Explore of Molecular Mechanism on Fungal Elicitors Regulating Shikonin Synthesis

doi:10.13304/j.nykjdb.2022.0622

Abstract

Abstract:

To explore the molecular mechanism of fungal elicitors regulating shikonin synthesis， RNA sequencing and analysis were performed on the roots of Arnebia euchroma sterile seedlings after bio-induction by elicitors prepared from Fusarium oxysporum and Rhizoctonia solani. The results showed that， compared with the control group， the Fusarium oxysporum experimental group differentially expressed 1 735 genes； the Rhizoctonia solani experimental group differentially expressed 1 043 genes. GO （gene ontology） analysis revealed that the differentially expressed genes of 2 experimental groups were mainly enriched in cellular processes in biological processes， membrane in cellular components， and catalytic activity in molecular functions. KEGG （kyoto encyclopedia of genes and genomes） analysis revealed that 2 experimental groups were enriched in a large number of differentially expressed genes in the pathways of plant-pathogen interaction， phytohormone signaling pathways and phenylpropanoid synthesis. The same transcription factors were differentially expressed in 2 experimental groups， mainly including bHLH， AP2/ERF-ERF and LOB. The expression of AeGHQH， AeDSH1， AeAP， AePAL， AeDI2， AePGT， AeHMGR and AeG10H genes， which were involved in the synthesis and positive regulation of shikonin， were found to be up-regulated in 2 experimental groups， with more significant up-regulation in the Fusarium oxysporum experimental group. Above results elaborated the response mechanism of Arnebia euchroma in the biological induction of fungal elicitors at the molecular level， which laid a theoretical foundation for the future application of fungal elicitors in the cultivation and production of Arnebia euchroma.

Key words: Arnebia euchroma, fungal elicitors, transcriptome, differentially expressed gene

摘要：

为探讨真菌诱导子调控紫草素合成的分子机制，以新疆紫草无菌苗为试材，经尖孢镰刀菌、立枯丝核菌制备成的诱导子生物诱导后，对其根部进行RNA测序和分析。结果表明，与对照组比较，尖孢镰刀菌试验组差异表达基因1 735个；立枯丝核菌试验组差异表达基因1 043个。GO（gene ontology）分析发现，2个试验组差异表达基因主要富集在细胞过程、细胞组分中膜和分子功能中催化活性等生物学过程。KEGG（kyoto encyclopedia of genes and genomes）分析发现，2个试验组在植物与病原菌互作、植物激素信号转导途径和苯丙素合成等通路均有大量差异表达基因富集。2个试验组差异表达情况相同的转录因子主要包括bHLH、AP2/ERF-ERF和LOB等，并发现参与紫草素合成及其正向调控的AeGHQH、AeDSH1、AeAP、AePAL、AeDI2、AePGT、AeHMGR、AeG10H基因在2个试验组均上调表达，其中尖孢镰刀菌试验组上调表达较显著。以上结果从分子水平探究了新疆紫草对真菌诱导子生物诱导的响应机制，为未来真菌诱导子应用于新疆紫草种植生产奠定了理论基础。

关键词: 新疆紫草, 真菌诱导子, 转录组, 差异表达基因

CLC Number:

S567.23+9

Xiangwu LI, Ziyang LIU, Yujun XU, Jianbo ZHU, Yanmin WU. Explore of Molecular Mechanism on Fungal Elicitors Regulating Shikonin Synthesis[J]. Journal of Agricultural Science and Technology, 2024, 26(1): 78-88.

李相吴, 刘自扬, 徐玉俊, 祝建波, 吴燕民. 真菌诱导子调控紫草素合成的分子机制探究[J]. 中国农业科技导报, 2024, 26(1): 78-88.

