侧孢短芽孢杆菌S2-31拮抗下细辛叶枯病菌转录组差异表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-0545

摘要/Abstract

摘要：

通过分析侧孢短芽孢杆菌拮抗作用下叶枯病菌的转录组学特征,研究差异表达基因（DEGs）和代谢通路的富集情况,初步探索侧孢短芽孢杆菌拮抗叶枯病菌的分子机制。首先利用S2-31与叶枯病菌的对峙培养观察其拮抗作用,然后利用转录组测序探究侧孢短芽孢杆菌拮抗下和正常生长下的叶枯病菌的基因表达水平差异,并进行RT-qPCR验证,最后,利用相关数据库对DEGs进行注释和富集分析。对峙培养后,叶枯病菌菌丝出现皱缩、扭曲等现象。测序后共得到3 681个DEGs,其中2 224个相对上调,1 457个相对下调。GO功能注释分析表明,上调和下调表达的DEGs均注释到生物过程8个亚群、细胞组分8个亚群和分子功能4个亚群。KEGG富集分析中,共有732个unigenes定位到115条生物学通路中,其中富集程度相对较高的通路有氨基糖和核苷糖代谢、糖基磷脂酰肌醇（GPI）锚定生物合成和过氧化物酶体;涉及差异基因较多的通路有淀粉和蔗糖代谢、剪接体和胞吞作用;上调表达的DEGs主要富集在代谢途径,下调表达的DEGs主要富集在遗传信息处理和细胞过程途径。从显著富集的通路中筛选出与菌丝生长发育相关的差异表达基因,结果表明侧孢短芽孢杆菌主要抑制病原菌菌丝过氧化物酶体的形成、胞吞过程和GPI锚定的合成等过程。差异表达基因的RT-qPCR验证结果与转录组测序的结果一致。在侧孢短芽孢杆菌S2-31的拮抗下抑制了叶枯病菌的生长,其转录组特征也发生显著变化,差异基因主要涉及代谢、细胞过程以及遗传信息处理等途径的相关通路。

关键词: 侧孢短芽孢杆菌, 细辛叶枯病, 槭菌刺孢, 生物防治, 转录组学

Abstract:

This work aims to explore the molecular mechanism of antagonistic action of Brevibacillus laterosporus against leaf blight pathogen Mycocentrospora acerina by analyzing the transcriptome characteristics of pathogen antagonized by Brevibacillus laterosporus and studying the enrichment of differentially expressed genes（DEGs）and metabolic pathway. First,the antagonistic effect of S2-31 on leaf blight pathogen was observed on confrontation culture. Then the differences of gene expression level of pathogen under antagonistic and normal growth conditions were conducted by RNA sequencing,and verified by RT-qPCR. Finally,DEGs was annotated and enriched by relevant databases. M. acerina mycelium appeared shrank and malformed after confrontation cultivation. A total of 3 681 DEGs were obtained after sequencing,among which 2 224 were up-regulated and 1 457 were down-regulated. GO enrichment showed that all DEGs were annotated into 8 subpopulations of biological process,8 subpopulations of cell components and 4 subpopulations of molecular functions. KEGG enrichment analysis showed that 732 unigenes were located at 115 biological pathways,and amino sugar,nucleoside sugar metabolism,glycosylphosphatidylinositol（GPI）anchor biosynthesis,and peroxisomes were the most pathways enriched by DEGs. There were a relatively large number of DEGs in starch and sucrose metabolism,spliceosome and endocytosis pathways. The up-regulated DEGs were mainly enriched in the metabolic pathways,while the down-regulated DEGs were mainly enriched in the genetic information processing and cell process pathways. Furthermore,DEGs related to mycelial growth were screened from the significantly enriched pathways,and the results revealed that B. laterosporus mainly inhibited the formation of peroxisomes,GPI anchors,and the process of endocytosis of pathogenic mycelial. The results of RT-qPCR on the selected DEGs were consistent with those of RNA sequencing. The antagonism of B. laterosporus S2-31 inhibited the growth of leaf blight pathogen in vitro and induced significant changes in its transcriptome characteristics,which were mainly related to the pathways of metabolism,cell process and genetic information processing.

Key words: Brevibacillus laterosporus, Asarum leaf blight, Mycocentrospora acerina, biological control, transcriptomics

刘亚苓, 于营, 鲁海坤, 雷慧霞, 隋昕, 郭靖. 侧孢短芽孢杆菌S2-31拮抗下细辛叶枯病菌转录组差异表达分析[J]. 生物技术通报, 2021, 37(2): 111-121.

LIU Ya-ling, YU Ying, LU Hai-kun, LEI Hui-xia, SUI Xin, GUO Jing. Transcriptome Differential Expression Analysis of Mycocentrospora acerina Under Antagonism by Brevibacillus laterosporus S2-31[J]. Biotechnology Bulletin, 2021, 37(2): 111-121.

