CN109666680B - A non-aflatoxin-producing strain ΔAflsnt2 and its application in preventing and controlling Aspergillus flavus contamination - Google Patents

Abstract

The present invention provides a non-aflatoxin-producing strain ΔAflsnt2 and a method for preventing and controlling Aspergillus flavus contamination, wherein the non-toxin-producing strain basically does not express the pathogenicity-related gene afllsnt2 of Aspergillus flavus. The invention is beneficial to fundamentally relieve the production and accumulation of aflatoxin in the early stage of aflatoxin pollution, thereby effectively controlling and reducing the infection rate of toxin-producing Aspergillus flavus on crops, and achieving the purpose of effectively controlling aflatoxin pollution. Compared with the above-mentioned aflatoxin contamination detection means and post-contamination detoxification technology, the present invention controls the contamination of Aspergillus flavus from the source, and the cost is significantly reduced.

Description

A non-aflatoxin-producing strain ΔAflsnt2 and its application in preventing and controlling Aspergillus flavus contamination

technical field

The invention belongs to the field of microbiology, and in particular relates to a non-aflatoxin-producing strain ΔAflsnt2 and an application for preventing and controlling Aspergillus flavus pollution.

Background technique

The genus Aspergillus currently includes more than 200 different species. Aspergillus flavus is one of the common asexual species, belonging to saprophytic fungi and the second largest pathogenic fungus after Aspergillus fumigatus ^[1] . Distributed in natural soil, air, water, plants and agricultural products. Aspergillus flavus is also a zoonotic pathogen, which can parasitize in food, food and feed for growth and reproduction, and produce aflatoxin, of which aflatoxin B1 (AFB1) is the most harmful, and it has been discovered so far. One of the most carcinogenic and toxic natural pollutants. According to the Food and Agriculture Organization of the United Nations (FOA), about 25% of the world's crops are contaminated with fungi and mycotoxins every year, causing economic losses in the hundreds of billions of dollars. Aflatoxin B1 (AFB1) is extremely toxic, 68 times that of arsenic and 10 times that of potassium cyanide, and the carcinogenic potential of AFB1 is several thousand times that of 3,4-benzopyrene. In 1993, the International Agency for Research on Cancer (IARC) has The aflatoxin AFB1 is listed as a class I carcinogen, and AFB1 is also stipulated by the National Quality Inspection Bureau as one of the mandatory inspection items for most food and dairy products. As a result, aflatoxins cause great harm to human and animal health. In my country, the contamination of peanuts and corn by Aspergillus flavus is very common, and the contamination of Aspergillus flavus in livestock and poultry feeds and aquatic feeds is even more serious. As a result, aflatoxins endanger human health through the food chain. Due to the stable physical and chemical properties of aflatoxin, it can be decomposed under the conditions of 237~299 °C, and even in daily cooking conditions or pasteurization, the pollution cannot be removed. Therefore, the pollution of Aspergillus flavus is very harmful, and it is of great significance to study the prevention and control of Aspergillus flavus pollution.

The ability of Aspergillus flavus to produce spores and toxins is closely related to its growth, spread, reproduction and toxicity, which affects the pathogenicity of Aspergillus flavus. With the completion of the whole genome sequencing of Aspergillus flavus, the screening and research of genetic factors related to its pathogenicity are increasing day by day. However, it has been found that even under the same genetic background, the production of toxins of Aspergillus flavus may vary greatly. This suggests that Aspergillus flavus is very sensitive to the influence of non-genetic factors. Among them, the research on the influence of external environmental factors such as light, nitrogen source, carbon source, temperature, nutrition, water activity, pH value, oxidation state, osmotic pressure and group sensibility on the morphology and toxin production of Aspergillus flavus has been carried out for many years. The study of non-genetic factors regulating the pathogenicity of A. flavus in vivo is still in its infancy.

The growth, reproduction, infection and secondary metabolism of Aspergillus flavus are intricate processes at the molecular level, involving many transcription factors regulating the expression of various genes. Studies have found that various transcription factors in Aspergillus flavus play a very important role in its life metabolic activities, especially in the formation of Aspergillus flavus conidia, the biosynthesis of secondary metabolites and the host infection process by Aspergillus flavus. All kinds of transcriptional regulators play a very important role in the control of A. flavus. Therefore, in-depth study of how transcription factors regulate these basic life metabolic activities will provide better ideas for scientific and rational control of Aspergillus flavus. There is no research report on the Snt2 transcription factor in Aspergillus flavus, so it is unknown whether Snt2 has an effect on the pathogenicity of Aspergillus flavus.

After extensive research, the inventors isolated an Aspergillus flavus pathogenicity-related gene for the first time in Aspergillus flavus, which the inventor named aflsnt2 gene. Therefore, the present invention discloses a non-toxigenic strain of Aspergillus flavus ⊿ aflsnt2 strain, in which the pathogenicity-related gene aflsnt2 of Aspergillus flavus is basically not expressed.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a non-aflatoxin-producing strain ΔAflsnt2 and the application of preventing and controlling Aspergillus flavus pollution, in which the aflsnt2 gene is not expressed or is under-expressed.

In a specific embodiment, the non-virulent strain:

(1) deletion of aflsnt2 gene or gene segment;

(2) Compared with wild-type Aspergillus flavus, the number of spores produced by the non-virulent strain decreased;

(3) Compared with wild-type Aspergillus flavus, the non-toxigenic strain does not produce aflatoxins; the aflatoxins include: aflatoxin B1 (Aflatoxin B1, AFB1) or aflatoxin B2 (Aflatoxin B2, AFB2).

(4) Compared with wild-type Aspergillus flavus, the expression of toxigenic-related genes in the non-toxigenic strains significantly decreased;

(5) Described toxigenic related genes include: AflR gene, AflS gene, AflA gene, AflB gene, AflC gene, AflD gene, AflE gene, AflF gene, AflG gene, AflH gene, AflI gene, AflJ gene, AflK gene , AflL gene, AflM gene, AflN gene, AflO gene, AflP gene or AflQ gene.

