Auricularia auricula-judae chlorogenic acid synthesis related gene and application thereof

Abstract

The application provides auricularia auricular syntheses related genes and application thereof, and the auricularia auricular syntheses related genes are AaHQT gene, AaHCT gene and AaC3H gene. The application adopts cultivation material to regulate the expression amount of the auricularia auricular syntheses related genes, so as to improve the production of chlorogenic acid of the fungus. The cultivation material is composed of wood chips, bran, bean powder, calcium carbonate, sucrose, superphosphate, potassium chloride and water. The application discloses the related genes involved in the synthesis of chlorogenic acid in auricularia auricular for the first time, and provides a theoretical basis for the application of the fungus in the production of chlorogenic acid. The application puts forward the application of the auricularia auricular syntheses related genes in the production of chlorogenic acid for the first time, the application adopts cultivation material to regulate the expression amount of the auricularia auricular syntheses related genes, and the operation is simple and easy to implement. The added substances of the cultivation material are safe and harmless, the price is low, the application is economical and practical, and large-scale production is facilitated.

Description

Technical Field

[0001] This invention belongs to the field of fungal metabolite technology, and relates to chlorogenic acid, specifically to genes related to chlorogenic acid synthesis in black fungus and their applications. Background Technology

[0002] Black fungus, belonging to the mesophilic fungus phylum, is widely distributed in temperate and subtropical regions. It is a large basidiomycete fungus used for both food and medicinal purposes, rich in protein, fat, carotene, B vitamins, polysaccharides, and minerals such as iron, calcium, and phosphorus. Black fungus is believed to have effects such as clearing the lungs and invigorating qi, nourishing blood and promoting blood circulation, calming and relieving pain, preventing coronary artery sclerosis, and anti-cancer properties. The fruiting body is a reddish-brown gelatinous substance, flaky (0.6–12 cm in size, 1–2 mm thick), with both ventral and dorsal surfaces. The ventral surface is smooth and dark in color, and when mature, it contains septate basidia, each producing 4 basidiospores. The dorsal surface is covered with hairs.

[0003] Chlorogenic acid is a phenylpropanoid produced during aerobic respiration in organisms. It possesses strong antioxidant capabilities and various other bioactive functions, such as enhancing immunity, improving plant stress resistance, inhibiting the growth of certain microorganisms, combating aging, preventing tumors, fighting viruses, and preventing cardiovascular diseases. Chlorogenic acid is widely found in higher dicotyledonous plants and ferns, such as honeysuckle, snow lotus, and citrus. Compared to higher plants, fungi have shorter growth cycles and require less land for cultivation, making them more suitable for large-scale production. However, the limited research on chlorogenic acid from fungi restricts their application in chlorogenic acid production. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the purpose of this invention is to provide genes related to the synthesis of chlorogenic acid in black fungus and their applications, thereby solving the technical problem that the application of fungi in the production of chlorogenic acid is limited in the existing technology.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0006] The genes related to chlorogenic acid synthesis in black fungus are specifically AaHQT, AaHCT, and AaC3H genes.

[0007]

[0008]

[0009]

[0010] The present invention also has the following technical features:

[0011] This invention also protects the application of chlorogenic acid synthesis-related genes in black fungus for chlorogenic acid production. This application increases the chlorogenic acid yield of fungi by regulating the expression level of chlorogenic acid synthesis-related genes in black fungus using cultivation materials.

[0012] The cultivation substrate is composed of the following raw materials: sawdust, wheat bran, soybean flour, calcium carbonate, sucrose, superphosphate, potassium chloride, and water.

[0013] Specifically, the cultivation material, by weight, is composed of the following raw materials: 800-900 parts sawdust, 50-200 parts wheat bran, 10-30 parts soybean flour, 10-30 parts calcium carbonate, 0.5-5 parts sucrose, 10-50 parts superphosphate, 0.5-5 parts potassium chloride, and 400-800 parts water.

[0014] Preferably, the cultivation material, by weight, is composed of the following raw materials: 860 parts sawdust, 100 parts wheat bran, 20 parts soybean flour, 20 parts calcium carbonate, 2.5 parts sucrose, 37.5 parts superphosphate, 2.5 parts potassium chloride, and 600 parts water.

[0015] Specifically, the fungus in question is black fungus.

[0016] Compared with the prior art, the present invention has the following technical effects:

[0017] (I) This invention reveals for the first time the genes involved in chlorogenic acid synthesis in black fungus, providing a theoretical basis for the application of fungi in chlorogenic acid production.

[0018] (II) This invention proposes for the first time the application of chlorogenic acid synthesis-related genes in black fungus for chlorogenic acid production. This application uses cultivation material to regulate the expression level of chlorogenic acid synthesis-related genes in black fungus. The operation is simple and easy to implement. The substances added to the cultivation material are safe and harmless, inexpensive, economical and convenient for large-scale production. Attached Figure Description

[0019] Figure 1 This is a line graph showing the effect of potassium chloride on the expression levels of genes related to chlorogenic acid synthesis in black fungus.

[0020] Figure 2 This is a line graph showing the effect of sucrose on the expression levels of genes related to chlorogenic acid synthesis in black fungus.

[0021] Figure 3 This is a line graph showing the effect of superphosphate on the expression levels of genes related to chlorogenic acid synthesis in black fungus.

