Effect of STC on mycelial growth and AFB1 production.
The effect of STC, at five concentrations (0.02, 0.1, 2.0, 10.0, 50.0 µg/mL), on mycelial growth of A. flavus and AFB1 production in PDB medium over 11 days are shown in Table 1. The dry weight of mycelia increasing with increasing time in culture. In the control A. flavus grew well in PDB medium with sustained growth over the 11 days. When STC was added to the medium, mycelial dry weights at the highest concentration of STC were significantly different to the control and the other treatments (p>0.05) after 3 and 4 days growth (except for 10.0 µg/mL in 3 days); From the 5th day, there was no significant difference in A. flavus dry weights amongst any of the STC concentrations and the control except in the 10 µg/mL treatment (p<0.05). This indicates that A. flavus growth was not affected by the presence of STC, even at high concentrations.
Table 1
Effect of different sterigmatocystin concentrations on dry weight of mycelium
Added concentration(µg/mL)
|
mycelial weights(mg)
|
3d
|
4d
|
5d
|
7d
|
9d
|
11d
|
CK
|
25.32±1.92a
|
26.97±3.42b
|
42.62±4.22bc
|
53.49±6.28b
|
60.27±3.77ab
|
75.81±8.16a
|
0.02
|
27.75±1.64a
|
33.63±2.87a
|
46.29±2.23bc
|
55.94±6.86ab
|
56.05±2.51b
|
78.91±3.57a
|
0.1
|
28.75±2.08a
|
35.30±2.33a
|
40.63±3.06c
|
61.21±4.13ab
|
65.83±6.40a
|
77.36±3.69a
|
2.0
|
20.10±2.31b
|
19.76±2.47c
|
44.59±3.95bc
|
61.18±1.57ab
|
69.29±1.70a
|
80.91±5.78a
|
10
|
16.54±2.58b
|
20.76±1.34c
|
54.90±4.29a
|
60.85±2.74ab
|
63.73±2.44ab
|
71.51±7.84a
|
50
|
18.55±0.64b
|
18.98±1.51c
|
50.32±1.20ab
|
65.84±2.20b
|
68.82±4.66a
|
77.48±4.13a
|
AFB1 production increased with increasing culture time in all treatments and the control, however the rate varied (Table 2). The relative ability to produce toxin did not show a linear trend in relation to STC concentration. For example, after 3 days AFB1 production was 17.18 µg/L, 0.38 µg/L, 2.94 µg/L and 33.36 µg/L at STC concentrations of 0.02 mg/L, 0.1 mg/L, 2 mg/L and 10 mg/L, respectively, while in the control it was 61.62 µg/L. Maximum AFB1 production reached 545.70 µg/L, 95.65 µg/L, 366.71 µg/L and 804.90 µg/L in the same four STC concentrations, and 1145.67 µg/L in the control. Hence, a certain concentration of STC inhibited AFB1 production, and at an STC concentration of 0.1 µg/mL AFB1 production was significantly different than the control (p>0.05). It is noteworthy that at an STC concentration of 50 mg/L AFB1 production was not significantly different to the control (p<0.05) between days 4 and 7 of growth.
Table 2
Effect of different sterigmatocystin concentrations on aflatoxin B1 production of Aspergillus flavus
Added concentration(µg/mL)
|
AFB1 production(µg/L)
|
3d
|
4d
|
5d
|
7d
|
9d
|
11d
|
CK
|
61.62±8.52a
|
255.79±9.98a
|
372.35±89.93a
|
759.72±61.02a
|
1199.85±203.76a
|
1145.67±71.44a
|
0.02
|
17.18±1.22c
|
108.06±22.45b
|
203.17±69.28b
|
419.09±32.51b
|
414.90±42.27c
|
545.70±155.63bc
|
0.1
|
0.38±0.10d
|
27.23±2.64c
|
39.00±3.97c
|
77.02±11.11d
|
66.68±10.18d
|
95.65±46.30d
|
2.0
|
2.94±0.79d
|
22.57±8.65c
|
81.27±27.23c
|
233.39±73.04c
|
365.99±82.04c
|
366.71±60.23c
|
10
|
33.36±6.12b
|
140.09±13.21b
|
310.18±19.74ab
|
674.41±52.34a
|
783.96±120.74b
|
804.90±131.15b
|
50
|
69.29±9.91a
|
131.09±22.47b
|
308.93±29.37ab
|
479.80±56.80b
|
966.24±138.27ab
|
1264.37±189.36a
|
Summary of RNA-seq data sets
Data output statistics and quality control.