(6) with respect to wild-type Aspergillus flavus, the ability of the non-virulent strain to infect the host changes;

(7) The host includes: grains and their products, beans and their products, nuts and their products, vegetable oils and their products, seasonings and spices and their products and feed;

(8) The ability to infect the host includes: colonizing the host to form hyphae, producing spores, and producing aflatoxin AFB1 or AFB2.

In another specific embodiment, the non-toxigenic strain is obtained by knocking out the aflsnt2 gene or gene fragment from the chromosome of Aspergillus flavus strain CA14 by homologous recombination, and the aflsnt2 gene:

(1) has the nucleotide sequence shown in SEQ ID NO: 1; or

(2) A truncated nucleotide sequence shown in SEQ ID NO: 1, and the gene does not encode AflSnt2 protein or encodes AflSnt2 with reduced activity (preferably reduced activity by 50%, more preferably reduced by 80%, more preferably inactive) protein or protein fragment.

The protein encoded by the aflsnt2 gene has the amino acid sequence shown in SEQ ID NO: 2.

The advantages of the present invention are:

The research on Aspergillus flavus contamination currently mainly focuses on the toxin production and toxin pathogenic mechanism of Aspergillus flavus, the improvement of the detection method of aflatoxin contamination of agricultural products, and the treatment of aflatoxin contamination. Little research has been done on contamination of crops, including feed and food. The invention is beneficial to fundamentally relieve the production and accumulation of aflatoxin in the early stage of aflatoxin pollution, thereby effectively controlling and reducing the infection rate of toxin-producing Aspergillus flavus on crops, and achieving the purpose of effectively controlling aflatoxin pollution. Compared with the above-mentioned aflatoxin contamination detection means and post-contamination detoxification technology, the present invention controls the contamination of Aspergillus flavus from the source, and the cost is significantly reduced. Therefore, the present invention has potentially enormous economic and social benefits.

Description of drawings

Figure 1. The strategy of knocking out aflsnt2 gene in Aspergillus flavus and its chromosomal restriction map (Figure 1A), Southern hybridization map (Figure 1B) and semi-quantitative RT-PCR detection of the expression level of aflsnt2 gene in ⊿ aflsnt2 knockout strains (Figure 1C).

Figure 2. Colony morphology (Figure 2A) and sporulation number statistics of Aspergillus flavus ⊿ aflsnt2 strain and control strain on PDA medium, and analysis of related regulatory genes (Figure 2B, 2C).

Figure 3. TLC analysis of AFB1 production by A. flavus ⊿ aflsnt2 strain and control strain in YES medium (Fig. 3A, 3B) and transcript level analysis of toxigenic genes (Fig. 3C).

Figure 4. Sclerotium production of Aspergillus flavus ⊿ aflsnt2 strain and control strain on WKM medium (Figure 4A), statistics of the number of sclerotia (Figure 4B), and analysis of the transcription level of sclerotium-related genes (Figure 4C).

Figure 5. Morphology of Aspergillus flavus ⊿ aflsnt2 strain and control strain after 5 days of infection of peanut and maize seeds (Figure 5A), statistical analysis of the number of spores produced (Figure 5B), and TLC analysis of aflatoxin content in seeds (Figure 5C) . According to the TLC analysis results, the toxin production of the ⊿ aflsnt2 strain was displayed in a bar graph (Fig. 5D).

Detailed ways

As used herein, the A. flavus aflsnt2 gene (SEQ ID NO: 1) is indicated by italic aflsnt2 , the non-toxigenic strain of A. flavus is indicated by ⊿ aflsnt2 , or the aflsnt2- deleted strain, and the complemented strain of A. flavus is indicated by ⊿ aflsnt2-C , the wild-type strain of Aspergillus flavus is represented by WT. Aspergillus flavus aflsnt2 protein (SEQ ID NO: 2) is represented by normal aflsnt2.

As used herein, "isolated" refers to the separation of a substance from its original environment (in the case of a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are isolated and purified if they are separated from other substances present in the natural state. .

As used herein, the words "comprising", "having" or "including" include "comprising", "consisting essentially of", "consisting essentially of", and "consisting of"; " Consists mainly of", "consisting essentially of" and "consisting of" are subordinate concepts of "contains", "has" or "includes".

The invention provides a non-toxigenic strain of Aspergillus flavus. The aflsnt2 gene is substantially not expressed in this non-toxigenic strain. The "substantially not expressed" means that the aflsnt2 gene is not expressed or is expressed at a low level in the non-toxigenic strain of Aspergillus flavus. Wherein, the low expression of the aflsnt2 gene means that the expression of the aflsnt2 gene in the non-virulent strain is lower than 20% of the wild-type Aspergillus flavus; preferably, it is lower than 10% of the wild-type Aspergillus flavus; more preferably, it is lower than the wild-type Aspergillus flavus. 5% or less of Aspergillus flavus, and the best is less than 2%. The non-toxin-producing strain of Aspergillus flavus can be used to study the effect of substantially no expression of the aflsnt2 gene on the expression of other genes in Aspergillus flavus, and to study the sporulation, toxin production and infection of Aspergillus flavus after the substantially no expression of the aflsnt2 gene. Pathogenic conditions such as the host.

Strains that basically do not express the aflsnt2 gene can be constructed by various techniques of gene suppression, gene silencing, and gene knockout. For example, the aflsnt2 gene can be knocked out from the chromosome by a gene knockout technique based on homologous recombination, so that the aflsnt2 gene is deleted; interfering RNA or antisense nucleotides can be designed for the aflsnt2 gene to suppress the expression of the aflsnt2 gene or silence.

As a specific embodiment of the present invention, a method for deleting the aflsnt2 gene is a gene knockout technique, and the aflsnt2 gene has the nucleotide sequence shown in SEQ ID NO: 1, and also includes a truncated form of aflsnt2 The gene (or called aflsnt2 gene fragment), the aflsnt2 protein has the amino acid sequence shown in SEQ ID NO: 2, and also includes the truncated form of the aflsnt2 protein. As long as the aflsnt2 gene fragment is knocked out, the aflsnt2 protein can be not expressed or abnormally expressed, or the activity of the expressed aflsnt2 protein fragment is reduced or inactive.

