A method for studying the mechanism of action of turmeric flavonoids on Aspergillus flavus

Abstract

The present invention discloses a research method for the mechanism of action of turmeric flavonoids on Aspergillus flavus, including RNA detection, library sequencing, on-machine sequencing, quality control of RNA sequencing data, comparison of transcriptome data with reference genome, quantitative gene expression and differential gene analysis, functional annotation and enrichment analysis of differentially expressed genes. The present invention uses sequencing technology and analyzes its related biological information to disclose for the first time the mechanism of action of turmeric flavonoids on Aspergillus flavus, providing a new path and idea for preventing and controlling Aspergillus flavus pollution, which is of great significance for the prevention and control of Aspergillus flavus pollution.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a method for studying the action mechanism of aromatic turmeric flavonoids on Aspergillus flavus. Background Art

[0002] Aspergillus flavus is a saprophytic aerobic fungus widely distributed in nature, with strong growth and diffusion capabilities. Aspergillus flavus can parasitize on grains, food and feed to grow and reproduce, and produce aflatoxins. Aflatoxin (AFT) is a group of derivative compounds with a chemical structure similar to difuranocoumarin. At present, there are about 20 types of aflatoxins and their derivatives discovered, including B1, B2, G1, G2, M1, M2, etc. The first four are directly produced after mold contamination and exist in plant food and feed. The latter two are heat-resistant hydroxylated metabolites produced by mammals eating aflatoxin-contaminated feed. Among them, B1 is the most harmful, with significant effects of causing cancer, deformity, inducing mutations and causing immunosuppression. At the same time, ingestion or inhalation of large amounts of this toxin can cause liver damage in humans and animals. Aflatoxins usually infect food crops in warm climates around the world, including corn, peanuts, tree nuts, coconut, spices, cotton seeds, etc. At present, 25% of agricultural crops in the world are contaminated by mycotoxins every year. In my country, aflatoxin contamination of peanuts and corn is relatively common, and aflatoxin contamination of livestock and poultry feed and aquatic feed is even more serious. As a result, aflatoxin endangers human health through the food chain.

[0003] Methods to prevent aflatoxin contamination include prevention and control of Aspergillus flavus and detoxification of aflatoxin. Aflatoxin detoxification methods include mechanical separation, density separation, thermal inactivation, microwave irradiation, solvent extraction, adhesive and adsorbent, chemical detoxification and biological detoxification. These methods are all processed after aflatoxin is produced, which may introduce new food safety issues or high prevention and control costs. In recent years, many studies have reported methods for preventing and controlling Aspergillus flavus.

[0004] Aromatic turmeric flavonoids are one of the main components of the volatile oil from the roots of ginger plants. The highest content is in the root of the traditional Chinese medicine turmeric, and it is one of the compounds that give turmeric oil or turmeric volatile oil a special smell. Aromatic turmeric flavonoids have the effects of resisting different tumor cells, inhibiting the formation of melanin, antifungal, antifertility, and antioxidant (Ji, M.; Choi, J.; Lee, J.; Lee, Y. Int. J. Mol. Med. 2004, 14 (2), 253-256.). Aromatic turmeric flavonoids can be synthesized by a simple three-step method, with low-cost synthetic raw materials and simple operation. The structural formula of aromatic turmeric flavonoids is shown below.

[0005]

[0006] At present, there is no research on the mechanism of action of aromatic turmeric flavonoids in inhibiting Aspergillus flavus. Summary of the invention

[0007] In order to understand the mechanism of action of turmeric flavonoids on Aspergillus flavus, sequencing technology is used to determine various gene changes, and provide important guidance and basis for the pollution prevention and control of Aspergillus flavus. The purpose of the present invention is to provide a research method for the mechanism of action of turmeric flavonoids on Aspergillus flavus.