Transcriptome sequencing of the six samples (two groups, three duplicates) of A. flavus CGMCC 3.2980 generated a total of 30.31 million raw reads; amongst the total read pairs, 29.38 million passed purity filtering standards. Through base composition and mass analysis, it was apparent that there were few low quality bases. This met the quality control standard of sequencing and requirements for subsequent bioinformatic analysis.
Reference sequence alignment analysis.
Comparative analysis of the genome of A. flavus was done using clean reads after filtering with TopHat 2.0 software. The results showed that over 85% of reads were uniquely mapped to the A. flavus genome and multiple mapped reads or fragments coincided with the standard of half-point ratio below 10% in multiple localization-sequencing. This meant that the reference genome was appropriate and three was no pollution in the experiment.
Gene expression level analysis.
Gene expression level is reflected in transcription abundance; the lower the transcription abundance, the lower the level of gene expression. The overall transcriptional activity of genes in our data were quantified by calculating the number of reads per kilobase of exon per million mapped reads (FPKM); it is generally recognized that an FPKM>1 means that the gene is expressed. Gene expression levels are shown in Table 3.
Table 3
Proportion of genes at different expression levels
Samples
|
FPKM Interval
|
0~1
|
1~3
|
3~15
|
15~60
|
>60
|
CK_1
|
233
|
399
|
1041
|
833
|
872
|
CK_2
|
220
|
407
|
1032
|
818
|
853
|
CK_4
|
251
|
423
|
1048
|
829
|
788
|
TJ_1
|
697
|
489
|
722
|
637
|
614
|
TJ_2
|
754
|
463
|
728
|
655
|
587
|
TJ_4
|
594
|
473
|
753
|
673
|
684
|
Identification and analysis of DEGs
Differential expression analysis.
To identify differences in molecular responses (based on read count) between experimental groups and the control group, we identified 3377 differentially transcribed (FDR<0.05, ∣log2 FC∣≥1) genes, of which 1182 genes (35.00%) were up-regulated and 2195 (65.00%) genes were down-regulated.
GO and KEGG analysis of DEGs.
Functional assignments were defined by Gene Ontology (GO) terms (http://www.genontology.org), which provided a broad functional classification of genes and gene products for various biological processes (BP), cellular components (CC) and molecular functions (MF). GO functional enrichment analysis revealed that: 13 MF terms were enriched, mainly involved with oxidoreductase activity, catalytic activity, structural constituent of ribosome and monooxygenase activity; 18 CC terms were enriched including intracellular organelle part, intracellular part, cell part and macromolecular complexes; 65 BP terms were enriched including cellular component organization or biogenesis, oxidation-reduction processes, single-organism metabolic processes, single-organism processes and cellular protein metabolic processes (Table 4).