In a specific embodiment of the present invention, the inventors searched the Aspergillus Comparative Database and used bioinformatics comparative analysis to find a new gene with unknown function in the genome sequence of Aspergillus flavus. The product encoded by the gene is similar to that of Aspergillus oryzae. The homologous protein has 99% similarity, so it is named A. flavus aflsnt2 gene. The present invention constructs the aflsnt2 gene knockout fragment in vitro, and replaces the 3.5 kb DNA homologous fragment in the Aspergillus flavus chromosome aflsnt2 gene with the Aspergillus fumigatus pyrG gene fragment by the method of homologous recombination, thereby knocking out the aflsnt2 gene on the chromosome.

The deletion of aflsnt2 slowed the growth rate of Aspergillus flavus, the colony diameter became smaller, the number of spores produced was reduced, no sclerotia was produced, and no aflatoxin AFB1 was produced, while the complementation of the aflsnt2 gene could restore the colony growth and the number of spores produced. , the number of sclerotia and the production of AFB1. In the seed infection experiment, the spores produced by the ⊿ aflsnt2 strain colonized on the seeds were significantly reduced, and AFB1 was not produced, while the complementation of the aflsnt2 gene could increase the number of spores colonized by Aspergillus flavus and the production of AFB1. These results indicate that aflsnt2 positively regulates the growth of A. flavus, positively regulates the spore production of A. flavus, positively regulates the sclerotia production of A. flavus, positively regulates the production of A. flavus, and also affects the pathogenicity of the fungus.

The aflsnt2 gene can be used as a marker for pathogenicity identification of Aspergillus flavus. For example, the pathogenicity of Aspergillus flavus can be determined by detecting the expression of aflsnt2 gene in Aspergillus flavus to be tested; The expression of the aflsnt2 gene in Aspergillus flavus is 20% or higher, and the better is 50% or higher), then the Aspergillus flavus has general pathogenicity; if compared with the wild-type Aspergillus flavus, the A. flavus aflsnt2 gene to be tested Low expression or no expression, the Aspergillus flavus has no pathogenicity.

In a specific embodiment, the method also includes: further Aspergillus flavus sporulation, toxin production inhibition or seed infection inhibition test is carried out on the obtained potential material, to further select and determine the effect of inhibiting the pathogenicity of Aspergillus flavus. useful substance.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to normal conditions such as people such as Sambrook, molecular cloning: laboratory guide (New York: Cold Spring Harbor Laboratory Press, 1989) conditions described in, or according to manufacturer's instructions. recommended conditions. Percentages and parts are by weight unless otherwise indicated.

I. Materials and Methods

1. Aflatoxin extraction and analysis

Inoculate the YES liquid medium with Aspergillus flavus spores to a concentration of 10 ⁶ /ml, continue to culture in the dark at 29°C for 6 days, add 2 mL of liquid medium to an equal volume of chloroform, mix by shaking, centrifuge at high speed for 5 min, and absorb the organic The phase liquid was dried and redissolved in 200 µl of chloroform and 5 µl was sampled for thin-layer chromatography (TLC) analysis.

2. Seed Infection Experiment

Peanut and corn seeds were carefully removed the germ, peeled the peanuts, soaked the intact seeds in 0.05% sodium hypochlorite for 3 min, transferred to sterile water for 30 s, soaked in 70% ethanol for 5 s, and then rinsed in sterile water for 1 min, soak the peanut cotyledons with 20 ml spore solution (final concentration 10 ⁵ /ml) after draining the water, soak the blank control with sterile water, 50 rpm, 30 min, place 20 cotyledons of similar weight in each petri dish, and cultivate Three layers of moistened filter paper were placed in the dish and incubated at 29°C for 5 days; the infected peanut cotyledons were placed in 10 mL of spore eluate containing 0.01% Tween 20, vortexed for 1 min, and 1 ml was taken. For spore counting, add an equal volume of chloroform to the rest of the solution, shake at 150 rpm for 30 min, stand at room temperature for 10 min, vortex to mix, centrifuge at 2000 rpm for 15 min, collect the lower organic phase liquid, and redissolve in 500 μl chloroform after drying. , take 5 μL of spot TLC plate.

II. Examples

Example 1. Knockout of aflsnt2 gene in Aspergillus flavus

To study the function of the A. flavus aflsnt2 gene in A. flavus morphogenesis and virulence expression, the aflsnt2 gene was first knocked out in A. flavus.

Figure 1A shows the gene knockout strategy. The aflsnt2 gene knockout fragment was constructed in vitro, and the 3.5 kb DNA fragment in the chromosomal aflsnt2 gene was replaced with AfpyrG by homologous recombination, thereby knocking out the aflsnt2 gene on the chromosome.

The specific method is as follows:

Using the 5' primer CTTCTCGAATTCCCCTTCATGACACTCTCC (SEQ ID NO: 3); and the 3' primer GCTAAATCAGGATGGGTTGGAGGGTGAC (SEQ ID NO: 4); the upstream fragment of about 1.2 kb was amplified by PCR from the genomic DNA of Aspergillus flavus CA14 strain;

Using the 5' primer GTCACCCTCCAACCCATCCTGATTTAGCGCCTCAAACAATGCTCTTCACCC (SEQ ID NO: 5); and the 3' primer ACACGGTCCAAACAACACAAAGGAGATGCGTCTGAGAGGAGGCACTGATGC (SEQ ID NO: 6); the pyrG gene fragment of about 1.9 kb was amplified from the genomic DNA of Aspergillus fumigatus by PCR;

