Application of feruloyl putrescine in preparation of deoxynivalenol

Abstract

The invention belongs to the technical field of bioengineering, and relates to application of feruloyl putrescine in preparation of deoxynivalenol. The invention verifies that when the feruloyl putrescine is used as the fusarium graminearum culture medium additive, the fusarium graminearum can be promoted to generate deoxynivalenol. According to the invention, more ways are provided for synthesis and acquisition of deoxynivalenol.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering and relates to application of feruloyl putrescine in preparation of deoxynivalenol. BACKGROUND

[0002] Deoxynivalenol (DON), also known as vomitoxin, is a class B mycotoxin of trichothecenes produced by Fusarium graminearum and other pathogenic fungi. Fusarium graminearum DON is one of the most common mycotoxins in cereals such as wheat and corn, which not only poses a serious threat to the health of livestock and humans, causing vomiting, diarrhea, immune suppression and other hazards, but also is an important virulence factor of Fusarium graminearum in the process of invading the host. In the fields of scientific research, toxicology evaluation and detection method development, a stable and reliable source of DON is needed. At present, DON is mainly obtained by extraction and purification from cereals naturally infected or artificially inoculated with Fusarium graminearum. However, this method is limited by factors such as crop growth cycle, climate conditions, uneven infection degree, resulting in unstable DON yield, high cost and complicated process, which is difficult to meet the application needs of large scale and standardization. Although it is theoretically possible to chemically synthesize DON, the synthesis path is complex, the steps are numerous, and the cost is extremely high, which is not an economically feasible preparation scheme at present.

[0003] The biosynthesis of DON in Fusarium graminearum is regulated by multiple genes in a gene cluster called TRI Among them, TRI5 is the key gene for starting toxin synthesis, TRI1 and TRI12 genes also play an important role in toxin modification and transport. Studies have shown that the synthesis of DON occurs in a subcellular structure called "toxin-producing organelle", and the formation of this structure is the premise of efficient toxin production. Environmental factors, especially signal molecules in the host plant, have an important inducing effect on the expression of TRI gene and the formation of toxin-producing organelle. Therefore, finding a key inducing factor that can effectively activate the toxin production mechanism of Fusarium graminearum is of great significance for developing a new and efficient DON preparation method.

[0004] In the prior art, there is still a lack of a culture medium additive or inducer that can specifically induce Fusarium graminearum to produce a large amount of DON. There is an urgent need in the field for a technical solution that can break away from the dependence on naturally infected cereals and achieve efficient and stable preparation of DON under controllable conditions. SUMMARY

[0005] The purpose of the present application is to provide more preparation methods for deoxynivalenol.

[0006] In one aspect, the present application relates to the use of feruloyl putrescine in the preparation of deoxynivalenol, which comprises: feruloyl putrescine promotes the expression of a gene of fusarium graminearum Fusarium graminearum to produce deoxynivalenol.

[0007] Further, in the use of feruloyl putrescine in the preparation of deoxynivalenol provided by the present application, feruloyl putrescine promotes the expression of a gene of fusarium graminearum Fusarium graminearum . TRI

[0008] Further, in the use of feruloyl putrescine in the preparation of deoxynivalenol provided by the present application, the TRI gene is TRI1 a gene, TRI5 a gene and TRI12 at least one of a gene.

[0009] TRI1 The coding sequence of the gene is shown in SEQ ID NO: 1, and is specifically as follows: ​

[0010] TRI5 The coding sequence of the gene is shown in SEQ ID NO: 2, as follows:

[0011] TRI12 The coding sequence of the gene is shown in SEQ ID NO: 3, as follows:

[0012] Further, in the application of feruloyl putrescine in preparing deoxynivalenol, the culture environment of Fusarium graminearum includes the ear of wheat. Fusarium graminearum

[0013] Further, in the application of feruloyl putrescine in preparing deoxynivalenol, the concentration of feruloyl putrescine in the culture environment of Fusarium graminearum is not less than 30 μM. Fusarium graminearum