Table 4
GO functional enrichment analysis of differentially expressed genes
GO ID
|
Number
|
Pop number
|
P value_uncorrected
|
FDR
|
Description
|
MF
|
GO:0016491
|
548
|
1573
|
4.81297E-10
|
0
|
Oxidoreductase activity
|
GO:0003824
|
1480
|
5098
|
6.51185E-10
|
0
|
catalytic activity oxidoreductase activity, acting on paired donors,
|
GO:0016705
|
119
|
287
|
9.20882E-09
|
0
|
with incorporation or reduction of molecular
oxygen
|
GO:0003735
|
6
|
113
|
1.43745E-08
|
0
|
structural constituent of ribosome
|
GO:0004497
|
119
|
291
|
1.88615E-08
|
0
|
monooxygenase activity
|
BP
|
GO:0071840
|
53
|
413
|
2.58209E-10
|
0
|
cellular component organization or biogenesis
|
GO:0055114
|
543
|
1538
|
4.10448E-10
|
0
|
oxidation-reduction process
|
GO:0044710
|
918
|
2926
|
5.87454E-10
|
0
|
single-organism metabolic process
|
GO:0044699
|
1217
|
4145
|
6.47599E-10
|
0
|
single-organism process
|
GO:0044267
|
76
|
504
|
1.37828E-09
|
0
|
cellular protein metabolic process
|
CC
|
GO:0044446
|
98
|
667
|
3.2679E-10
|
0
|
intracellular organelle part
|
GO:0044422
|
98
|
672
|
3.71546E-10
|
0
|
organelle part
|
GO:0032991
|
168
|
996
|
4.08468E-10
|
0
|
macromolecular complex
|
GO:0044424
|
456
|
2232
|
5.59664E-10
|
0
|
intracellular part
|
GO:0044464
|
538
|
2507
|
9.22963E-10
|
0
|
cell part
|
Note: FDR: P value_correctedNote: FDR: P value_corrected |
To further investigate the biological functions and interactions amongst genes, pathway-based analysis was done using the Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www. genome.ad.jp/kegg) pathway database. Results showed that 3377 DEGs were involved in 110 pathways. The top ten down- and up-regulated genes enriched in KEGG is shown in Table 5. For down-regulated genes, the most enriched were involved in aflatoxin biosynthesis, followed by starch and sucrose metabolism and tyrosine metabolism; this explains why gene expression involved in aflatoxins synthesis and glucose metabolic pathway was suppressed in the experiment group. For up-regulated genes, valine, leucine and isoleucine biosynthesis was the most enriched, followed by alanine, aspartate and glutamate metabolism; this accounts for why genes participating in amino acid metabolism in the experimental groups were highly expressed. KEGG metabolic pathway analysis showed that enrichment of valine, leucine and isoleucine biosynthesis and aflatoxin biosynthesis was significant at p≤0.05.
Table 5
Top 10 KEGG enrichments for down- and up-regulated genes
Ko id
|
Term
|
Number
|
P value_uncorrected
|
|
Genes down -egulated
|
|
|
map00254
|
Aflatoxin biosynthesis
|
13
|
0.0000
|
map00500
|
Starch and sucrose metabolism
|
20
|
0.0011
|
map00350
|
Tyrosine metabolism
|
18
|
0.0012
|
map00650
|
Butanoate metabolism
|
12
|
0.0018
|
map00380
|
Tryptophan metabolism
|
15
|
0.0024
|
map00071
|
Fatty acid degradation
|
12
|
0.0058
|
map02010
|
ABC transporters
|
5
|
0.0066
|
map04213
|
Longevity regulating pathway - multiple species
|
8
|
0.0070
|
map00591
|
Linoleic acid metabolism
|
3
|
0.0114
|
map00061
|
Fatty acid biosynthesis
|
7
|
0.0125
|
|
Genes up-regulated
|
|
|
map00290
|
Valine, leucine and isoleucine biosynthesis
|
14
|
0.0000
|
map00250
|
Alanine, aspartate and glutamate metabolism
|
16
|
0.0002
|
map04113
|
Meiosis - yeast
|
18
|
0.0003
|
map00770
|
Pantothenate and CoA biosynthesis
|
11
|
0.0003
|
map00970
|
Aminoacyl-tRNA biosynthesis
|
15
|
0.0004
|
map00670
|
One carbon pool by folate
|
8
|
0.0007
|
map00300
|
Lysine biosynthesis
|
8
|
0.0011
|
map03008
|
Ribosome biogenesis in eukaryotes
|
18
|
0.0015
|
map00230
|
Purine metabolism
|
23
|
0.0020
|
map00260
|
Glycine, serine and threonine metabolism
|
18
|
0.0036
|
Analysis of gene expression in secondary metabolism gene clusters of A. flavus.