图/表 11

参考文献 32

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Sample	Clean reads	Error rate/%	Q30/%	GC content/%
CK-1	41 693 598	0.0279	91.52	53.76
CK-2	41 493 354	0.0267	92.53	53.31
CK-3	42 041 596	0.0267	92.59	54.02
S-1	40 579 598	0.027	92.26	53.33
S-2	42 027 532	0.0276	91.78	53.2
S-3	41 786 006	0.027	92.28	52.81

Sample	Clean reads	Error rate/%	Q30/%	GC content/%
CK-1	41 693 598	0.0279	91.52	53.76
CK-2	41 493 354	0.0267	92.53	53.31
CK-3	42 041 596	0.0267	92.59	54.02
S-1	40 579 598	0.027	92.26	53.33
S-2	42 027 532	0.0276	91.78	53.2
S-3	41 786 006	0.027	92.28	52.81

Pathway	Gene ID	Gene description	FC（S/CK）	P value
Peroxisome	TRINITY_DN5187_c2_g3	PEX19	0.148	6.239E-04
	TRINITY_DN4909_c1_g15	alpha/beta-hydrolase	7.412	1.452E-06
	TRINITY_DN2942_c0_g1	PEX16	0.317	2.685E-03
Endocytosis	TRINITY_DN5265_c0_g11	Arf GTPase activating protein	0.165	1.759E-05
	TRINITY_DN5050_c1_g5	vacuolar-sorting protein-like protein snf7	0.159	1.382E-04
	TRINITY_DN4927_c2_g3	GTP binding	0.214	9.932E-04
	TRINITY_DN5015_c8_g6	f-actin-capping protein subunit alpha	0.304	3.159E-06
	TRINITY_DN5245_c3_g6	actin cytoskeleton organization	0.463	0.0098
GPI-anchor biosynthesis	TRINITY_DN5325_c0_g1	GPI：protein transamidase	0.291	1.858E-03
	TRINITY_DN4901_c0_g4	PIG-A	0.298	0.00012
	TRINITY_DN4400_c0_g1	PIG-C	0.385	0.0005
	TRINITY_DN4854_c1_g10	PIG-H	0.418	0.0012
	TRINITY_DN5334_c1_g6	PIG-L	0.446	0.0021
Spliceosome	TRINITY_DN4979_c1_g13	ATP binding	0.156	1.290E-10
	TRINITY_DN5195_c2_g7	pre-mRNA splicing factor	0.282	1.466E-05
	TRINITY_DN5299_c2_g2	splicing factor 3b	0.390	1.489E-04
	TRINITY_DN5211_c1_g1	spliceosomal tri-snRNP complex assembly	0.468	0.015

Pathway	Gene ID	Gene description	FC（S/CK）	P value
Peroxisome	TRINITY_DN5187_c2_g3	PEX19	0.148	6.239E-04
	TRINITY_DN4909_c1_g15	alpha/beta-hydrolase	7.412	1.452E-06
	TRINITY_DN2942_c0_g1	PEX16	0.317	2.685E-03
Endocytosis	TRINITY_DN5265_c0_g11	Arf GTPase activating protein	0.165	1.759E-05
	TRINITY_DN5050_c1_g5	vacuolar-sorting protein-like protein snf7	0.159	1.382E-04
	TRINITY_DN4927_c2_g3	GTP binding	0.214	9.932E-04
	TRINITY_DN5015_c8_g6	f-actin-capping protein subunit alpha	0.304	3.159E-06
	TRINITY_DN5245_c3_g6	actin cytoskeleton organization	0.463	0.0098
GPI-anchor biosynthesis	TRINITY_DN5325_c0_g1	GPI：protein transamidase	0.291	1.858E-03
	TRINITY_DN4901_c0_g4	PIG-A	0.298	0.00012
	TRINITY_DN4400_c0_g1	PIG-C	0.385	0.0005
	TRINITY_DN4854_c1_g10	PIG-H	0.418	0.0012
	TRINITY_DN5334_c1_g6	PIG-L	0.446	0.0021
Spliceosome	TRINITY_DN4979_c1_g13	ATP binding	0.156	1.290E-10
	TRINITY_DN5195_c2_g7	pre-mRNA splicing factor	0.282	1.466E-05
	TRINITY_DN5299_c2_g2	splicing factor 3b	0.390	1.489E-04
	TRINITY_DN5211_c1_g1	spliceosomal tri-snRNP complex assembly	0.468	0.015

Gene ID	Gene description	Primer sequences（5'-3'）
TRINITY_DN4979_c1_g13	ATP binding	CGAACGAACCACCAAACAT	GGAACCAGTAGGGGCACAG
TRINITY_DN5195_c2_g7	pre-mRNA splicing factor	CGAAACATCCCTTACCATT	AGTACGACCAGCACGACCG
TRINITY_DN5265_c0_g11	Arf GTPase activating protein	CGTGAAGAAGCGGAGCGTA	GCGACAGGTGTGGGGGATA
TRINITY_DN5050_c1_g5	vacuolar-sorting protein-like protein snf7	CCCGTCTCAAACCATCTACAAC	ATTATTTGGGAGCTGGTCTGTT
TRINITY_DN4873_c1_g11	chitin binding	TGGCAGGTGCGGTAAGGAGT	TGTGGTGGTGGAAGGTGGTC
TRINITY_DN4909_c1_g15	alpha/beta-hydrolase	CTCCGCACCAAGCAACAAAC	AGCCCCAGTCATGCCCAATC
TRINITY_DN5416_c0_g1	gamma-glutamyltranspeptidase-like protein	CTCTTCCCTGGTCCACTGTT	TGTTGCGTTCTGATTTGCTG