[0014] Further, in the application of feruloyl putrescine in preparing deoxynivalenol, the concentration of feruloyl putrescine in the culture environment of Fusarium graminearum is 30 μM to 300 μM. Fusarium graminearum

[0015] Further, in the application of feruloyl putrescine in preparing deoxynivalenol, the feruloyl putrescine promotes the production of virulence body by the recombinant bacteria containing the gene. TRI1

[0016] Further, in the application of feruloyl putrescine in preparing deoxynivalenol, the feruloyl putrescine is the single nitrogen source in the culture environment of Fusarium graminearum. Fusarium graminearum

[0017] Further, in the application of feruloyl putrescine in preparing deoxynivalenol, Fusarium graminearum produces deoxynivalenol in a liquid virulence culture medium. Fusarium graminearum 1L of the liquid virulence culture medium contains 0.3 g of plant gel, 30 g of sucrose, 1 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.5 g of KCl, 10 mg of FeSO4·7H2O, 264.3 mg of feruloyl putrescine, and 20 μL of trace elements. 5 g of citric acid, 5 g of ZnSO4·7H2O, 0.25 g of CuSO4·5H2O, 50 mg of MnSO4·H2O, 50 mg of H3BO3, and 50 mg of NaMoO4·2H2O are contained in 100 mL of the trace elements.

[0018] Compared with the prior art, the technical scheme provided by the application has at least the following beneficial effects or advantages: ​​​​​​This invention is the first to discover and verify that the plant-derived metabolite ferulic acid putrescine can act as a highly efficient inducer, significantly promoting the synthesis of deoxynivalenol (DON) by Fusarium graminearum. Compared to traditional methods that rely on naturally infected grains for extraction, this invention achieves stable and significant increases in DON yield by adding ferulic acid putrescine to a controlled culture medium in the laboratory, eliminating dependence on crop growth cycles and infection environments, and opening up more avenues for the standardized and large-scale preparation of DON. This invention not only verifies the effect of ferulic acid putrescine on increasing the final DON yield, but also further reveals its deeper mechanism of action: namely, by specifically upregulating key components in Fusarium graminearum. TRI Genes (including) TRI1 , TRI5 and TRI12 The expression of feruloyl putrescine is significantly promoted, and the formation of the toxin-producing body structure responsible for DON synthesis is significantly enhanced. This invention optimizes and provides specific liquid toxin-producing culture medium formulations and culture conditions, clarifies the effective concentration range of ferulic acid putrescine (e.g., 30 μM~300 μM), and confirms its effectiveness as a single nitrogen source, providing a reliable guarantee for subsequent scientific research, preparation of detection standards, and other applications. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 To promote the synthesis of deoxynivalenol by ferulic acid putrescine, TRI Diagrams showing gene expression and toxin-producing body formation. In diagram A, DON production is shown in liquid toxin-producing media using ammonium nitrate, putrescine, or ferulic acid putrescine as a single nitrogen source; in diagram B, DON production is shown in liquid toxin-producing media using putrescine and ferulic acid putrescine as single nitrogen sources. TRI The effect of gene expression; the effect of liquid toxin-producing medium with putrescine and ferulic acid putrescine as single nitrogen sources on the formation of toxin-producing bodies (C); DIC indicates observation under field conditions; TRI1 -GFP refers to the fusion protein of green fluorescent protein (GFP), which contains the coding sequence of the TRI1 gene; NH4NO3 is ammonium nitrate, Put is putrescine, and Ferput is ferulic acid putrescine; scale bar = 20 µm. Detailed Implementation

[0021] The technical solutions of the present application are described below in combination with examples, but the present application is not limited to the following examples. The experimental methods and detection methods described in each example are all conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified. In the following examples, % is mass percentage unless otherwise specified. In the following examples, the ratio is mass ratio unless otherwise specified.

[0022] Example 1 This example provides that the metabolomics detects that feruloylputrescine is significantly increased in wheat spikes infected by Fusarium graminearum.