To investigate the effect of STC on secondary metabolism synthesis of A. flavus, we used CIFR (Center for Integrated Fungal Research) information (http://weir.statgen.ncsu.edu/aspergillus/chromosomes.php) and SMURF (http://www.jcvi.org/smurf) software to analyze 55 secondary metabolic gene clusters from A. flavus. Expression of most gene clusters was not affected by STC. Comparative analysis of secondary metabolism gene expression demonstrated that in 639 secondary metabolism genes there were 162 genes with significantly different expression (q-value≤0.001), of which 17 were backbone genes: AFLA_004450, AFLA_010020, AFLA_064560, AFLA_069330 and AFLA_139490 encoded mainly non-ribosomal peptide synthetase (NRPS); AFLA_006170, AFLA_10545, AFLA_128060, AFLA_137870 and AFLA_139410 are responsible for polyketide synthase, PKS; AFLA_017840, AFLA_023020, AFLA_079400, AFLA_105190 encode NRPS-like synthase and AFLA_028660, AFLA_066890 and AFLA_139480 encode for mitochondrial translation initiation factor IF-2, cytochrome P450 and hybrid PKS/NRPS enzymes, respectively.
Most secondary metabolic gene clusters were down-regulated, with only the minority being up-regulated. In the 2# gene cluster, backbone gene AFLA_004450, which is responsible for encoding dimethylallyl transferase, was up-regulated, but AFLA_004370 and AFLA_004400 were down-regulated. In the 11# gene cluster, only AFLA_028660 was up-regulated while there was no significant difference in expression of other genes in this cluster. Similarly, the genes AFLA_082220, AFLA_082230 and AFLA_082250 in the 27# gene cluster and AFLA_101690 and AFLA_101710 in the 33# gene cluster were all up-regulated to varying degrees. Amongst the up-regulated genes, AFLA_112890 and AFLA_066740 encode major facilitator superfamily transporters (MFS), which have an important role in transfer of some metabolites. Furthermore, genes involved in amino acid synthesis were differentially expressed to varying degrees; for example, AFLA_005510, AFLA_041550, AFLA_083270 and AFLA_126970, which encode amino acid permease, cysteine β-lyase, GABA permease and arginine permease, respectively, were up-regulated, but AFLA_038620 (encoding branched amino acid transferase) and AFLA_062910 (encoding specific proline permease) were down-regulated.
AFB1 synthesis genes were located on the 54# gene cluster. In this study, 30 genes were all down-regulated to varying degrees; norB was completely repressed in the experimental group (Table 6). However, there was no difference in expression of the global regulatory genes of secondary metabolite, laeA and veA,. Also, brlA, which is involved with growth was down-regulated.
Table 6
Analysis of genes expressed in the biosynthesis of aflatoxins
Gene name
|
RPKM
|
Log2
|
q value
|
DGE
|
Annotated gene function
|
CK
|
TJ
|
TJ/CK
|
AFLA_139200
|
152.1003
|
0.0890
|
-10.7389
|
1.076E-83
|
down
|
aflQ/ ordA/ ord-1/ oxidoreductase
/ cytochrome P450 monooxigenase
|
AFLA_139210