Using the 5' primer TAGATCACCCAGCGGGCCACAA (SEQ ID NO: 7); and the 3' primer GACTCAAATGGAAATCCCGTCGTGCC (SEQ ID NO: 8); a downstream fragment of about 1.4 kb was amplified by PCR from the genomic DNA of Aspergillus flavus CA14 strain;

The above three fragments were ligated together by fusion PCR method to construct aflsnt2 knockout fragment, which was introduced into Aspergillus flavus CA14 strain, and positive transformants were screened on medium without uracil and uridine. The aflsnt2 gene on the chromosome is replaced by the homologous recombination of the two homologous fragments upstream and downstream of the knockout fragment and the homologous fragments upstream and downstream of the aflsnt2 gene on the chromosome, thereby knocking out the aflsnt2 gene on the chromosome. The genotypes of correctly inserted transformants were determined by Southern blotting. The genomic DNA of these strains was digested with HindIII and hybridized with the probe (SEQ ID NO: 9). The hybridization results showed that the wild-type strain showed a hybridization band of about 5.3 kb, and the ⊿ aflsnt2 deletion strain showed a hybridization band of about 2.8 kb. Figure 1B shows a map of Southern blot analysis of each strain during aflsnt2 knockout. At the same time, the inventors further verified the transcription level of aflsnt2 gene in these strains by semi-quantitative qRT-PCR (Fig. 1C).

Example 2. The effect of knockout of aflsnt2 gene on sporulation of Aspergillus flavus

The aflsnt2 gene was knocked out in Aspergillus flavus by homologous recombination, and Southern blot analysis proved that the knockout was successful. In order to test whether the deletion of the aflsnt2 gene will affect the sporulation of Aspergillus flavus, the inventors cultured them on PDA medium at 37°C (Fig. 2A) in the dark for 5 days, and observed the sporulation of the following strains. The number of green spores produced by the ⊿ aflsnt2 strain was significantly reduced compared to the wild-type strains WT and apoplectic strains, as demonstrated by statistical analysis of the data (Fig. 2B).

The results indicated that the deletion of aflsnt2 gene could affect the sporulation of Aspergillus flavus.

Example 3. The effect of knockout of aflsnt2 gene on toxigenicity of Aspergillus flavus

In order to detect whether the deletion of the aflsnt2 gene will affect the toxin production of Aspergillus flavus, the inventors inoculated the spores in the YES liquid medium to a final concentration of 10 ⁶ /ml, and cultured them for 6 days under continuous dark conditions at 29°C to extract the toxins. Toxicity of each strain was analyzed by TLC. The results showed that the WT strain produced a large amount of aflatoxins AFB1 and AFB2, while ⊿ aflsnt2 obviously did not produce AFB1 and AFB2, which was also demonstrated by statistical analysis of the data (Fig. 3A, 3B).

The present inventors simultaneously detected the transcription levels of the aflatoxin biosynthesis pathway regulatory genes AfllR and AfllS , as well as part of the structural genes AfllC and AfllO by qRT-PCR, and used actin as the internal reference for transcription analysis. Compared with the WT strain, the transcription levels of the above genes were significantly down-regulated in the aflsnt2- deficient strain, and the data trend was consistent with the results of TLC (Fig. 3C).

The above results indicate that the deletion of aflsnt2 gene will affect the toxigenic production of Aspergillus flavus.

Embodiment 4. The knockout of aflsnt2 gene affects the sclerotia production of Aspergillus flavus

In order to detect whether the deletion of aflsnt2 gene will affect the sclerotia production of Aspergillus flavus, the inventors inoculated 1 μl of spore liquid with a concentration of 10 ⁶ cells/ml in WKM medium, and cultured them for 7 days under continuous dark conditions at 37°C. Observe the sclerotia production of the following strains. The results showed that the wild-type strain WT produced a large number of sclerotia, while the aflsnt2- deleted strain obviously did not produce sclerotia, which was also demonstrated by statistical analysis of the data. (Figure 4A, 4B).

The inventors simultaneously detected the transcription levels of Aspergillus flavus nuclear synthesis-related genes nsdC , nsdD and sclR by qRT-PCR, and used actin as an internal reference for transcription analysis. Compared with the WT strain, the transcription levels of the above genes were significantly down-regulated in the ⊿ aflsnt2 strain (Fig. 4C).

The above results indicate that the deletion of aflsnt2 gene can significantly affect the sclerotia production of Aspergillus flavus.

Embodiment 5, the impact of knockout of aflsnt2 gene on the pathogenicity of Aspergillus flavus

The ability to produce spores and toxins is closely related to the pathogenicity of Aspergillus flavus, and the pathogenicity of strains that cannot produce spores or toxins will be lost or greatly reduced.

The ⊿ aflsnt2 strain constructed by the inventors has defects in the ability of sporulation and toxin production, so the inventors tested the pathogenicity of ⊿ aflsnt2 through peanut and corn seed infection tests. Using the wild-type strain WT as a positive control and sterile water as a blank control, the pathogenicity of ⊿ aflsnt2 was detected.

The intact seeds after degermination were sterilized with sodium hypochlorite and alcohol, soaked in 20 ml of spore solution (final concentration 10 ⁵ /ml) at 29 °C, and cultivated in the dark for 5 days; the infected peanuts were eluted and counted, and the yellow seeds were extracted. Aspergillus toxin was detected by TLC. The WT strain could produce dense green spores after 5 days of infection. In contrast, the ⊿ aflsnt2 strain could produce more dense green spores after 5 days of infection, and the spore number statistics also showed that the number of spores was significantly reduced (Fig. 5A). , 5B). Similarly, the WT strain was able to produce toxin normally, while ⊿ aflsnt2 could not produce aflatoxin (Fig. 5C, 5D). These results indicated that the pathogenicity of ⊿ aflsnt2 strain was significantly lower than that of wild-type WT strain.

Therefore, AflSnt2 regulates the pathogenicity of Aspergillus flavus and is an important pathogenic factor.

The above descriptions are only preferred embodiments of the present invention, and all equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope of the present invention.

