Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (12): 2079-2091.doi: 10.3864/j.issn.0578-1752.2019.12.006

• PLANT PROTECTION • Previous Articles     Next Articles

Molecular Regulation of Trichoderma harzianum ACCC32527 Response to NaCl Based on Transcriptome and Metabolome Analysis

XIANG Jie,CHEN JingShi,XIA XinXin,LIU Kuai,LI ShiGui,GU JinGang()   

  1. Key Laboratory of Microbial Resources Collection and Preservation, Ministry of Agriculture and Rural Affairs, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2019-01-21 Accepted:2019-03-18 Online:2019-06-16 Published:2019-06-22
  • Contact: JinGang GU E-mail:gujingang@caas.cn

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Abstract:

【Objective】The objective of this study is to mine the key functional differentially expressed genes (DEGs) and secondary metabolites by analyzing the data of Trichoderma harzianum ACCC32527 differential transcriptomes and metabolomes under NaCl stress.【Method】The transcriptomes and metabolomes of ACCC32527 with the treatment of 0 (T1), 0.4 (T2), 0.6 (T3) mol?L -1 NaCl were performed by RNA-seq and GC-TOF-MS, respectively, and the annotation, screening and classification of DEGs (|log2fold change|>1 & FDR<0.01) and secondary metabolites (P-value≤0.05 & VIP>1) were completed by related softwares and databases (GO, COG and KEGG pathway). Finally, RT-qPCR was conducted to validate RNA-seq data.【Result】In this study, after NaCl stress treatments, the growth of ACCC32527 was inhibited and the reproductive rate was significantly decreased, 637 and 1 570 DEGs were screened from the T1 vs T2 and T1 vs T3 comparison groups, respectively. Trend analysis result showed that 1 921 DEGs were assigned to 16 significant expression patterns, including 9 up-regulated trends (950 genes), 3 down-regulated trends (662 genes) and 4 irregular trends (309 genes). GO enrichment analysis result showed that each tread was shared by catalytic activity, binding, organelle, cell membrane part, cell membrane, cell part, cell, single-organism process and metabolic process, and accounted for 61%-94%. Among them, the classification with great difference in gene proportion before and after treatment was catalytic activity. The enrichment analysis of KEGG metabolic pathway displayed that the classification and numbers of pathways treated with different NaCl concentrations were significantly different, there were 225 and 535 DEGs enriched in 20 KEGG pathways, respectively, including metabolic pathways, biosynthesis of antibiotics, biosynthesis of secondary metabolites and so on. Among them, 12 and 18 related genes of ribosome biosynthesis pathway were suppressed under T2 and T3 treatments, respectively. A total of 73 genes were screened from the DEGs including ROS scavenging, ion transport and cell wall structure, and most of them were up-regulated. In addition, intracellular small molecule metabolites showed significant change in response to NaCl stress, and there were 30 metabolites with increased accumulation, including amino acids and their derivatives, sugars and their derivatives and alcohols, while 53 metabolites were reduced including fatty acids and organic acids. Among them, glycerol, a known important osmoregulation substance of fungi, was increased under T3 treatment, which may be involved in the process of cell osmotic adjustment. RT-qPCR verified the expression changes of 7 DEGs, which were consistent with the RNA-seq analysis, indicating the reliability of transcriptome sequencing results.【Conclusion】Under NaCl stress, a large number of genes and secondary metabolites of T. harzianum ACCC32527 were changed, which were related to NaCl stress regulation. These processes act together to reduce the toxic effect to the cells, and the results can provide important information for the study of salt-tolerant mechanism of Trichoderma spp..

Key words: NaCl, Trichoderma harzianum, transcriptome, metabolome, salt-tolerant mechanism

Table 1

Gene selection and primer design for RT-qPCR"

基因ID Gene ID 引物序列Primer sequence (5′→3′)
c55075.graph_c0 F-5′ ATTCCCGTCTTAGGGTTCG 3′; R-5′ TGTTGGCTGCTACGCTTT 3′
c56173.graph_c0 F-5′ CCAGCCTTCCTTCTCCTC 3′; R-5′ ATGGTGCCACAGTCTATTCC 3′
c44870.graph_c0 F-5′ TGCTGAAGCGTGAGGTTG 3′; R-5′ AAGCCCGAGTCTTGTTGC 3′
c57043.graph_c0 F-5′ ATTGGCAGGTAGAGTCGT 3′; R-5′ TCATTGATAAACTGGTCCTT 3′
c60715.graph_c3 F-5′ GCGGGAAATCTGCCTTAG 3′; R-5′ GCTGGGTGACGCATAGCT 3′
c60689.graph_c1 F-5′ TACGACCAAACGAGTGCG 3′; R-5' GGCGTCAAGCAGGTGTCT 3′
c55738.graph_c0 F-5′ CTGCCGATTGTTCCGTAT 3′; R-5' GCTTATTAGCCAAGTTGTTTCT 3′
UCE F-5′ GTGGCGGCAGCACTTGTTAT 3′; R-5'ATGACGAACGAAAAGCACCG 3′