[0023] Fusarium graminearum wild-type strain PH-1 was activated to PDA culture medium (purchased from Beijing Aoboxing Biotechnology Co., Ltd.) in a culture dish and cultured in a 25°C constant temperature incubator in the dark for 3 days. A 60mm puncher was used to punch the fungus cake, and 3 fungus cakes were inoculated into 50ml CMC liquid medium, 1L CMC liquid medium containing 1g yeast extract (purchased from OXOID), 1g NH4NO3 (purchased from Shanghai Chemical Reagent Factory), 1g KH2PO4 (purchased from Guangdong Guanghua Technology Co., Ltd.), 0.5g MgSO4·7H2O (purchased from Guangdong Guanghua Technology Co., Ltd.), and 15g carboxymethyl cellulose (purchased from Aldrich). After 3 days, spores were collected and the spore concentration was adjusted to 1×10 6 Ten μL of spore solution was inoculated into the glume of Norm wheat in the flowering stage, and water was inoculated into the control group. After 24h of bagging and moisturizing, the wheat spikes were collected and quickly frozen in liquid nitrogen.

[0024] The sample was placed in a freeze dryer (Scientz-100F) for vacuum freeze drying for 63h, and then ground into powder using a grinder (MM400, Retsch) at 30Hz for 1.5min. 50mg of sample powder was weighed using an electronic balance (MS105DM), and 1200ml of 70% methanol pre-cooled at -20°C was added. The sample was vortexed every 30min for 30s, and vortexed a total of 6 times. After centrifugation (12000rpm for 3min), the supernatant was aspirated, the sample was filtered with a microporous filter (0.22μm), and stored in an injection vial for UPLC-MS / MS analysis.

[0025] The chromatographic column was Agilent SB-C18 (1.8 µm × 2.1 mm × 100 mm), the mobile phase A was 0.1% formic acid / water, and B was 0.1% formic acid / acetonitrile. The elution gradient was 0.0 min with a B phase ratio of 5%, linearly increased to 95% within 9.0 min, and maintained at 95% for 1 min, 10.00-11.10 min, the B phase ratio decreased to 5%, and balanced at 5% for 14 min, the flow rate was 0.35 mL / min, the column temperature was 40°C, and the injection volume was 2 µL. The ion source temperature of electrospray was 500°C; the ion spray voltage was 5500V (positive ion mode) / -4500V (negative ion mode); the ion source gas I (GSI), gas II (GSII) and curtain gas (CUR) were set to 50, 60 and 25 psi respectively, and the collision-induced ionization parameters were set to high. QQQ scanning used MRM mode, and the collision gas (nitrogen) was set to medium. Through further optimization of the declustering voltage (DP) and collision energy (CE), the DP and CE of each MRM ion pair were completed. According to the metabolites eluted in each period, a specific group of MRM ion pairs was monitored in each period. The compounds were compared based on the self-built database MWDB (metware database) of Wuhan Metware Metabolic Biotechnology Co., Ltd. The results are shown in Table 1.

[0026] Table 1: Compound categories significantly increased in wheat spikes infected by F. graminearum

[0027] After comparison, the phenolic amine compounds were the most increased category in the wheat spikes infected by F. graminearum, and feruloyl putrescine was enriched in the wheat spikes infected by F. graminearum.

[0028] Example 2 This example provides that feruloyl putrescine promotes the synthesis of deoxynivalenol (DON).

[0029] Toxicogenic culture conditions: 2 mL of liquid toxicogenic culture medium was added to a 24-well plate. 1 L of liquid toxicogenic culture medium contained 0.3 g of plant gel, 30 g of sucrose, 1 g of KH2PO4, 0.5 g of MgSO4·7H2O, 0.5 g of KCl, 10 mg of FeSO4·7H2O, 80.043 mg of ammonium nitrate (or 88.151 mg of putrescine, or 264.3 mg of feruloyl putrescine), and 20 µL of trace elements. Each 100 mL of trace elements contained 5 g of citric acid, 5 g of ZnSO4·7H2O, 0.25 g of CuSO4·5H2O, 50 mg of MnSO4·H2O, 50 mg of H3BO3, and 50 mg of NaMoO4·2H2O, which were all purchased from Sigma. The pH was adjusted to 5.4-5.6. The final concentration of 1 × 10 4The spore suspension was prepared at 1 spore / mL and cultured in a constant temperature incubator at 25℃ in the dark for 7 days. Then, the bacterial film on the surface of the culture medium was quickly placed in liquid nitrogen, freeze-dried for 24 hours, and weighed.

