CN111334460A - Method for inhibiting temperature tolerance of aspergillus flavus - Google Patents
Method for inhibiting temperature tolerance of aspergillus flavus Download PDFInfo
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Abstract
A method for inhibiting temperature tolerance of Aspergillus flavus is disclosed, which can obviously inhibit the temperature tolerance of Aspergillus flavus by inhibiting the expression level of ergosterol synthetic gene and transcription factor srbA gene or ergosterol synthetic and transcription factor SrbA protein in Aspergillus flavus, or inhibiting the content of ergosterol in Aspergillus flavus. After the expression level of ergosterol synthesis genes or ergosterol synthesis and transcription factor SrbA protein is inhibited, 5mM hydrogen peroxide is additionally added under the condition of normal temperature, so that aspergillus flavus can be obviously inhibited and even killed. The method for inhibiting the temperature tolerance of the aspergillus flavus can effectively inhibit the pollution of the aspergillus flavus to a plurality of agricultural products, and achieves the effects of preventing and controlling in advance and reducing economic loss.
Description
Technical Field
The invention relates to genetic engineering, in particular to a method for inhibiting aspergillus flavus temperature tolerance.
Background
Aspergillus flavus is a widely existing soil saprophytic aspergillus fungus, is used as a common pathogenic bacterium of plant animals, not only causes great harm to agricultural production, feed industry and livestock industry, but also brings serious threat to human health. Aspergillus flavus can infect many important crops, such as peanuts, corn, cotton, etc., all of which can contaminate the crop before and after harvest, causing significant economic losses to agricultural production around the world. According to the food and agriculture organization of the united nations, about 25% of grains are contaminated with fungi each year, the most important of which is aspergillus flavus. China is a serious disaster area of aflatoxin pollution, aflatoxin pollution is detected in corns and peanuts stored in a plurality of provinces in China, and aflatoxin is detected in processed products such as soy sauce, aquatic feeds and the like. Therefore, the pollution of the aspergillus flavus causes great economic loss and potential safety hazard to human beings, and the research on the prevention and control of the pollution of the aspergillus flavus has very important significance.
Ergosterol is an important component of fungal cell membrane and plays an important role in ensuring cell viability, membrane fluidity, membrane-bound enzyme activity, membrane integrity, and intracellular material transport. Many antifungal drugs reported so far, such as fluconazole and the like, use ergosterol synthesis as a drug target to inhibit the growth of fungi. At present, ergosterol is only reported in fungus high temperature stress resistance, and the role of ergosterol synthesis related genes in aspergillus flavus is not reported.
Disclosure of Invention
The invention aims to provide a method for inhibiting temperature tolerance of aspergillus flavus.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a method of inhibiting aspergillus flavus temperature tolerance comprising:
s 1: the expression level of an ergosterol synthetic gene and a transcription factor srba gene or an ergosterol synthetic and transcription factor srba protein in aspergillus flavus is reduced by means of gene knockout, rna interference or drug treatment and the like;
s 2: the aspergillus flavus with low expression level of ergosterol synthetic gene and transcription factor srba gene or ergosterol synthetic gene coding protein and transcription factor srba protein grows under the environment that the temperature is 42 ℃ or more than 42 ℃.
s 3: under the condition of room temperature, 5mm of hydrogen peroxide is added into aspergillus flavus with low expression level of ergosterol synthetic gene and transcription factor srba gene or ergosterol synthetic gene coding protein and transcription factor srba protein.
Further, in the step s2, the expression level of the ergosterol synthesis gene and its regulatory gene or its encoded protein in Aspergillus flavus is inhibited by molecular genetic manipulation or an ergosterol synthesis inhibitor.
Further, in the step s2, the culture temperature of the aspergillus flavus is 42 ℃ to over 42 ℃.
Further, in the step s3, after the step s1 is met, 5mm of strong oxidant, namely hydrogen peroxide is added, and the culture temperature of the aspergillus flavus is the room temperature condition
The invention has the beneficial effects that: the inventor finds out for the first time that the temperature tolerance of the aspergillus flavus can be obviously inhibited by inhibiting the expression level of an ergosterol synthetic gene of the aspergillus flavus and a srba gene or a coding protein thereof. Further, the hydrogen peroxide with the thickness of 5mm is additionally added under the normal temperature condition, so that the aspergillus flavus can be obviously inhibited and even killed. The method for inhibiting the temperature tolerance of the aspergillus flavus can effectively inhibit the pollution of the aspergillus flavus to a plurality of agricultural products, and achieves the effects of preventing and controlling in advance and reducing economic loss.