SEQUENCE LISTING

<110> Fujian Agriculture and Forestry University

<120> A non-aflatoxin-producing strain ΔAflsnt2 and its application in preventing and controlling Aspergillus flavus contamination

<130> 9

<160> 9

<170> PatentIn version 3.3

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caggagacca caaaaaagaa gggacgcacc aacgataaag acagtggcgc gacttcgact 3060

gctacatccg tggaaccggc gccaaagaag aagccagcac cagagaaggc cccggaaccg 3120

gcaccgattg tccccgatcc acccaaggct aagacacttc cttgtgccgt ctgtaacaag 3180

atggaaccta tgggtgatca acatttgtcc tgtagggact gtcggctaac cgttcaccga 3240

agctgctatg gagtcagccc gtcacggaat tgcgttaaat ggctgtgcga catgtgtacc 3300

aatgacagaa atccgttgtt ctcgacatgt tacgagtgtg ttctatgccc tgtgacctgg 3360

acagagcatg aattgatgga agctccacgg tcgacgcaca agaagaagac tgagcgggat 3420

cgggaaaagg agaggttaga gaaggagatg gttagtgaag ctatcaaact ctatcgacaa 3480

aggcaggaag ccgtaggcaa gccgatcggt cctcgggaac cgctgaagcg gactgatggt 3540

aacaactggg tccatgtcgc ttgtgcggtt tggactcccg agatcaagtt tgccaacgcg 3600

aaagagcttg aacctgctga aggatttgca cttatttccg cagacaagta ccgtgaggtt 3660

tgcaaaattt gcaagtcaaa caatggtgct tgtgtaccat gccatttcag tggctgcaat 3720

gttcagttcc atgttggttg cgcgttccag gcgcagtaca cgtttggctt cgatatcacg 3780

ccgttaaga gctctaggag agataccgtg cagagtgtgc ggctcaagga tgaggttgga 3840

gtggcctctg cgggaatctg gtgccctcat catacagtac cgtctgttgt tcatgcagta 3900

ggcgagccta cggaggaaga aggcatcaat gccctccagc gattcgtcca aaactacaaa 3960

caagccgacc tttccttgac tgggactcta cgtagagcgg cgtatgttca gcagtctatt 4020

atcgcatcac aacacagtgc gacatcggcc ggacaccgac gggcatctgc tgtcaacggc 4080

gttaccgctc ctccgcctac aacgaaggat acctctaaga atacaggggt gtcccctgag 4140

gaagctacgg acgagatggc cattgattcc gaaaatcatg caccgaccca ggtcactggc 4200

acagatgcaa caagaaaatg tgcgcgttgc tctaccgcat acacccctcg gtggtggcct 4260

atcgataagt cacgacgaac aacagctgct gatcacaggc cacctctact caacggagct 4320

gggatgaatg aaccaagatt tccgcctgcg gcctcagcaa gccctccata tattcatcga 4380

aacccctctc aacattccct catgaagctt aacggtgaat tcaattctgc agacaagaat 4440

ggactagcga gtaatcccga agcgtcattg gaccaacaga accaggactc atacgagtgt 4500

cataaatgcc atctcaaaca gatacctgcc cagccctctc cagagcctcg gccgtctcct 4560

tactcggctc agcggccagt cctccccgcc ccccgtcttc cagaatatca caatcattct 4620

tacggccctc atgcccatcc tccacaaacc ggggtgctgc ctaggcctct tggtccgccg 4680

ccttccaatg ggcctgaatg gtatccggga tatgaacagc gtcccggcga ttatggggat 4740

aagcttcgca atggcattcc agttgctagc taccgcggag gacctccacc acccccaccg 4800

caccatatga atggcttcca gcccgctcct tcacaccatg ccccgccgca tcattacacg 4860

agtggagctc atcctcctcc acctccccag ccgtttccta ctcatcagag cccctatggg 4920

ccagtctcta taccctctcc tcatctttct cacccagctg ggcctcgacc atacgcccca 4980

tcggcatcac ctccggatgt gcactctaca atggttcgac actccccaca gcactcactc 5040

agtgcattga acgggggacc accggcacgt gtctattctg tcgatcgcgt tcttagtgcg 5100

ccaacgcagt cacctcccgt ttcgcaagca catgttgatc cacgaggtcg gacgccaccc 5160

ggaaagttag acgatgcacc tgtcaccgcg cccgcaggtc cgacgagcag catcaggcat 5220

acgaatgtca acgggacaaa tggcggctcc ggcgctagcg caagcccatc tctcaagaat 5280

ttactctctt aa 5292

<210> 2

<211> 1713

<212> PRT

<213> Artificial sequences

<400> 2

Met Ala Ser Asp Arg Ser Pro Pro Gln Lys Ser Gln Gln His His Val

1 5 10 15

Arg Pro Asn Ser Ser Ser Ser Ala Ala Ala Thr Ala Ala Gly Gln Arg

20 25 30

Arg Met Pro Thr Pro Gly Gln Ser Ser Arg Thr Gly Ser Ala Asp Ser

35 40 45

Pro Thr Pro Gln Gly Leu Ala Thr Leu Asn Glu Arg Gln Ser Gln Pro

50 55 60

Met Thr Thr Ser Thr Ser Ala Ser Glu Val Ala Gly Thr Glu Pro Pro

65 70 75 80

Ser Ala Ser Ala Thr Pro Ala Pro Tyr Gly Thr Arg Ser Arg Gly Arg

85 90 95

Asn Ala Ala Pro Arg Pro Asn Tyr Ala Glu Asp Arg Asp Ile Asp Met

100 105 110

Asp Leu Glu Ile Ala Gln Pro Ala Thr Lys Ala Ala Lys Arg Thr Asn

115 120 125