[0030] DON toxin extraction: Take 400 μL of the liquid toxin-producing medium after 7 days of culture, add 1600 μL of methanol and mix well. Pass the resulting extract through a C18 SPE solid-phase extraction column. Take 800 μL of the filtrate, concentrate under vacuum to dryness, add 50 μL of silanizing reagent (trimethylsilylimidazolium to trimethylchlorosilane, volume ratio 100:1, purchased from Sigma), vortex thoroughly to ensure the silanizing reagent and sample are fully mixed, and shake on a shaker for 10 min. Add 800 μL of isooctane, gently invert and mix thoroughly, then add 800 μL of ultrapure water, gently invert and allow to stand for separation, transfer the supernatant to a sample vial.

[0031] GC-MS detection: Chromatographic conditions: Rxi-5MS column (30m×0.230-1000μM×0.25μm); split ratio 50:1; injection port temperature 260℃; carrier gas: helium, flow rate 1mL / min; temperature program: initial column temperature 150℃, hold for 1min, increase to 280℃ at 30℃ / min, and hold for 15min.

[0032] Mass spectrometry conditions: mass spectrometer interface temperature 280℃; ion source temperature 250℃; injection volume 1μL; qualitative analysis of DON was performed using m / z 295, 235, and 193 in SIM mode, and quantitative analysis was performed using m / z 235. Mass spectrometry acquisition time was 5–10 min. The effect of ferulic acid putrescine on DON synthesis is as follows: Figure 1 As shown in A, the content of deoxynivalenol was significantly higher than that of ammonium nitrate and putrescine, indicating that ferulic acid putrescine can promote the synthesis of DON.

[0033] Example 3 This embodiment provides the effect of ferulic acid putrescine on Fusarium graminearum. TRI Effects of gene expression exist TRI In genes, TRI5 Catalyzing the cyclization of farnesyl pyrophosphate into trichothecene is the first-step catalytic enzyme in the synthesis of DON toxin. TRI1 The encoded protein catalyzes the formation of C8 trichothecene from lecithin and is localized to the toxin-producing body. TRI12 The encoded transporter protein is involved in the transport of DON toxin.

[0034] Add 50 mL of liquid toxin-producing medium to an Erlenmeyer flask, then add spores of wild-type Fusarium graminearum strain PH-1 to bring the final spore concentration to 1 × 10⁻⁶. 40.5 mL. The mycelia were collected after 36 h of incubation at 30℃ with shaking at 90 r / min in the dark and immediately frozen in liquid nitrogen. The mycelial RNA was extracted, followed by DNA digestion and RNA reverse transcription. The quantitative primers of the target gene and the internal reference gene were designed for quantitative PCR, and the data were analyzed and processed, and the results are shown in Fig. 2B. TRI Fig. 2B shows the results of quantitative PCR of the target gene and the internal reference gene. Figure 1 TRI The quantitative primers of the target gene and the internal reference gene are shown in Table 2.

[0035] Table 2: Specific primers of the internal reference gene actin and the TRI gene

[0036] As shown in Fig. 2B, feruloylputrescine promoted the expression of Figure 1 , TRI1 , TRI5 and TRI12 , and the expression levels were all increased by more than 6 times.

[0037] Example 4 This example provides the effect of feruloylputrescine on the formation of toxin corpuscles of F. graminearum.