Drawings
FIG. 1 is a graph of the effect of temperature on the growth of Aspergillus flavus, (A) colony morphology of Aspergillus flavus at different temperatures and different media; (B) the influence of different temperatures on the growth inhibition rate of aspergillus flavus on a gmm culture medium is shown, and the inhibition rate is compared with that under the condition of 29 ℃;
FIG. 2 is a graph showing the effect of different temperatures on the expression of genes involved in ergosterol synthesis in Aspergillus flavus, (A) the expression of genes involved in ergosterol synthesis in Aspergillus flavus under different temperature conditions; (B) the expression condition of an ergosterol synthesis regulatory factor srba gene in aspergillus flavus under different temperature conditions;
FIG. 3 is a schematic diagram of the knockout and complementation of the ergosterol synthesis regulator encoding gene srba and the results of the validation, where (A) the knockout schematic diagram illustrates the replacement of the Aspergillus flavus srba gene with the Aspergillus flavus argb gene; (B) verifying a gene knockout strain by a pcr method, amplifying an ap fragment by using the target genes orf, p1 and argb/f amplified by of or and amplifying the bp fragment by using the argb/r and p6 amplified by using the target genes orf, p1 and argb/f amplified by of or to verify that the target gene knocked out is correctly replaced by argb;
FIG. 4 shows the effect of the knockout of the gene srba encoding the ergosterol synthesis regulatory factor on the synthesis of ergosterol in Aspergillus flavus, (A) UV spectrophotometry was used to detect the ergosterol synthesis of srba knockout mutants at 29 ℃; (B) detecting the ergosterol synthesis condition of the srba knockout mutant at 37 ℃ by using an ultraviolet spectrophotometry; (C) quantification of ergosterol component of srba knockout mutants at 29 ℃; (D) quantification of ergosterol composition of srba knockout mutants at 37 ℃;
FIG. 5 shows the effect of the knockdown of srba gene on the high temperature resistance of Aspergillus flavus, (A) the growth phenotype of a knockout mutant of the gene srba encoding the ergosterol synthesis regulatory factor in Aspergillus flavus under different temperature conditions; (B) growth of the srba knockout mutant after addition of hydrogen peroxide at 37 ℃; (B) growth inhibition rate of knock-out mutants of srba in hydrogen peroxide containing medium.
The specific implementation mode is as follows:
through a large amount of researches, the inventor discovers that the expression level of an ergosterol synthetic gene in aspergillus flavus is obviously up-regulated after high-temperature induction, and identifies an ergosterol synthesis regulatory factor srba in aspergillus flavus for the first time. After the srba gene in the aspergillus flavus strain is knocked out by a homologous recombination method, the ergosterol content in aspergillus flavus bacteria cells is obviously reduced at different growth temperatures, the growth of a defective strain is inhibited to a certain extent at a high temperature, and the aspergillus flavus can be killed at room temperature by further adding a strong oxidant.
The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, such as molecular cloning, a laboratory manual, such as sambrook, or conditions as recommended by the product instructions.
Examples
1. Fresh spore collection
Respectively sucking 2 mul of wild aspergillus flavus spore liquid, inoculating the wild aspergillus flavus spore liquid on a pda solid culture medium, and culturing the wild aspergillus flavus spore liquid in the dark at the temperature of 37 ℃ for 7 d; the spores were eluted with 0.001% tween 20 spore eluent, the hyphae were filtered through four layers of mirror paper and the conidia were collected in 10ml centrifuge tubes. The spores are diluted and counted, and the concentration of the spore liquid is uniformly diluted to 106One per ml.
2. Growth of Aspergillus flavus under different temperature conditions
2 mul of the newly collected aspergillus flavus spores are respectively inoculated into the middle of yes, ygt and gmm culture media, and are put into four temperature gradient incubators to be cultured for five days, and as shown in figure 1, the growth of aspergillus flavus at different temperatures has obvious difference. The result shows that the aspergillus flavus grows fastest at 37 ℃, and can generate yellow-green conidiospore pigment in different culture media; aspergillus flavus, although growing faster at 29 ℃, produced less conidiospore pigment; moreover, the growth of the aspergillus flavus is still allowed to grow under the temperature condition of 42 ℃ although the growth is inhibited.