Gly Val Pro Asn Gln Ser Ala Asn Gly Thr Lys Thr Asp Gly Glu Lys

130 135 140

Ser Ala Pro Ser Ser Asn Ser Arg Lys Ser Gln Thr Ala Val Asn Gly

145 150 155 160

Thr Ser Pro Ala Ser Ala Ala Lys Asp Ser Ile Pro Gly Thr Ser Ser

165 170 175

Phe Ser Ala Lys Leu Glu Glu Ala Asn Gly Ala Ser Asn Ser Arg Lys

180 185 190

Arg Lys Gln Pro Ala Ser Ala Thr Thr Ser Ser Ser Ala Asn Gly Ser

195 200 205

Ala Ser Lys Lys Leu Phe Thr Thr Pro Gly Ala Ser Gln Gly His

210 215 220

Asn Asp Ser Asn Ser Asn Met Val Ser Phe Glu Asn Arg Gly Ala His

225 230 235 240

Leu Glu Asp Gly Lys Leu Thr Ala Asp Asp Gly Thr Thr Phe Ser Ile

245 250 255

Asn Asp His Val Tyr Leu Ile Cys Glu Pro Pro Gly Glu Pro Tyr Tyr

260 265 270

Leu Ala Arg Ile Met Glu Phe Ile Pro Asn Lys Asp Val Pro Ser Gly

275 280 285

Pro Ile Glu Ala Val Arg Val Asn Trp Tyr Tyr Arg Pro Arg Asp Ile

290 295 300

Gln Arg Lys Val Ala Asp Thr Arg Leu Val Phe Ala Ser Met His Ser

305 310 315 320

Asp Thr Cys Pro Leu Thr Ser Leu Arg Gly Lys Cys Gln Ile Lys His

325 330 335

Leu Ser Glu Ile Asp Asp Leu Glu Glu Glu Tyr Arg Lys Thr Arg Asp Cys

340 345 350

Phe Trp Tyr Asp Lys Met Phe Asp Arg Tyr Ile His Arg Tyr Tyr Glu

355 360 365

Val Ile Pro Thr Lys Arg Val Ile Asn Val Pro Ala Asn Val Lys Arg

370 375 380

Val Leu Asp Asp Arg Trp Lys Phe Val Leu Val Glu Ile Gly Lys Arg

385 390 395 400

Lys Glu Leu Thr Ser Ala Val Lys Thr Cys Lys Arg Cys Ser Leu Tyr

405 410 415

Ala Ala Ser Thr Asp Ser Val Asp Cys Ala Val Cys His Asp Thr Tyr

420 425 430

His Met Tyr Cys Val Arg Pro Val Leu Thr Lys Lys Pro Ala Arg Gly

435 440 445

Phe Ala Trp Ala Cys Ala Ala Cys Ser Arg Ala Gln Glu Arg Lys Leu

450 455 460

Glu Ala Arg Asn Thr Pro Ile Leu Gly Glu Ser Gln Ala Glu Val Glu

465 470 475 480

Glu Glu Val Val Glu Glu Glu Glu Glu Glu Glu Pro Asn Gly Ala Asn Gly

485 490 495

Thr Ser Ser Ser Thr Pro Ala Ile Val Glu Glu Glu Ala Pro Arg Pro

500 505 510

Ala Thr Glu Glu Gln Val Ala Gln Ala Arg Met Trp Pro Tyr Arg Tyr

515 520 525

Leu Gly Ile His Cys Arg Val Glu Asp Ala Leu Asp Tyr Asp Asp Arg

530 535 540

Ile Tyr Pro Arg Ala Ser Ser Arg Leu Gly Pro Arg Phe Gln Ala Ile

545 550 555 560

Val Asn Pro Trp Pro Gly Arg Pro Val Glu Tyr Val Lys Pro Thr Asp

565 570 575

Ile Lys Lys Lys Tyr Met Lys Ser Ser Gly Gly Arg Lys Asp Ser Lys

580 585 590

Leu Ser Lys Glu Ala Leu Ala Ala Leu Glu Ala Ala Lys Gln Glu Lys

595 600 605

Ala Asn Arg Pro Lys Trp Val Met Asp Glu Pro Gln Gly Tyr Val Arg

610 615 620

Arg Gly Glu Asp Glu Pro Val Thr Val Asn Gly Lys Gln Val Arg Thr

625 630 635 640

Ala Glu Leu Met Phe Lys Met Pro Thr Ala Thr Gln Ile Pro Ser Arg

645 650 655

Gly Glu Asp Asp Ala Pro Gly Ala Asp Leu Ser Ala Ala Asp Arg Glu

660 665 670

Arg Phe Ile Asp Asp Tyr Met Ala Arg Ala Lys Glu Ile Ala Pro Asp

675 680 685

Leu Gly Val Glu Lys Tyr Ser Thr Asn Phe Leu Asp Lys Ala Leu Glu

690 695 700

Leu Leu Tyr Ala Asn Ser Phe Asp Val Glu Thr Ala Leu Ser Lys Leu

705 710 715 720

Lys Gln Met Asn Lys Tyr Lys Asp Leu Lys Glu Pro His Leu Arg Pro

725 730 735

Glu Glu Val Lys Ala Phe Glu Gln Gly Val Ala Lys Tyr Gly Ser Glu

740 745 750

Trp Arg Asn Leu Thr Lys His Val Gly Thr Val Pro His Tyr Gln Ile

755 760 765

Val Arg Phe Tyr Tyr Met Trp Lys Lys Thr Ala Arg Gly His Gln Ile

770 775 780

Trp Asp His Tyr Glu Gly Arg Arg Gly Lys Lys Glu Ala Lys Arg Asn

785 790 795 800

His Thr Ala Lys Leu Val Asp Asp Val Ala Asp Asp His Asp Asp Ser

805 810 815

Ala Tyr Asp Asn Glu Lys Ala Val Glu Lys Lys Arg Gly Phe Gln Cys

820 825 830

Lys Phe Cys Ser Thr Arg Thr Ser Arg Gln Trp Arg Arg Ala Pro Gly

835 840 845

Ile Pro Pro Gly Thr Thr Thr Pro Ser Glu Pro Ser Ser Lys Gln Arg

850 855 860

Asp Lys Gly Pro Gln Leu Thr Val Ala Leu Cys Leu Arg Cys Ala Leu

865 870 875 880

Leu Trp Arg Lys Tyr Gly Ile Gln Trp Glu Asn Val Asp Glu Val Ala