Fig. 2

Go classification of DEGs"

Fig. 3

Enriched KEGG terms of DEGs"

Fig. 4

Speculated mechanism of T. harzianum ACCC32527 resistance to NaCl"

Fig. 5

PLS-DA score map of samples"

Table 2

The differential metabolites of T. harzianum ACCC32527"

Category 种类 分析物 Analyte 保留时间RT (min) log2 FC P-value VIP Regulated
氨基酸及其衍生物Amino acids and their derivatives 天冬氨酸Aspartic acid 13.568 0.6834 0.0247 1.3218 Up
谷氨酸Glutamic acid 14.744 1.2283 0.0017 1.5026 Up
谷氨酸盐Glutamine 16.503 3.2743 0.0443 1.4131 Up
异亮氨酸Isoleucine 10.588 1.1547 0.0232 1.3751 Up
蛋氨酸Methionine 13.578 0.9800 0.0227 1.3712 Up
N-甲酰-L-蛋氨酸N-formyl-L-methionine 15.729 -0.4150 0.0463 1.3166 Down
羟脯胺酸Oxoproline 13.649 0.6648 0.0402 1.3251 Up
鸟氨酸Ornithine 16.915 1.2580 0.0489 1.3506 Up
酪氨酸Tyrosine 18.255 1.3905 0.0027 1.4861 Up
苯基丙氨酸Phenylalanine 14.860 0.8506 0.0268 1.3273 Up
L-组氨酸甜菜碱N(alpha), N(alpha)-dimethyl-L-histidine 17.136 1.2927 0.0064 1.4160 Up
糖类及其衍生物
Sugars and their derivatives		 赤藓糖Erythrose 12.687 -0.6572 0.0008 1.5139 Down
塔格糖Tagatose 17.197 1.1699 0.0181 1.4103 Up
6-脱氧-D-葡萄糖6-deoxy-D-glucose 16.175 2.1378 0.0091 1.4189 Up
葡萄糖-1-磷酸Glucose-1-phosphate 16.379 0.9139 0.0438 1.2903 Up
葡萄糖-6-磷酸Glucose-6-phosphate 21.850 -1.6865 0.0154 1.4795 Down
脂肪酸
Fatty acid		 豆蔻酸Myristic acid 17.327 2.6436 0.0109 1.5117 Up
花生四烯酸Arachidonic acid 22.200 -1.9118 0.0413 1.3907 Down
有机酸
Organic acid		 柠檬酸Citric acid 9.8766 -0.7485 0.0009 1.5087 Down
马来酸Maleic acid 10.698 -0.4605 0.0464 1.2939 Down
3-羟丁酸3-hydroxybutyric acid 8.7102 -1.5905 0.0214 1.4670 Down
3-羟基-3-甲基戊二酸3-hydroxy-3-methylglutaric acid 14.526 -1.2892 0.0037 1.5065 Down
3-邻羟基苯乙酸3-hydroxyphenylacetic acid 14.727 -1.1634 0.0010 1.5084 Down
β-甘露糖基甘油酸β-mannosylglycerate 20.685 2.0396 0.0442 1.3879 Up
莽草酸Shikimic acid 16.753 1.9183 0.0215 1.4382 Up
醇类
Alcohol		 丙三醇Glycerol 10.320 0.5890 0.0249 1.3618 Up
丁三醇2-deoxyerythritol 10.509 -0.5241 0.0464 1.2975 Down
核酸及其衍生物Nucleic acids and their derivatives 2-脱氧尿苷2-deoxyuridine 10.419 -0.8792 0.0008 1.5304 Down
尿嘧啶Uracil 11.224 1.9685 0.0212 1.4209 Up
胞苷-1-磷酸Cytidine-monophosphate 18.965 -0.6101 0.0481 1.2821 Down
其他
Others		 2-羟基莠去津Atrazine-2-hydroxy 17.975 2.6033 0.0285 1.3645 Up
N-环已基甲酰胺N-cyclohexylformamide 10.544 -0.4960 0.0233 1.4012 Down
21-羟基孕烯醇酮21-hydroxypregnenolone 27.105 -1.8590 0.0395 1.3045 Down
1,2,4-苯三酚1,2,4-benzenetriol 14.634 -1.0837 0.0006 1.5169 Down

Fig. 6

Enriched column map of KEGGThe red numbers represent the cumulative up-regulated metabolites, and the black numbers represent the cumulative down-regulated metabolites"

Fig. 7

Quantitative real-time PCR validation of relative expression levels of DEGs"

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