The above shows that the aspergillus flavus temperature has higher tolerance.
3. Expression level of ergosterol synthesis related gene under different temperature conditions
Get 107Adding the freshly collected aspergillus flavus spore liquid into a 6 bottle of 50ml gmm liquid culture medium, and continuously culturing at 29 ℃ and 180r/min for 24 hours; 3 of these flasks were transferred to a shaker at 37 ℃ and incubated for 24 hours with continued shaking at 180 r/min. Collecting mycelium with sterile four-layer gauze, washing with sterile water for 3 times, quickly freezing the mycelium with liquid nitrogen, freeze-drying in a freeze-drying instrument for 24 hr, mincing the mycelium with toothpick, extracting rna, and performing reverse transcription. Respectively taking the reverse-transcribed cdna as templates, and carrying out fluorescent quantitative pcr detection on the afla _036130 gene transcript by utilizing an upstream primer cacgccagtctttggctccg (seq id No.1) and a downstream primer ttgcaatgctcgaccggcag (seq id No. 2);
fluorescent quantitative pcr detection of afla _111350 gene transcripts was performed using the forward primer agtcttcgcactcggcctgt (seq id No.3) and the reverse primer tggattgcagccagtcccca (seq id No. 4);
fluorescence quantitative pcr detection of afla _051080 gene transcripts was performed using the upstream primer cttggccgtaactggcgtgg (seq id No.5) and the downstream primer ccggcaaatccagtgggcag (seq id No. 6);
carrying out fluorescent quantitative pcr detection on the afla _030250 gene transcript by using an upstream primer cgactgggtcagcactggg (seq id No.7) and a downstream primer gtgccccaggctagggtgaa (seq id No. 8);
fluorescent quantitative pcr detection of afla _028640 gene transcripts was performed using the forward primer aatgtgcgtctccgcaacgg (seq id No.9) and the reverse primer atccgggtcgaaggtgtccg (seq id No. 10).
Fluorescent quantitative pcr detection of srba gene transcripts was performed using the forward primer tgtcgtccagcaacttccg (seq id No.11) and the reverse primer cggttccaggtgagatttgc (seq id No. 12).
The fluorescent quantitative pcr result shows (fig. 3), the genes related to ergosterol synthesis and the regulatory gene srba thereof in aspergillus flavus are all up-regulated after high temperature induction at 37 ℃, which indicates that the genes related to ergosterol synthesis and the regulatory gene srba thereof can play a potentially important role in the temperature tolerance of aspergillus flavus.
Construction of a knockout Strain of srba Gene
Searching the ncbi database (https:// www.ncbi.nlm.nih.gov/gene /), a bioinformatics method was used to identify a novel gene with unknown function in the genome of Aspergillus flavus, which is numbered afla _058610(seq id No.13), and which is homologous to the srba gene of Aspergillus fumigatus and thus named srba gene, the srba protein having the amino acid sequence shown in seq id No. 14. In order to research the function of the srba gene in aspergillus flavus in the temperature tolerance of the aspergillus flavus, a fusion fragment knocked out by the srba gene is constructed in vitro, and a 3452bpdna homologous fragment in the aspergillus flavus genome is replaced by an aspergillus flavus argb gene fragment by a homologous recombination method, so that the srba gene in the aspergillus flavus genome is knocked out. FIG. 3A shows the principle of the gene knockout.