[0008] In order to solve the above technical problems, a technical solution adopted by the present invention is:

[0009] A method for studying the mechanism of action of turmeric flavonoids on Aspergillus flavus comprises the following steps:

[0010] S1. RNA detection: Take the Aspergillus flavus hyphae treated with aromatic turmeric flavonoids as the test group sample, and the Aspergillus flavus hyphae not treated with aromatic turmeric flavonoids as the control group sample, and perform RNA detection;

[0011] S2, library construction and sequencing;

[0012] S3, on-chip sequencing: single-stranded circular DNA molecules are replicated through rolling circles to form DNA nanoballs, and the resulting DNA nanoballs are added to the mesh holes on the chip for sequencing;

[0013] S4. Quality control of RNA sequencing data;

[0014] S4, alignment of transcriptome data with reference genome;

[0015] S5. Gene expression quantification and differential gene analysis: Calculate the number of mapped reads and transcript lengths in the sample for horizontal comparison between different samples and groups, and perform differentially expressed gene analysis between sample groups;

[0016] S6. Functional annotation and enrichment analysis of differentially expressed genes.

[0017] In some embodiments, the aromatic turmeric flavonoids are derived from the following preparation route:

[0018]

[0019] In some embodiments, the Aspergillus flavus is cultured using PDA medium.

[0020] In a specific embodiment of the present invention, the Aspergillus flavus is cultured at 28° C. for 7 days.

[0021] PDA medium refers to potato dextrose agar medium.

[0022] In some embodiments, the cultured Aspergillus flavus is prepared into an Aspergillus flavus spore suspension.

[0023] In a specific embodiment of the present invention, the Aspergillus flavus spore suspension is 1×10 6 CFU / mL.

[0024] In some embodiments, the method for constructing a library for sequencing is as follows: opening the secondary structure of the digested total RNA sample, fragmenting the mRNA, configuring a synthetic reaction PCR to synthesize a single-strand cDNA and a second-strand cDNA respectively, repairing the ends and connecting the adapters, and then performing PCR amplification, denaturing the PCR product into a single strand, and then configuring a circularization reaction system to react to obtain a single-stranded circular product, digesting the linear DNA molecules that have not been circularized, and obtaining the final library.

[0025] In some embodiments, the RNA detection in step S1 includes detecting sample concentration, RIN / RQN value, and 28S / 18S.

[0026] In some embodiments, the library construction and sequencing in step S2 includes library construction and mRNA purification.

[0027] In a specific embodiment of the present invention, oligo-dT magnetic beads are used for purification of the mRNA.

[0028] In some embodiments, the DNA nanoballs obtained in step S3 are added to the mesh holes on the chip using high-density DNA nanochip technology, and sequencing is performed using a combined probe anchor polymerization technology.

[0029] In some embodiments, in step S4, Fastp software is used to perform quality control on the data, filter the raw data, check the sequencing error rate, and check the GC content distribution.

[0030] In a specific embodiment of the present invention, HISAT2 is used in step S5 to compare the clean reads with the sequence of the reference genome.

[0031] In a specific embodiment of the present invention, the reference genome version is JCVI-AFL1-v3.0.

[0032] In a specific embodiment of the present invention, FPKM is used in step S6 to calculate the number of mapped reads and transcript length in the sample, and FPKM<1 is regarded as no expression; DESeq2 is used to perform differentially expressed gene analysis between sample groups, and the conditions for screening differentially expressed genes are |log2FC|≥1, FDR<0.05.

[0033] In some embodiments, the calculation formula of the FPKM value is as follows:

[0034]

[0035] In some embodiments, the differential gene expression fold, ie, fold change, is calculated by the FPKM.

[0036] Differential gene expression refers to the phenomenon that during cell differentiation, the expression of some luxury genes results in the production of one type of differentiated cells, while the expression of another set of luxury genes results in the appearance of another type of differentiated cells.

[0037] In some embodiments, step S6 further includes:

[0038] The FDR was calculated by correcting the hypothesis testing probabilities for multiple hypothesis testing;

[0039] The differential expression fold was calculated according to the FPKM value.

