真菌诱导子调控紫草素合成的分子机制探究

doi:10.13304/j.nykjdb.2022.0622

中国农业科技导报 ›› 2024, Vol. 26 ›› Issue (1): 78-88.DOI: 10.13304/j.nykjdb.2022.0622

真菌诱导子调控紫草素合成的分子机制探究

李相吴¹^,²(), 刘自扬²^,³, 徐玉俊², 祝建波¹(), 吴燕民²^,³()

^1.石河子大学生命科学学院，新疆石河子 832003
^2.中国农业科学院生物技术研究所，北京 100081
^3.兰州大学草地农业科技学院，兰州大学农业农村部草牧业创新重点实验室，兰州 730020

收稿日期:2022-07-23 接受日期:2022-09-28 出版日期:2024-01-15 发布日期:2024-01-08
通讯作者: 祝建波,吴燕民
作者简介:李相吴 E-mail：1003786451@qq.com；
基金资助:
中央级公益性科研院所基本科研业务费专项(1610392019004)

Explore of Molecular Mechanism on Fungal Elicitors Regulating Shikonin Synthesis

Xiangwu LI¹^,²(), Ziyang LIU²^,³, Yujun XU², Jianbo ZHU¹(), Yanmin WU²^,³()

^1.School of Life Sciences，Shihezi University，Xinjiang Shihezi 832003，China
^2.Biotechnology Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081，China
^3.Key Laboratory of Grassland Livestock Industry Innovation of Ministry of Agriculture and Rural Affairs，College of Pastoral Agriculture Science and Technology，Lanzhou University，Lanzhou 730020，China

Received:2022-07-23 Accepted:2022-09-28 Online:2024-01-15 Published:2024-01-08
Contact: Jianbo ZHU,Yanmin WU

摘要/Abstract

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

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

中图分类号:

S567.23+9

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.

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.

图/表 9

表1 样品测序数据评估

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

表2 测序数据与组装结果的比对

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

图 1 真菌诱导子诱导新疆紫草根部差异表达基因

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

表3 差异基因功能注释统计

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

图2 差异基因GO功能注释注释：1—细胞过程；2—代谢过程；3—单一有机体过程；4—生物调节；5—刺激反应；6—细胞成分组织或生物合成；7—定位；8—发育过程；9—多细胞生物过程；10—信号；11—再生过程；12—多生物过程；13—再生；14—生长；15—免疫系统过程；16—节律过程；17—细胞运动；18—生物附着；19：解毒作用；20细胞聚合；21—细胞；22—细胞组分；23—细胞膜；24—细胞器；25—膜成分；26—细胞器组分；28—膜封闭腔；29—胞外区；30—胞外区组分；31—细胞连接；32—共生体；33—其他有机体；34—其他有机体成分；35—超分子复合物；36—内核；37—结合；38—催化活动；39—转运活动；40—核酸结合转录因子活性；41—结构分子活性；42—电子载体活性；43—抗氧化活动；44—分子转导活性；45—信号转导活性；46—转录因子活性，蛋白结合；47—分子功能调控。

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.

图3 差异基因KEGG功能注释图（前20条通路）

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

图4 差异表达的转录因子

Fig. 4 Differentially expressed transcription factor

表4 部分差异表达的转录因子（前20）

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

图5 参与紫草素合成及调控相关基因表达聚类分析热图

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

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真菌诱导子调控紫草素合成的分子机制探究

Explore of Molecular Mechanism on Fungal Elicitors Regulating Shikonin Synthesis

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