885 890 895

Lys Lys Ile Ser Gln Ser Gly Asn Lys Ser Trp Arg Arg Arg Val Asp

900 905 910

Glu Glu Leu Leu Thr Gln Leu Leu Ile Ser Thr Glu Thr Pro Ile Ser

915 920 925

Ile Asn Ser Ala Thr Ala Ala Thr Ala Ala Ser Ile Gly Val Pro Val

930 935 940

Ala Ala Asn Pro Gln Val Gln Glu Thr Thr Lys Lys Lys Gly Arg Thr

945 950 955 960

Asn Asp Lys Asp Ser Gly Ala Thr Ser Thr Ala Thr Ser Val Glu Pro

965 970 975

Ala Pro Lys Lys Lys Pro Ala Pro Glu Lys Ala Pro Glu Pro Ala Pro

980 985 990

Ile Val Pro Asp Pro Pro Lys Ala Lys Thr Leu Pro Cys Ala Val Cys

995 1000 1005

Asn Lys Met Glu Pro Met Gly Asp Gln His Leu Ser Cys Arg Asp

1010 1015 1020

Cys Arg Leu Thr Val His Arg Ser Cys Tyr Gly Val Ser Pro Ser

1025 1030 1035

Arg Asn Cys Val Lys Trp Leu Cys Asp Met Cys Thr Asn Asp Arg

1040 1045 1050

Asn Pro Leu Phe Ser Thr Cys Tyr Glu Cys Val Leu Cys Pro Val

1055 1060 1065

Thr Trp Thr Glu His Glu Leu Met Glu Ala Pro Arg Ser Thr His

1070 1075 1080

Lys Lys Lys Thr Glu Arg Asp Arg Glu Lys Glu Arg Leu Glu Lys

1085 1090 1095

Glu Met Val Ser Glu Ala Ile Lys Leu Tyr Arg Gln Arg Gln Glu

1100 1105 1110

Ala Val Gly Lys Pro Ile Gly Pro Arg Glu Pro Leu Lys Arg Thr

1115 1120 1125

Asp Gly Asn Asn Trp Val His Val Ala Cys Ala Val Trp Thr Pro

1130 1135 1140

Glu Ile Lys Phe Ala Asn Ala Lys Glu Leu Glu Pro Ala Glu Gly

1145 1150 1155

Phe Ala Leu Ile Ser Ala Asp Lys Tyr Arg Glu Val Cys Lys Ile

1160 1165 1170

Cys Lys Ser Asn Asn Gly Ala Cys Val Pro Cys His Phe Ser Gly

1175 1180 1185

Cys Asn Val Gln Phe His Val Gly Cys Ala Phe Gln Ala Gln Tyr

1190 1195 1200

Thr Phe Gly Phe Asp Ile Thr Pro Val Lys Ser Ser Arg Arg Asp

1205 1210 1215

Thr Val Gln Ser Val Arg Leu Lys Asp Glu Val Gly Val Ala Ser

1220 1225 1230

Ala Gly Ile Trp Cys Pro His His Thr Val Pro Ser Val Val His

1235 1240 1245

Ala Val Gly Glu Pro Thr Glu Glu Glu Gly Ile Asn Ala Leu Gln

1250 1255 1260

Arg Phe Val Gln Asn Tyr Lys Gln Ala Asp Leu Ser Leu Thr Gly

1265 1270 1275

Thr Leu Arg Arg Ala Ala Tyr Val Gln Gln Ser Ile Ile Ala Ser

1280 1285 1290

Gln His Ser Ala Thr Ser Ala Gly His Arg Arg Ala Ser Ala Val

1295 1300 1305

Asn Gly Val Thr Ala Pro Pro Pro Thr Thr Lys Asp Thr Ser Lys

1310 1315 1320

Asn Thr Gly Val Ser Pro Glu Glu Ala Thr Asp Glu Met Ala Ile

1325 1330 1335

Asp Ser Glu Asn His Ala Pro Thr Gln Val Thr Gly Thr Asp Ala

1340 1345 1350

Thr Arg Lys Cys Ala Arg Cys Ser Thr Ala Tyr Thr Pro Arg Trp

1355 1360 1365

Trp Pro Ile Asp Lys Ser Arg Arg Thr Thr Ala Ala Asp His Arg

1370 1375 1380

Pro Pro Leu Leu Asn Gly Ala Gly Met Asn Glu Pro Arg Phe Pro

1385 1390 1395

Pro Ala Ala Ser Ala Ser Pro Pro Tyr Ile His Arg Asn Pro Ser

1400 1405 1410

Gln His Ser Leu Met Lys Leu Asn Gly Glu Phe Asn Ser Ala Asp

1415 1420 1425

Lys Asn Gly Leu Ala Ser Asn Pro Glu Ala Ser Leu Asp Gln Gln

1430 1435 1440

Asn Gln Asp Ser Tyr Glu Cys His Lys Cys His Leu Lys Gln Ile

1445 1450 1455

Pro Ala Gln Pro Ser Pro Glu Pro Arg Pro Ser Pro Tyr Ser Ala

1460 1465 1470

Gln Arg Pro Val Leu Pro Ala Pro Arg Leu Pro Glu Tyr His Asn

1475 1480 1485

His Ser Tyr Gly Pro His Ala His Pro Pro Gln Thr Gly Val Leu

1490 1495 1500

Pro Arg Pro Leu Gly Pro Pro Pro Ser Asn Gly Pro Glu Trp Tyr

1505 1510 1515

Pro Gly Tyr Glu Gln Arg Pro Gly Asp Tyr Gly Asp Lys Leu Arg

1520 1525 1530

Asn Gly Ile Pro Val Ala Ser Tyr Arg Gly Gly Pro Pro Pro Pro

1535 1540 1545

Pro Pro His His Met Asn Gly Phe Gln Pro Ala Pro Ser His His

1550 1555 1560

Ala Pro Pro His His Tyr Thr Ser Gly Ala His Pro Pro Pro Pro

1565 1570 1575

Pro Gln Pro Phe Pro Thr His Gln Ser Pro Tyr Gly Pro Val Ser

1580 1585 1590

Ile Pro Ser Pro His Leu Ser His Pro Ala Gly Pro Arg Pro Tyr

1595 1600 1605

Ala Pro Ser Ala Ser Pro Pro Asp Val His Ser Thr Met Val Arg

1610 1615 1620