[0040] The beneficial effects of the present invention are:

[0041] The research method of the action mechanism of turmeric flavonoids on Aspergillus flavus described in the present invention adopts RNA sequencing technology, as well as differential gene analysis, differentially expressed gene function annotation and enrichment analysis, Aspergillus flavus growth and aflatoxin biosynthesis pathway related gene expression analysis, and discloses the action mechanism of turmeric flavonoids on Aspergillus flavus for the first time, providing a new path and idea for preventing and controlling Aspergillus flavus pollution, which is of great significance for the prevention and control of Aspergillus flavus pollution.

[0042] The following abbreviations have the following meanings:

[0043] PDA medium indicates potato dextrose agar;

[0044] DNB indicates DNA nanoball;

[0045] cPAS stands for combined probe-anchored polymerization technique;

[0046] DEGs indicate differentially expressed genes;

[0047] DGEs indicates differential gene expression;

[0048] FC indicates the fold difference in gene expression;

[0049] FDR stands for false discovery rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It is a volcano plot of differential gene expression analysis of different groups in the present invention;

[0051] Figure 2 This is a bar graph of GO biological function enrichment of differentially expressed genes in the present invention;

[0052] Figure 3 This is the KEGG pathway diagram of the differentially expressed genes in the present invention. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment.It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.In addition, if not clearly stated, all reagents adopted in the following embodiments are commercially available, or can be synthesized according to this article or known methods, and for the reaction conditions not listed, they are also easily available to those skilled in the art.

[0054] The technical solution is clearly and completely described below in conjunction with the accompanying drawings.

[0055] Example 1

[0056] Synthesis of turmeric flavonoids

[0057] Lithium diisopropylamide (30 mmol, 3 eq) and 4-methyl-3-pentene-2-one (15 mmol, 1.5 eq) were placed in a reaction bottle containing 2.5 mL of dry tetrahydrofuran. After reacting at 0°C for 1.5 hours, anhydrous cerium trichloride (12 mmol, 1.2 eq) and 10 mL of dry tetrahydrofuran were added. After stirring for 1.5 hours, p-methylacetophenone (10 mmol, 1 eq) and 2.5 mL of dry tetrahydrofuran were slowly added dropwise, and stirred at 0°C for 15 hours. After the reaction was completed, the mixture was acidified with 10% HCl aqueous solution, extracted with methyl tert-butyl ether, and the organic phase was collected and dried with anhydrous sodium sulfate. Column chromatography (stationary phase was 300-400 mesh silica gel, petroleum ether: ethyl acetate = 100:1) was performed to obtain 6-hydroxy-2-methyl-6-(4-methylphenyl)-2-heptene-4-one as a light yellow oil. The yield of compound 2 was calculated to be about 68%.

[0058] Trimethylsilyl chloride (35mmol, 7eq), sodium iodide (35mmol, 7eq), acetonitrile (35mmol, 7eq) were placed in a reaction bottle, 6-hydroxy-2-methyl-6-(4-methylphenyl)-2-heptene-4-one (5mmol, 1eq) and 12.5mL of n-hexane were added, and the mixture was reacted at room temperature for 24 hours. After the reaction, anhydrous potassium carbonate (3.025mmol, 60.5%eq) and 25mL of tetrahydrofuran were added, and the mixture was reacted at room temperature for 20 hours. After the reaction, water was added to quench the mixture, and the mixture was extracted with methyl tert-butyl ether. The organic phase was washed with saturated sodium thiosulfate solution and saturated sodium chloride solution, and dried with anhydrous sodium sulfate. The light yellow-green oily liquid was obtained by column chromatography (the stationary phase was 300-400 mesh silica gel, petroleum ether: ethyl acetate = 200:1), and the yield of aromatic turmeric flavonoids was about 56%.