Figures/Tables 9

Table 1 sample sequencing data evaluation

处理 Treatment	样品编号 Sample ID	总碱基数 Base number	GC含量 GC content/%	Q30/%
F20	F20-1	6 293 089 734	43.61	92.39
	F20-2	6 422 595 670	43.56	93.06
	F20-3	7 834 038 640	43.62	92.46
R20	R20-1	6 872 640 242	43.61	93.02
	R20-2	7 140 087 048	43.71	93.05
	R20-3	6 946 752 472	43.66	92.70
CK	CK-1	7 792 349 390	43.66	92.82
	CK-2	6 870 409 240	43.68	92.81
	CK-3	5 930 234 288	43.68	92.44

Table 2 Comparison of sequencing data with assembly results

处理 Treatment	样品编号 Sample ID	Read数 Reads number	匹配Read数 Mapped reads number	匹配率 Mapped ratio/%
F20	F20-1	21 096 427	15 558 872	73.75
	F20-2	21 504 765	16 195 509	75.31
	F20-3	26 236 242	19 430 135	74.06
R20	R20-1	23 005 518	17 277 331	75.10
	R20-2	23 913 025	17 644 691	73.79
	R20-3	23 283 171	17 129 464	73.57
CK	CK-1	26 093 101	19 723 787	75.59
	CK-2	23 036 789	17 376 776	75.43
	CK-3	19 879 659	14 903 996	74.97

Fig. 1 Differentially expressed genes in the leaves of Arnebia euchroma

Table 3 Numbers of functional annotated DEGs

数据库名称Database name	被注释到的差异基因数量 Annotated number of DGEs
数据库名称Database name	F20/CK	R20/CK
COG	517	317
GO	1 276	785
KEGG	1 063	651
KOG	758	439
Pfam	1 294	775
Swiss-Prot	1 188	731
NR	1 555	943

Fig. 2 GO functional annotation of differential expressed genesNote：1—Cellular process； 2—Metabolic process； 3—Single-organism process； 4—Biological regulation； 5—Response to stimulus； 6—Cellular component organization or biogenesis； 7—Localization； 8—Developmental process； 9—Multicellular organismal process； 10—Signaling； 11—Reproductive process； 12—Multi-organism process； 13—Reproduction； 14—Growth； 15—Immune system process； 16—Rhythmic process； 17—Locomotion； 18—Biological adhesion； 19—Detoxification； 20—Cell aggregation； 21—Cell； 22—Cell part； 23—membrane； 24—Organelle； 25—Membrane part； 26—Organelle part； 27—Macromolecular complex； 28—Membrane-enclosed lumen； 29—Extracellular region； 30—Extracellular region part； 31—Cell junction； 32—Symplast； 33—Other organism； 34—Other organism part； 35—Supramolecular complex； 36—Nucleoid； 37—Binding； 38—Catalytic activity； 39—Transporter activity； 40—Nucleic acid binding transcription factor activity； 41—Structural molecule activity； 42—Electron carrier activity； 43—Antioxidant activity； 44—Molecular transducer activity； 45—Signal transducer activity； 46—Transcription factor activity protein binding； 47—Molecular function regulator.

Fig. 3 KEGG functional annotation of differential expressed genes（top 20 pathways）

Fig. 4 Differentially expressed transcription factor

Table 4 Some differentially expressed transcription factor （top 20）

家族 Family	基因ID Gene ID	log₂FC
家族 Family	基因ID Gene ID	F20/CK	R20/CK
TRAF	c65742.graph_c0	2.51	2.01
Tify	c54302.graph_c0	1.59	0.96
SRS	c54737.graph_c1	-4.53	-2.95
MYB-related	c58018.graph_c0	4.26	2.80
MYB	c62808.graph_c0	1.59	1.15
LOB	c48779.graph_c0	1.71	1.28
HB-other	c47461.graph_c0	-2.71	-1.85
HB-BELL	c58247.graph_c0	1.70	1.54
GARP-G2-like	c65836.graph_c0	-1.95	-1.23
GARP-G2-like	c53563.graph_c1	-1.78	-1.82
C2H2	c56397.graph_c0	-2.06	-1.52
C2C2-YABBY	c28651.graph_c0	-3.50	-2.26
C2C2-Dof	c53061.graph_c0	1.71	1.50
bHLH	c59392.graph_c0	2.16	1.43
bHLH	c54786.graph_c0	1.65	1.66
bHLH	c53656.graph_c0	-6.60	-3.19
bHLH	c47522.graph_c0	-2.99	-1.71
AP2/ERF-ERF	c60825.graph_c0	-1.78	-1.17
AP2/ERF-ERF	c57468.graph_c0	2.29	1.93
AP2/ERF-ERF	c55718.graph_c0	-1.68	-1.33

Fig. 5 Heat map of cluster analysis of gene expression involved in shikonin synthesis and regulation

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