|
917.6813
|
0.2213
|
-12.0176
|
7.73E-142
|
down
|
aflP/ omtA/ omt-1/
O-methyltransferase A
|
AFLA_139220
|
2925.1377
|
0.5760
|
-12.3101
|
5.21E-174
|
down
|
aflO/ omtB/ dmtA/
O-methyltransferase B
|
AFLA_139230
|
99.5160
|
0.1137
|
-9.7740
|
1.589E-45
|
down
|
aflI/ avfA/ cytochrome P450
monooxygenase
|
AFLA_139240
|
1044.9193
|
0.2383
|
-12.0981
|
2.354E-65
|
down
|
aflLa/ hypB/ hypothetical protein
|
AFLA_139250
|
799.4160
|
0.1230
|
-12.6661
|
2.64E-125
|
down
|
aflL/ verB/ desaturase/ P450
monooxygenase
|
AFLA_139260
|
786.0533
|
0.1383
|
-12.4723
|
3.22E-117
|
down
|
aflG/ avnA/ ord-1/ cytochrome P450 monooxygenase
|
AFLA_139270
|
1428.8010
|
1.4530
|
-9.9416
|
5.055E-67
|
down
|
aflNa/ hypD/ hypothetical protein
|
AFLA_139280
|
227.1293
|
0.0113
|
-14.2907
|
5.76E-61
|
down
|
aflN/ verA/ monooxygenase
|
AFLA_139290
|
821.2367
|
0.1913
|
-12.0675
|
7.899E-43
|
down
|
aflMa/ hypE/ hypothetical protein
|
AFLA_139300
|
6524.7297
|
1.3467
|
-12.2423
|
1.72E-134
|
down
|
aflM/ ver-1/ dehydrogenase/ ketoreductase
|
AFLA_139310
|
1071.7900
|
0.2320
|
-12.1736
|
3.66E-130
|
down
|
aflE/ norA/ aad/ adh-2/ NOR reductase/ dehydrogenase
|
AFLA_139320
|
1900.3200
|
0.4837
|
-11.9399
|
2.15E-124
|
down
|
aflJ/ estA/ esterase
|
AFLA_139330
|
3153.5033
|
1.8543
|
-10.7318
|
7.72E-131
|
down
|
aflH/ adhA/ short chain alcohol dehydrogenase
|
AFLA_139340
|
560.8290
|
3.6423
|
-7.2666
|
3.33E-124
|
down
|
aflS/ pathway regulator
|
AFLA_139360
|
92.4453
|
4.6113
|
-4.3253
|
5.357E-37
|
down
|
aflR / apa-2 / afl-2 / transcription activator
|
AFLA_139370
|
143.3517
|
3.2357
|
-5.4694
|
2.01E-161
|
down
|
aflB / fas-1 / fatty acid synthase beta subunit
|
AFLA_139380
|
117.8963
|
5.8397
|
-4.3355
|
3.961E-70
|
down
|
aflA / fas-2 / hexA / fatty acid synthase alpha subunit
|
AFLA_139390
|
2307.8407
|
5.6377
|
-8.6772
|
1.382E-74
|
down
|
aflD / nor-1 / reductase
|
AFLA_139400
|
2940.4380
|
1.0157
|
-11.4994
|
1.607E-86
|
down
|
aflCa / hypC / hypothetical protein
|
AFLA_139410
|
196.8083
|
4.3753
|
-5.4913
|
4.01E-164
|
down
|
aflC / pksA / pksL1 / polyketide synthase
|
AFLA_139420
|
333.8647
|
4.3997
|
-6.2457
|
1.04E-112
|
down
|
aflT / aflT / transmembrane protein
|
AFLA_139430
|
178.0950
|
0.3763
|
-8.8864
|
1.018E-60
|
down
|
aflU / cypA / P450 monooxygenase
|
AFLA_139440
|
25.7907
|
0.0000
|
_
|
1.613E-06
|
down
|
aflF / norB / dehydrogenase
|
AFLA_139450
|
11.8450
|
0.0000
|
_
|
3.169E-06
|
down
|
conserved hypothetical protein
|
AFLA_139460
|
558.1760
|
1.0897
|
-9.0007
|
1.47E-48
|
down
|
MFS multidrug transporter, putative
|
AFLA_139470
|
320.0510
|
1.5767
|
-7.6653
|
1.088E-14
|
down
|
FAD dependent oxidoreductase, putative
|
AFLA_139480
|
493.3330
|
3.8730
|
-6.9930
|
2.24E-48
|
down
|
dimethylallyl tryptophan synthase,
putative
|
AFLA_139490
|
8.2883
|
0.0330
|
-7.9725
|
3.695E-43
|
down
|
hybrid PKS/NRPS enzyme, putative
|
AFLA_139500
|
0.8250
|
0.0257
|
-5.0064
|
3.439E-05
|
down
|
conserved hypothetical protein
|