[0038] Toxin corpuscles are considered to be a kind of circular proteins for DON synthesis. The TRI1 -GFP-F (5'-AGGGAACAAAAGCTGGGTACCGCTATACTCGGCAGTCCTTTGC-3') and TRI1 -GFP-R (5'-GAACAGCTCCTCGCCCTTGCTCACGGCGTCATTTGGGCTTGAGATAG-3') primers were used for PCR amplification to amplify the TRI1 gene sequence of F. graminearum. The pKNTG plasmid was double-digested with KpnI / HindIII, and the TRI1 -GFP fragment was introduced into the cut pKNTG plasmid. PCR verification was performed using the pKNTG seq / F (5'-CCCCAGGCTTTACACTTTATGCT-3') and TRI1 -GFP-R primers, and the plasmid was sent to a company for sequencing to determine whether there was a mutation before being introduced into the protoplast of PH-1. The TRI1 -GFP transformed strain of F. graminearum was obtained by PCR detection. The mycelial blocks of the TRI1 -GFP transformed strain were shaken in CMC liquid medium for 3 days (25℃, 200 rpm), filtered with filter cloth and centrifuged, and the supernatant was discarded. 50 mL of liquid toxin culture medium was added to a conical flask, and the spore solution of the Figure 1 -GFP strain was added to make the final concentration of spores 1×10 4 ​The mycelia were collected by centrifugation at 3000rpm for 10 min, and the concentration of the mycelia was adjusted to 1 g / mL. The mycelia were incubated in the dark for 36 h (25℃, 90 rpm), and the formation of toxin corpuscles was observed under a fluorescence microscope after the mycelia were washed with sterile water. The results are shown in Table 1. ​ As shown in Table 1, compared with the control group, a large number of toxin corpuscles were formed in the mycelia of F. graminearum in the toxin production medium with feruloylamine as the single nitrogen source, indicating that the treatment of feruloylamine significantly promoted the formation of toxin corpuscles.

[0039] The above-described embodiments are only some of the embodiments of the present application, rather than all the embodiments. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but merely represents the preferred embodiment of the application. All other embodiments obtained by those of ordinary skill in the art based on the relevant deductions and substitutions made under the concept of the present application, without making creative efforts, fall within the scope of the present application.

Claims

1. Use of feruloylputrescine in the preparation of deoxynivalenol, characterized in that, Comprising: feruloyl putrescine promotes fusarium graminearum Fusarium graminearum producing deoxynivalenol.

2. Use of feruloylputrescine according to claim 1 for the preparation of deoxynivalenol, characterized in that, feruloyl putrescine promotes fusarium graminearum Fusarium graminearum gene expression. TRI gene expression.

3. Use of feruloylputrescine according to claim 2 for the preparation of deoxynivalenol, characterized in that, The TRI genes are TRI1 genes, TRI5 genes and TRI12 at least one of the genes.

4. Use of feruloylputrescine according to claim 1 for the preparation of deoxynivalenol, characterized in that, f. fusarium cerealis Fusarium graminearum spikes of wheat.

5. Use of feruloylputrescine according to claim 1 for the preparation of deoxynivalenol, characterized in that, fusarium cerealis Fusarium graminearum The concentration of feruloylamine in the culture environment of fusarium cerealis is not less than 30 μM.

6. Use of feruloylputrescine according to claim 1 for the preparation of deoxynivalenol, characterized in that, f. fusum Fusarium graminearum The concentration of feruloylamine in the culture environment of f. fusum is 30 μM-300 μM.

7. Use of feruloylputrescine according to claim 1 for the preparation of deoxynivalenol, characterized in that, feruloyl putrescine promotes the production of TRI1 toxophore by recombinant bacteria containing the gene.

8. Use of feruloylputrescine according to claim 1 for the preparation of deoxynivalenol, characterized in that, Feruloyl putrescine is a Fusarium graminearum species. Fusarium graminearum A single nitrogen source in the culture environment.

9. Use of feruloylputrescine according to claim 1 for the preparation of deoxynivalenol, characterized in that, f. graminearum Fusarium graminearum Deoxynivalenol was produced in liquid toxinogenic medium; 1L of the liquid toxin production medium contains 0.3g plant gel, 30g sucrose, 1g KH2PO4, 0.5g MgSO4·7H2O, 0.5g KCl, 10mg FeSO4·7H2O, 264.3mg feruloyl putrescine, 20μL trace elements; 5g citric acid, 5g ZnSO4·7H2O, 0.25g CuSO4·5H2O, 50mg MnSO4·H2O, 50mg H3BO3, 50mg NaMoO4·2H2O per 100mL of the trace elements.

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