The specific method comprises the following steps:
amplifying an upstream homologous arm about 0.7kb fragment from the genome of wild type Aspergillus flavus by pcr using an upstream primer gcttagcaccgaatcttgg (seq id No.15) and a downstream primer ttctaccgaactcatcaccaccgggaattgacaacgaaggtgggag (seq id No. 16);
amplifying a fragment of about 1.0kb of a downstream homology arm from the genome of wild type Aspergillus flavus by pcr using an upstream primer tggtgcccgcattcacatgtcacggggttgatgtcgctgtctaagg (seq id No.17) and a downstream primer caggtgaagattgcgtaaagg (seq id No. 18);
amplifying an Aspergillus flavus argb fragment of about 1.8kb from the genome of wild type Aspergillus flavus with pcr using an upstream primer tcccggtggtgatgagttc (seq id No.19) and a downstream primer cccgtgacatgtgaatgcg (seq id No. 20);
upstream primer catcgctgtcaggcaccac (seq id No.21) and downstream primer ctttcaagatgacgaagtggc (seq id No.22) are used, upstream and downstream homologous arm fragments of aspergillus flavus srba gene are connected with aspergillus flavus argb gene fragment by overlapping pcr to construct a knockout fragment, the knockout fragment is simultaneously introduced into protoplast of aspergillus flavus tjes20.1 delta ku70 delta argb strain by using a homologous recombination method (yang, k.et al. viral, 2018, aug 23,9(1): 1273-once 1286), argb is used as a screening marker, and the knockout positive transformant is screened and identified by pcr. As shown in FIG. 1B, since the wild type Aspergillus flavus wt contains the srba target gene, orf fragments can be amplified by using the internal upstream and downstream primers of (seq id No.11) and or (seq id No.12) of the srba gene, and ap and bp fragments cannot be amplified by using the primers p1(seq id No.15) and argb downstream primer (seq id No.20), and argb upstream primer (seq id No.19) and p6(seq id No. 18). On the other hand, the δ srba knockout strain cannot amplify an orf fragment because it does not have the target gene, but it contains an argb fragment and can amplify ap and bp fragments, respectively (fig. 3).
Effect of srba Gene knockout on Erigerstol Synthesis in Aspergillus flavus
Ergosterol is an indispensable component of fungal cell membranes as an isoprenoid derivative, and is structurally and functionally similar to cholesterol in mammalian cell membranes. As the main sterol component in fungal cell membranes, it is said to be important for the stability of fungal plasma membranes and for the normal functioning of intracellular and extracellular signal transduction. The research detects the ergosterol synthesis situation of an ergosterol synthesis regulation gene srba knockout mutant in aspergillus flavus under different temperature conditions by an ultraviolet spectrophotometry, the result is shown in figure 4, the normal synthesis of ergosterol in the aspergillus flavus cell membrane after the srba gene knockout is blocked, and the ergosterol content is obviously reduced.
6 Effect of srba Gene knockout on Aspergillus flavus temperature tolerance
In order to further study the influence of the synthesis of ergosterol on the temperature tolerance of aspergillus flavus after being hindered, the growth condition of the srba knockout mutant under different temperature conditions is observed in the study. As a result, it was found that, after the srba gene was knocked out, the colony growth of the delta srba knocked-out strain was inhibited compared with that of wild type Aspergillus flavus at 42 ℃ (FIG. 5A). To further improve the sensitivity of the δ srba knockout strain to temperature, the present study found that by adding exogenous 5mm hydrogen peroxide to the medium, even at 29 ℃, the δ srba knockout strain could be significantly inhibited or even killed (fig. 5B).