[0059] Example 2

[0060] Preparation of sequencing samples

[0061] Aflatoxin was inoculated into PDA medium and cultured at 28°C for 7 days. After that, 10 mL of 1% Tween-20 sterile aqueous solution was added. The conidia on the surface were gently scraped with a sterile inoculation loop and dissolved in Tween aqueous solution. The conidia were counted using a hemocytometer and the conidia suspension of Aflatoxin was adjusted to 1×10 6 CFU / mL.

[0062] Weigh an appropriate amount of turmeric flavonoids, add 2 mL of 5% Tween-20 sterile aqueous solution to prepare a turmeric flavonoids suspension and pour it into a culture dish to which 18 mL of PDA medium has been added, so that the final concentration of turmeric flavonoids in the culture medium is 0.00 μg / mL (control group) and 25.00 μg / mL (test group), and sterilize and solidify by ultraviolet irradiation in a clean bench. Use a sterile inoculation loop to dip 10 μL of Aspergillus flavus spore suspension and inoculate it in the center of the sterile culture medium, and culture it at 28°C for 7 days.

[0063] Example 3

[0064] A method for studying the mechanism of action of turmeric flavonoids on Aspergillus flavus

[0065] 1 Experimental methods

[0066] 1.1 RNA sequencing

[0067] The mycelia of the control group and the test group cultured for 7 days in Example 2 were scraped off, quickly frozen in liquid nitrogen, and stored at -80°C for testing. The Aflatoxin samples of the control group and the test group were thawed on ice, centrifuged and mixed, and the sample concentration, RIN / RQN and 28S / 18S were detected using Agilent2100.

[0068] 1.2 Library construction and sequencing

[0069] Open the secondary structure of the digested Total RNA sample, use oligo-dT magnetic beads to enrich mRNA, and fragment it. Prepare the first-strand and second-strand synthesis reaction systems respectively, and PCR to synthesize the first-strand cDNA and second-strand cDNA. Perform end repair and adapter connection. Then PCR amplification. After the PCR product is denatured into a single strand, prepare the circularization reaction system, react at an appropriate temperature to obtain a single-stranded circular product, digest the linear DNA molecules that have not been circularized, and obtain the final library. Finally, use Agilent 2100Bioanalyzer to detect the fragment size and concentration of the library.

[0070] 1.3 Sequencing

[0071] High-density DNA nanochip technology is used to replicate single-stranded circular DNA molecules through rolling circles to form DNA nanoballs (DNBs) and add them to the mesh holes on the chip. Sequencing is performed using combined probe anchor polymerization technology (cPAS) to obtain a sequencing read length of 150bp.

[0072] 1.4 Sequencing data analysis

[0073] Data quality control: Fastp software was used to perform data quality control. The quality control results are shown in Table 1. The quality control data were obtained after raw data filtering, sequencing error rate check and GC content distribution check. The data volume of all samples was greater than 9G, and the base quality Q20 / Q30 was higher than 93%.

[0074] Table 1 Quality control results of sequencing data of each group

[0075]

[0076] Sequence alignment: Alignment of transcriptome data with reference genome HISAT2 was used to compare clean reads with the sequences of the reference genome, the reference genome version was JCVI-AFL1-v3.0, downloaded from https: / / www.ncbi.nlm.nih.gov / genome / ?term=+Aspergillus+flavus. The genome alignment results of each group are shown in Table 2. The percentage of mapped reads to clean reads in all groups reached more than 94.22%, the species tested were consistent with the reference genome, and there was no contamination in the related experiments.

[0077] Table 2 Genome comparison results of each group

[0078]

[0079]

[0080] 1.5 Quantification of gene expression and differential gene analysis

[0081] FPKM was used to calculate the number of mapped reads and transcript length in the sample to obtain the true gene expression level for horizontal comparison between different samples and groups. DEGS analysis between sample groups was performed using DESeq2. FDR was calculated by correcting the hypothesis test probability (p-value) for multiple hypothesis testing. FC was calculated based on the FPKM value. During the study, FPKM<1 was considered as no expression. The conditions for screening differentially expressed genes were |log2FC|≥1, FDR<0.05, and FPKM was calculated according to formula (1).

