID	转录因子家族 Transcription factor family	功能 Function
MEturquoise	COL13	QYY_transcript_15942	C2C2-CO-like	拟南芥：与HY5和COL3形成HY5-COL3-COL13模块调控拟南芥下胚轴的伸长^[22] A. thaliana: COL13 forms a HY5-COL3-COL13 module with HY5 and COL3 to regulate the elongation of hypocotyls in A. thaliana
	COL5-1	QYY_transcript_19756	C2C2-CO-like	拟南芥：过表达COL5可诱导短日照生长拟南芥开花^[23] A. thaliana: Overexpression of COL5 can induce short-day growth of A. thaliana
	COL5-2	QYY_transcript_20889	C2C2-CO-like	拟南芥：过表达COL5可诱导短日照生长拟南芥开花^[23] A. thaliana: Overexpression of COL5 can induce short-day growth of A. thaliana
	HB2	QYY_transcript_18216	HB-HD-ZIP	拟南芥：负向调控茶树花青素合成酶基因CsANS，同时AtHB2可能与AtPAP1在结合AtTTG1上存在竞争，进而影响AtPAP1参与的类黄酮合成^[24] A. thaliana: AtHB2 negatively regulates the tea tree anthocyanin synthetase gene CsANS, and AtHB2 may compete with AtPAP1 for binding to AtTTG1, which in turn affects the flavonoid synthesis involved in AtPAP1
	MYBH	QYY_transcript_19945	MYB-related	拟南芥：通过增强生长素积累来调节下胚轴伸长^[25] A. thaliana: MYBH regulates hypocotyl elongation by enhancing auxin accumulation
	NAC22	QYY_transcript_20070	NAC	水稻：提高对干旱和盐害的耐性^[26] O. Sativa: Increased tolerance to drought and salinity
	BBX19	QYY_transcript_22992	DBB	拟南芥：BBX19与PRR相互作用以协调昼夜节律^[27] A. thaliana: BBX19 interacts with PRR to harmonize circadian rhythms
	ARF6	QYY_transcript_3258	B3-ARF	拟南芥：ARF6与ARF8共同促进茉莉酸的产生和花的成熟^[28] A. thaliana: ARF6 and ARF8 work together to promote the production of jasmonic acid and the maturation of flowers
	PRR95	QYY_transcript_6993	Pseudo ARR-B	水稻：作为转录抑制因子负调控编码叶绿体定位的Mg²⁺转运蛋白的OsMGT3的节律表达^[29] O. sativa: As a transcriptional repressor, it negatively regulates the rhythmic expression of OsMGT3, which encodes a chloroplast-localized Mg²⁺ transporter
	ZHD1	QYY_transcript_15114	zf-HD	水稻：过表达OsZHD1可诱导水稻叶片轴向卷曲和下垂^[30] O. sativa: Overexpression of OsZHD1 induces axial curling and drooping of rice leaves
	-	QYY_transcript_20217	其他Others	未知功能 Unknown features
MEgreen	HB13	QYY_transcript_20535	HB-HD-ZIP	拟南芥：参与生物和非生物抗性途径^[31] A. thaliana: Involved in biotic and abiotic resistance pathways

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基因/基因ID Gene/Gene ID	上游引物 Forward primer sequence (5′-3′)	下游引物 Reverse primer sequence (5′-3′)
CHS5	GATCATCGGAGCAGACCCTG	ACCAGGATGAGCGATCCAGA
CHS8	TCATCGGAGCAGACCCTGAT	TACAGTGGAGTGGGCTTTGG
CYP75B1	TACAGTGGAGTGGGCTTTGG	GGAAGGGAAAGTGGCGTTGA
FLS	GTTGCCGCTCGAAGAAAAGG	CTCCTCGTTTGCTTCCCTGT
CCoAOMT1	CAGCAATCGACGTTAGTCGG	CGATGCCACCAACTCTCAAC
HIDH1	GGCAGCGTTAAACTGGGTTC	TTCAGGGTTAGTTCCGGCTC
HB2	CTTCTCGGAGCGGTGGTAAT	ACTTGGTCCTTGCCCTTCTG
HCT3	CTGATGGAGCGTCTGGTCTC	AGTGTGTTGAGCTGGTCTCG
QYY_transcript_10050	TGACGCACAAATTGCATCCG	TCACAACAGCATCTCCGTCC
QYY_transcript_10042	CAGGTCGTTTTGGTGTGACG	CTTTGCCCTGGGATTGACCT
QYY_transcript_10301	TGACGGAAGCACTCAGGATG	CTCCACAGCAGCCTTTGTTG
QYY_transcript_10319	TGGCCAAGACATTTGAGGGT	GACACCTCCAACTACGGCAT
GAPDH	TTCCGTGTTCCTACCGTTGA	TCCCTTCATCTTACCCTCGG

样品 Sample	原始序列 Raw reads	过滤序列 Clean reads	过滤碱基 Clean base (G)	错误率 Error rate (%)	Q30 (%)	GC含量 GC content (%)
QYY1-P	48823172	47635926	7.15	0.03	92.59	41.23
QYY2-P	46197126	44753484	6.71	0.03	92.39	41.18
QYY3-P	48061136	46960540	7.04	0.03	92.97	41.03
QYY1-R	47342122	45955834	6.89	0.03	92.82	41.81
QYY2-R	46427954	45210284	6.78	0.03	93.45	41.65
QYY3-R	47136742	45743182	6.86	0.03	93.42	42.13
QYY1-S	47689492	46405540	6.96	0.03	93.03	41.98
QYY2-S	47008018	45681508	6.85	0.03	92.3	41.62
QYY3-S	48115292	46505116	6.98	0.03	93.61	41.83
QYY1-L	43879048	42694332	6.4	0.03	93.92	42.89
QYY2-L	47482914	46435172	6.97	0.03	93.46	42.1
QYY3-L	47789072	46216224	6.93	0.03	93.27	42.7
总计 Total	565952088	550197142	82.52	-	-	-

数据库 Database	注释到的基因数量 Number of genes	占比 Percentage (%)
KEGG	16322	81.26
Nr	18891	94.06
SwissProt	16369	81.50
TrEMBL	19109	95.14
KOG	12894	64.20
GO	12894	64.20
Pfam	17683	88.04
至少在一个数据库中被注释 Annotated in at least one Database	20085	100.00
合计Unigenes数 Total Unigenes	20085	100.00

模块 Module	基因 Gene	基因 Gene ID	基因 Gene	基因 Gene ID
MEturquoise	CHS1	QYY_transcript_7862	CYP75B1	QYY_transcript_13636
	CHS2	QYY_transcript_16509	HCT1	QYY_transcript_16740
	CHS3	QYY_transcript_17459	FLS	QYY_transcript_21239
	CHS4	QYY_transcript_24260	CCoAOMT1	QYY_transcript_23710
	CHS5	QYY_transcript_18432	HIDH1	QYY_transcript_21081
	CHS6	QYY_transcript_19383	HIDH2	QYY_transcript_20408
	CHS7	QYY_transcript_17766	HIDH3	QYY_transcript_21805
	CHS8	QYY_transcript_18116	F3H	QYY_transcript_19623
	CHS9	QYY_transcript_20289	CHI1	QYY_transcript_23390
MEgreen	CHI2	QYY_transcript_21688	CCoAOMT2	QYY_transcript_22442
	HCT2	QYY_transcript_16576	CCoAOMT3	QYY_transcript_21971
	HCT3	QYY_transcript_17410	CYP73A	QYY_transcript_12639