The important role of ergosterol synthesis regulatory protein srba in aspergillus flavus in the regulation of ergosterol synthesis to respond to the temperature stress process is shown.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Sequence listing
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Met Ala Met Pro Gly Met Gly Ser Asp Phe Gln Leu Phe Ser Pro Ala
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Gln Ser Ser Asp Arg Arg Glu Ser Gln Thr Asp Ser Met Ala Phe Pro
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Gln Ser Ser Pro Glu Asp Ser Gly Gln Asp Trp Thr Gln Trp Met Arg
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Trp Asp Asp His Ser Phe Pro Glu Thr Thr Asn Glu Ile Thr Gln Ser
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Pro Phe Asp Phe Pro Phe ThrSer Pro Gly Thr Ser Ile Gly Lys Gln
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Ser Leu Gly Ala Val Gln Lys Ser Glu Phe Ser Pro Asp Ile Ser Leu
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Asp Cys Val Thr Asn Leu Ser Phe Asp Ser Phe Leu Gly Gln Asp Gly
100 105 110
Gly Gly Gly Leu Pro Pro His Arg Ser Ser Asn Ala Gly Thr Asp Met
115 120 125
Gln Ser Ala Gln Ser Thr Thr Ser Gly Ser Pro Met Ser Leu Ala Gly
130 135 140
Ala Lys Arg Lys Ser Gly Ser Asp Asp Asp Gly Ser Thr Val Ser Gly
145 150 155 160
Leu Ala Pro Pro Gly Lys Lys Met Pro Ser Lys Lys Arg Ala His Asn
165 170 175
Val Ile Glu Lys Arg Tyr Arg Ala Asn Leu Asn Glu Lys Ile Ala Glu
180 185 190
Leu Arg Asp Ser Val Pro Ser Leu Arg Ala Ser Arg His Leu Val Asp
195 200 205
Glu Asp Asp Gly Glu Gly Val Thr Pro Ala Asn Lys Leu Asn Lys Ala
210 215 220
Ser Ile Leu Ser Lys Ala Thr Glu Tyr Ile Arg His Leu Glu Ile Arg
225 230 235 240
Asn Lys Arg Leu Glu Asp Glu Asn Thr Ala Leu Lys Asn Arg Leu Arg
245 250 255
Gln Leu Asp Lys Ala Val Asp Gln Asn Val Thr Ser Ala Ala Ser Val
260 265 270
Ser Ser Pro Ser Asn Tyr Thr Glu Ser Gly Ala Ser Ser Ser Pro Ser
275 280 285
Val Phe Ser Asn Ala Glu Asp Val Pro Ser Asp Pro Ser Pro Thr Ser
290 295 300
Leu His Pro Pro Glu Gly Met Ile Lys Val Pro Asp Ala Phe Lys Arg
305 310 315 320
Met Arg Ala Ala Ala Pro Arg Asp Glu Ser Trp Ser Gln Ser Tyr Ile
325 330 335
Gln Tyr Pro Ser Ser Gly Asn Ser Ser Pro Gln Pro Gly Ser Arg Lys
340 345 350
Arg Ser His Tyr Pro Asn Lys Tyr Met Leu Gly Ala Leu Ala Gly Leu
355 360 365
Met Val Phe Glu Gly Met Ser Ser Glu Lys Lys Thr Glu Ser Thr Ala
370 375 380
Lys Gly Leu Leu Ala Ile Pro Val Asn Phe Phe Asn Asn Leu Gln Leu
385 390 395 400
Pro Pro Ala Gly Tyr Trp Val Ala Trp Ala Arg Ser Phe Trp Tyr Ser
405 410 415
Trp His Ala Arg Ala Ile Ser His Phe Leu Ile Leu Ala Thr Leu Val
420 425 430
Val Gly Ser Ala Phe Ile Val Phe Val Tyr Leu Phe Asn Ser Gly Gly
435 440 445
Pro Gly Leu Gln Tyr Ser Ser Ser Lys Ser Ala Ser Ala Thr Leu Ser
450 455 460
Ser Ser Asn Phe Arg Arg Gln Ala Trp Leu Thr Ser Ile Gln Arg Val
465 470 475 480
Gly Val Pro Arg His Thr Phe Phe His Glu Trp Tyr Val Val Thr Ser
485 490 495
Arg Cys Phe Glu Tyr Val Leu Arg Cys Leu Leu Gly Trp Lys Leu Tyr
500 505 510
Ser Trp Ala Thr Gly Ile Thr Glu Glu Asp Glu Lys Gly Arg Val Lys
515 520 525
Thr Trp Asp Ile Ala Ile Asp Ala Gln Leu Ala Gly Gly Asp Ala Glu
530 535 540
Val Ser Lys Ser Arg Leu Val Leu Thr Ile Phe Ala Ala Gly Thr Leu
545 550 555 560
Pro Arg Ser Pro Met Arg Met Met Leu Lys Ala Leu His Val Arg Ile
565 570 575
Leu Leu Trp Arg Val Gly Asp Pro Gly Ser Trp Thr Phe Asn Val Ser
580 585 590
Asn Asp Val Ala Arg Ser Leu Ala Arg Tyr Gln Trp Asp Leu Ala Arg
595 600 605
Lys Met Asn Ser Ser Leu Pro Glu Asp His Pro Asp Val Leu Pro Ser
610 615 620
His Leu Ser Ala Leu Leu Glu Leu Asp Ser Glu Glu Val Met Ile Asp
625 630 635 640
Ser Val Ile Gln Arg Ala Ala Asn Leu Thr Trp Asn Arg Pro Thr Gln
645 650 655
Glu Gly Thr Asp Gly Asp Glu Ala Leu Leu Asp Val Val Glu Glu Asp
660 665 670
Pro Ala Ile Gln Ser Ser Leu Asp Ala Leu Ala Ala Trp Trp Ser Ser