[0082] 1.6 Functional annotation and enrichment analysis of differentially expressed genes

[0083] First, the differentially expressed genes were annotated with GO, which included molecular function (MF), biological process (BP), and cellular component (CC). The three different characteristic levels can provide a comprehensive description of the effects of differentially expressed genes. Secondly, KEGG pathway annotation was performed, which can help the study obtain the corresponding signal pathways regulated by the differentially expressed genes. Finally, differential functional enrichment analysis was performed on the differentially expressed genes based on GO and KEGG classifications.

[0084] 2. Results

[0085] 2.1 Quantification of gene expression and differential gene analysis

[0086] The study found that there was a significant difference in gene expression levels between the control group and the test group. Among the 12,563 genes in the transcriptional sequencing, 3,686 annotated differentially expressed genes were found, of which 1,668 were upregulated and 2,018 were downregulated. The specific changes are as follows: Figure 1 As shown in the volcano plot, 9.95% of the differentially changed genes had gene expression suppressed by more than 100 times.

[0087] 2.2 Functional annotation and enrichment analysis of differentially expressed genes

[0088] The study found that there were 2457 differentially expressed genes corresponding to 2665 GO functional categories, of which 1793 differentially expressed genes affected biological processes; 536 differentially expressed genes affected molecular functions; and 336 differentially expressed genes affected cell composition. The top 20 genes with significant differences in the functional categories of the three developmental stages in GO enrichment analysis were as follows: Figure 2 A total of 128 KEGG pathways were found, of which 8 were the most significant, mainly involving ribosomes, oxidative phosphorylation, metabolic pathways, tricarboxylic acid cycle, biosynthesis of secondary metabolites, carbon metabolism, glycolic acid and dicarboxylic acid metabolism, and glycolysis. The top 20 KEGG pathways enriched in differentially expressed genes are as follows Figure 3 shown.

[0089] 2.3 Analysis of gene expression related to Aspergillus flavus growth and aflatoxin biosynthesis pathway

[0090] The regulation of turmeric on the expression of genes related to oxidative stress response by turmeric flavonoids was revealed by analyzing the representative genes encoding transcription factors, antioxidant enzymes and oxidized lipoproteins. The expression of specific representative differential genes is shown in Table 3. catA and ppoC were significantly downregulated after turmeric flavonoids treatment, 13077 times and 66799 times of the control group, respectively. The transcription factor atfB involved in stress response decreased by 13.42 times, while other genes such as sod1, mnsod and atfA did not change much. The gene laeA that regulates the production of secondary metabolites of Aspergillus flavus was upregulated by 0.26 times, the gene veA that produces Aspergillus flavus sclerotia did not change significantly, and the gene fluG that regulates the germination of Aspergillus flavus spores was downregulated by 3.89 times, resulting in an overall downward trend. The expression levels of erg4, erg6, and erg7 genes related to ergosterol biosynthesis were significantly downregulated, downregulated by 5.07, 467.4, and 699.91 times, respectively, while erg3 and erg26 were upregulated by 0.14 and 0.28 times, respectively. In general, the inhibitory effect of turmeric flavonoids on ergosterol biosynthesis was much greater than the upregulation. A total of 9 genes related to aflatoxin biosynthesis changed in expression, among which aflA, aflE, aflO, and aflV were downregulated, and aflatoxin biosynthesis was significantly inhibited. The enzymes involved in aflatoxin synthesis are mainly encoded by aflA, aflB, aflC, and hypC. Turmeric flavonoids downregulated the expression of aflA by 12.7 times, upregulated the expression of aflB by 0.18 times, and there was no significant difference in the expression of aflC and hypC. Under the action of aromatic turmeric flavonoids, the expression of aflE was downregulated by 3.84 times, the expression of aflF was upregulated by 0.67 times, and the expression of aflD had no significant difference. Overall, this may lead to the accumulation of deoxyaflatoxins. aflO, aflV, and aflW are essential for encoding the formation of aflatoxin bifuran rings. Aromatic turmeric flavonoids significantly inhibited the expression of aflO and aflV, while aflW was slightly upregulated. aflS and aflR are essential for regulating the biosynthesis pathway of aflatoxin. Under the action of aromatic turmeric flavonoids, the expression of aflR and aflS increased slightly, but the aflatoxin content decreased. Comprehensively evaluated, the expression of most genes related to biosynthesis was significantly downregulated. Genes related to carbon metabolism include creA, creB, creC and creD. Compared with the control group, the expression of creA in the test group did not change much under the action of aromatic turmeric flavonoids, but creB and creD were inhibited to a certain extent, thereby inhibiting the biosynthesis of aflatoxin by inhibiting the expression of creB and creD. arceA is a regulatory factor related to nitrogen source metabolism and absorption. There was no obvious change in Aspergillus flavus treated with aromatic turmeric flavonoids, indicating that it is not sensitive to the regulation of nitrogen sources.