His Ser Pro Gln His Ser Leu Ser Ala Leu Asn Gly Gly Pro Pro

1625 1630 1635

Ala Arg Val Tyr Ser Val Asp Arg Val Leu Ser Ala Pro Thr Gln

1640 1645 1650

Ser Pro Pro Val Ser Gln Ala His Val Asp Pro Arg Gly Arg Thr

1655 1660 1665

Pro Pro Gly Lys Leu Asp Asp Ala Pro Val Thr Ala Pro Ala Gly

1670 1675 1680

Pro Thr Ser Ser Ile Arg His Thr Asn Val Asn Gly Thr Asn Gly

1685 1690 1695

Gly Ser Gly Ala Ser Ala Ser Pro Ser Leu Lys Asn Leu Leu Ser

1700 1705 1710

<210> 3

<211> 30

<212> DNA

<213> Artificial sequences

<400> 3

cttctcgaat tccccttcat gacactctcc 30

<210> 4

<211> 28

<212> DNA

<213> Artificial sequences

<400> 4

gctaaatcag gatgggttgg agggtgac 28

<210> 5

<211> 51

<212> DNA

<213> Artificial sequences

<400> 5

gtcaccctcc aacccatcct gatttagcgc ctcaaacaat gctcttcacc c 51

<210> 6

<211> 51

<212> DNA

<213> Artificial sequences

<400> 6

acacggtcca aacaacacaa aggagatgcg tctgagagga ggcactgatg c 51

<210> 7

<211> 22

<212> DNA

<213> Artificial sequences

<400> 7

tagatcaccc agcgggccac aa 22

<210> 8

<211> 26

<212> DNA

<213> Artificial sequences

<400> 8

gactcaaatg gaaatcccgt cgtgcc 26

<210> 9

<211> 1500

<212> DNA

<213> Artificial sequences

<400> 9

cttctcgaat tccccttcat gacactctcc acgagtcctc ctggctttat ccccttccac 60

cgagccccat cctcgtccta attgatcctt tccatggccc ctcccgggta tcaacatcat 120

tcttcacatc gggtcgctcg gcccacacaa tccctttcta acgagaccag cgacaagcat 180

ccatgatatt gctaaaatgt attcccgcta aacccagtaa ccacgtagat cacccagcgg 240

gccacaacgc cgcgttaata gagtaagggg aacaaagtaa tggctatact ataggcacta 300

ctacccaccc caagaaaaaa gaaaaaagaa aaaagaaaaa aacctttagc ggcgcgggaa 360

ggctagccag agcaccggga cactgggcca tgtattaaac cataaaacga aagggaaagg 420

agagtaatcg cgattttttct ctggttcttg gtggatggag gatccgtcat attaacatat 480

cgggactagg ggcgaggacg tggactggtc gggatcctgg acggctttcc tgattcttct 540

tgtctctttc tctcttccct cttctttact ctctctttct ctgggcttgt cgcgtttccc 600

attcttgtcc cacttcttcc tctgcttgtt tccctcgtcc cgattctact tatgttagtc 660

taccatcttc cgttcttctc ttctcccttt cctgcgccta tatcatttcg gccctcgtcg 720

tctcggtagc cctggtcaaa ctctctaact acggccgaac tacgaactag ttccgtcctg 780

ttggttacga cctacttatc cattttctac ctggagcgct caccccacga gtccgcccta 840

gtggcttgat tcttctaccg ccgctacctt catttgacta ttttgacagc ttcagcgtcc 900

cttctgtctc tccttctctt cgtacctcct tgattgccat tcggtttctt caccttatcg 960

caatctgctt ctctacgtct tccacacatt agtgttccgc ttcccctact gttgccacgg 1020

acttctttta cgtccactta gtagtctatt gtcatccttc ggggcctgtc gatcttgata 1080

ttcctcccgc acatctggag catttcattt tctgacattg gcacaaccac tgattttcca 1140

aactcccttg cgggaataag aggagtctta acgggtaaaa aaaaagaaaa gaggaaagtc 1200

actgctgaca actgggatcc gccgtcaagt atcactttca gtccttagtg cggtcaaccg 1260

caccacaggg ccttctcgtc tcccttccga ctttacgttt tcccccttca tattccattc 1320

ggttgatcca tacgaccatt acctccgccc catttgcaaa agcaacgctt atgagggttc 1380

gactaaccgc cacggcttct tgctgaattt cacatcaccg ccttgtaagc agttttgcaa 1440

ggcctggacg ccgccacttg agggggttat atgtcaccct ccaacccatc ctgatttagc 1500

Claims (3)

1. A non-aflatoxin-producing strain ΔAflsnt2 , characterized in that, in the non-toxin-producing strain, the aflsnt2 gene is not expressed or underexpressed; the aflsnt2 gene, its nucleotide sequence is as shown in SEQ ID NO.1 ; The amino acid sequence of the protein encoded by the aflsnt2 gene is shown in SEQ ID NO: 2. 2 . The non-aflatoxin-producing strain ΔAflsnt2 according to claim 1 , wherein it is obtained by knocking out the aflsnt2 gene or gene fragment from the chromosome of Aspergillus flavus strain CA14 by means of homologous recombination. 3 . 3. The application of a non-aflatoxin-producing strain ΔAflsnt2 in the prevention and control of Aspergillus flavus pollution as claimed in claim 1.

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