675 680 685
His Leu Leu Gln Arg Ala Leu Leu Arg Tyr Phe Glu Ala Ser Ala Gly
690 695 700
Gly Gln Asp Arg Arg Lys Ser Arg Asp Ala Phe Lys Ser Lys Leu Arg
705 710 715 720
Thr Ala Leu Asn Val Ala Pro Gln Pro Ser Ala Ala His Thr Arg Ala
725 730 735
Leu Val Met Gln Ala Val Phe Phe Glu Gln Asp Arg Val Ala Asn Ile
740 745 750
Asn Met Val Leu Ala Ala Leu Pro Lys Asp Lys Gly Asn Thr Lys Lys
755 760 765
Ala Gln Ala Ser Asn Phe Leu Asp Ser Ser Leu Pro Val Ser Val Arg
770 775 780
Asp Glu Ile Asn Ile Ala Val Arg Cys Ala Met Ile Ala Ala Ile Phe
785 790 795 800
Thr Ala Arg Thr Thr Gly Asp Thr Ser Leu Pro Ala Ser Phe Thr Met
805 810 815
Gln Lys Ala Val Ser Trp Phe Asn Gln Leu Pro Leu Asp Pro Val Glu
820 825 830
Leu Thr Leu Leu Gly Phe Ala Ala Thr Tyr His Leu Leu His Val Leu
835 840 845
Ala Ser Asp Thr Asp Tyr Met Asn Ser Ser Asp Ser Ser Val Pro Ser
850 855 860
Ser Pro Val Ser Arg His Arg Ser Leu Ser Asp Asn Glu Ser Asp Arg
865 870 875 880
Pro Ala Pro Ser His Asn Pro Lys Cys Ser Pro Leu Ile Pro Asn Leu
885 890 895
Ser Arg Val Ala Ser Glu Leu Ser Tyr Trp Ala Lys Asn Ala Tyr Asn
900 905 910
Pro Ala Phe Tyr Gly Phe Thr Ser His Leu Val Gly Val Val Asp Ala
915 920 925
Glu Cys Lys Thr Leu Cys His Ser Ala Gly Val Asp Leu Ile Asp Tyr
930 935 940
Ser Arg Ile Gln Glu Glu Arg Ser Lys Ala Ile Arg Asn Lys Asp Glu
945 950 955 960
Lys Lys Lys Lys Lys Pro Arg Lys Lys Ser Asn Glu Asp Ser Arg Gln
965 970 975
Thr Arg Trp Ala Thr Ser Asn Ser Lys Pro Ala Arg Cys Pro Ser Pro
980 985 990
Glu Ser Ser Arg Glu Val Glu Ala
995 1000
<210>15
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
<210>16
<211>46
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
ttctaccgaa ctcatcacca ccgggaattg acaacgaagg tgggag 46
<210>17
<211>46
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
tggtgcccgc attcacatgt cacggggttg atgtcgctgt ctaagg 46
<210>18
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
caggtgaaga ttgcgtaaag g 21
<210>19
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
tcccggtggt gatgagttc 19
<210>20
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
<210>21
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
catcgctgtc aggcaccac 19
<210>22
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
ctttcaagat gacgaagtgg c 21
Claims (4)
1. A method for inhibiting temperature tolerance of Aspergillus flavus, which is characterized by comprising the following steps:
s1: the expression level of an ergosterol synthesis gene and a transcription factor srbA gene or an ergosterol synthesis and transcription factor SrbA protein in aspergillus flavus is reduced by means of gene knockout, RNA interference or drug treatment and the like;
s2: the aspergillus flavus with low expression level of ergosterol synthetic gene and transcription factor srbA gene or ergosterol synthetic gene coding protein and transcription factor SrbA protein grows at the temperature of 42 ℃ over 42 ℃.
S3: at room temperature, 5mM of hydrogen peroxide is added into aspergillus flavus with low expression level of ergosterol synthesis gene and transcription factor srbA gene or ergosterol synthesis gene coding protein and transcription factor SrbA protein.
2. The method as claimed in claim 1, wherein the step S2 is carried out by suppressing the expression level of ergosterol synthesis gene and its regulatory gene or its encoded protein in Aspergillus flavus by molecular genetic manipulation or ergosterol synthesis inhibitor.
3. The method as set forth in claim 1, wherein the Aspergillus flavus is cultured at a temperature of 42 ℃ to over 42 ℃ in step S2.
4. The method as claimed in claim 1, wherein in the step S3, 5mM of strong oxidant, hydrogen peroxide, is added after the step S1 is satisfied, and the culture temperature of Aspergillus flavus is room temperature.
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