[0091] Table 3 Expression of representative differentially expressed genes related to Aspergillus flavus growth and aflatoxin synthesis under curcumin treatment

[0092]

[0093]

[0094] The present invention is described in detail above through specific and preferred embodiments, but those skilled in the art should understand that the present invention is not limited to the embodiments described above, and any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for studying the mechanism of action of turmeric flavonoids on Aspergillus flavus, comprising the following steps: S1. RNA detection: Take the Aspergillus flavus hyphae treated with aromatic turmeric flavonoids as the test group sample, and the Aspergillus flavus hyphae not treated with aromatic turmeric flavonoids as the control group sample, and perform RNA detection; S2, library construction and sequencing; S3, on-chip sequencing: single-stranded circular DNA molecules are replicated through rolling circles to form DNA nanoballs, and the resulting DNA nanoballs are added to the mesh holes on the chip for sequencing; S4. Quality control of RNA sequencing data; Fastp software was used to perform data quality control, filter raw data, check sequencing error rates, and examine GC content distribution; S5, alignment of transcriptome data with reference genome; Use HISAT2 to compare the clean reads with the sequence of the reference genome; the reference genome version is JCVI-AFL1-v3.0; S6. Gene expression quantification and differential gene analysis: Calculate the number of mapped reads and transcript lengths in the sample for horizontal comparison between different samples and groups, and perform differentially expressed gene analysis between sample groups; In step S6, FPKM is used to calculate the number of mapped reads and transcript length in the sample, and FPKM < 1 is considered as no expression; DESeq2 is used to analyze differentially expressed genes between sample groups, and the conditions for screening differentially expressed genes are |log2FC| ≥ 1, FDR < 0.05; S7. Functional annotation and enrichment analysis of differentially expressed genes.

2. The research method according to claim 1, characterized in that: The method for library construction and sequencing in step S2 is as follows: opening the secondary structure of the digested RNA sample, fragmenting the secondary structure, using PCR to synthesize the first-strand and second-strand cDNA, repairing the ends, connecting the adapters, performing PCR amplification, and denaturing the PCR product into a single strand. Then, a cyclization reaction system is used to react to obtain a single-stranded circular product, and the linear DNA molecules that have not been cyclized are digested to obtain the final library.

3. The research method according to claim 1, characterized in that: The RNA detection in step S1 includes detecting sample concentration, RIN / RQN value, and 28S / 18S.

4. The research method according to claim 1, characterized in that: The library construction and sequencing described in step S2 includes library construction and mRNA purification.

5. The research method according to claim 1, characterized in that: In step S3, the obtained DNA nanoballs are added to the mesh holes on the chip using high-density DNA nanochip technology, and sequencing is performed using a combined probe anchoring polymerization technology.

6. The research method according to claim 1, characterized in that: Step S6 also includes: The FDR was calculated by correcting the hypothesis testing probabilities for multiple hypothesis testing; The differential expression fold was calculated according to the FPKM value.

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