[0022] In the diagram: 1 to 16 represent the numbers of different components and ratios of the cultivation material.

[0023] The specific content of the present invention will be further explained in detail below with reference to the embodiments. Detailed Implementation

[0024] In this invention:

[0025] The AaHQT gene refers to the quinic acid hydroxycinnamoyl transferase gene in black fungus, also known as hydroxycinnamon coenzyme A or hydroxycinnamoyl transferase gene.

[0026] The AaHCT gene refers to the coumarin-CoA acyltransferase gene in black fungus.

[0027] The AaC3H gene refers to the coumaric acid-3-hydroxylase gene in black fungus.

[0028] It should be noted that all reagents used in this invention, unless otherwise specified, are reagents known in the art.

[0029] The following are specific embodiments of the present invention. It should be noted that the present invention is not limited to the following specific embodiments. All equivalent modifications made based on the technical solutions of this application fall within the protection scope of the present invention.

[0030] Example 1:

[0031] This embodiment provides genes related to chlorogenic acid synthesis in black fungus, specifically the AaHQT gene, the AaHCT gene, and the AaC3H gene.

[0032]

[0033] It should be noted that the nucleotide sequence of the AaHQT gene mentioned above contains multiple cds regions. The full-length amino acid sequence encoded by the cds regions is: 5'-MTASSIFEQAEVTITAVRTVHCANNSSHAHAPTHLGPLDSTVWAVIPIAVV FVYRRLDADAELMPIDRVQRAMELVLDHYPHLTGRLHIDSTNGARTFDRIGSGAELVMAECNAPLPKENFTFHDLPDAANALLPAIVTSLDEFCRNPLLSIKHTRFACASVALGVRLPHTVCDADGFFQFVRDLAEVYRTSKLSNPPHIQPYLADLYLSNSPEDRAAALAFQPSMYTVEPLSSAPSIPSASPPPPVIGRSLRFSA RQLAQLKEKYTPTSGWISTFDALTAHLYDRVLAARAAAGLSPFTDILTSVNLRPAGRLGLPSRYFPNALLAPYLTVTPDTPTALTVHALAECAKDRDEIEKIARWIAAQPDQRKIRFTFGGAGLMTSAWSRFDIYEGVAFDNAPALVVPPFTAISNIDGLAYYLPVPPNTPGGGLDVYVPFSSPVWGILERDPEWTRFLDV*-3'.

[0034]

[0035] It should be noted that the nucleotide sequence of the AaHCT gene mentioned above contains multiple cds regions. The full-length amino acid sequence encoded by the cds regions is: 5'-MQQSSSIVSRARVFPAHTKDGAVHDTPLSIIDATVARFSPASATWMFNRPH DSDLIDTDKLRQSLVVTLDAYPQLAGQLRWACFDASGRGGHKRRYGRLMLHYGAQDDPGVELVIAHSPHALADVVPDGETRRAAGAWDARIVPTLGLLPDDPPVALHDLKTFEGLPCTIVQLTAFAVIQFARDWASVHRALAANAPLPSLQPVFDPQRLDACAAGDIDTDEPDTELVRQARALPMHRYDWWAGDSSAVPAELASHASLEPPGRALPWAEWDLAAP VVHTLVYFAPQEIARMSHAAKAPGVSRLDALLAHVWAAIVRARGLAEKTEVYLDVTLGLRHRVSPPLPESFMGSPIVLTHAAVPAQTITTDPSKTALAIREAMALFTPSAIAA LLHERAFASCPARYWDAFLGARHTIVTSWLRLDVYELDFGFGSGKARYVDALMPEVDGCMHIMEAGPRVGNGDKWYDEAVCVNLHLAADVMARVLADPLLRVYRNEDA*-3'.

[0036]

[0037] It should be noted that the nucleotide sequence of the AaC3H gene mentioned above contains multiple CDS regions. The full-length CDS region encodes the following amino acid sequence: 5'-MFNVSNELLIYFAFGLAFVAVLVKLQSGTRRLPPGPAGHFIS GNAHQFPTGPGGRQYFAWSKTLGDLIHLRNYSRHVIVLNSERAIRDLIVRRADIYSDRAQTTMYCDLVGRGKTVFSSNYGDRFRQYSKLLHQALSKTAIQDYQSAQDDATNRMLRSMLTNPDAFVQHIRTFAGRIIMRVAYGYELQGEDDYFIGLVQQSIALGNEAFKPGRWLVDSFPLLRYVPSWMPAAGFKKWAAAAKSKLDEMTRAPHDMVKAQMAAGTAVPSFTSRNLVDND GAPVSTEREEIVLWVSAALYVGAADTTVAALSTFILLMVRHPEVQRKAQKEIDALLGTSRLPNLEDRERLPYVEAVMKECMRYNTIGPLALGHSLRREDNYNGFTLPKGSTVYMNLWA VTRDEGIYSRPETFRPERFMPDENGAVERDPWDIVFGYGLRRCPGRHLADATLFMAIAKMLAAFSFRPECDEKGEERVPPEEWTPGPNCGPMPFRCRILPRSEDMKSLVEESSR*-3'.

[0038] In this embodiment, the gene related to chlorogenic acid synthesis in black fungus was obtained by transcriptome sequencing of black fungus mycelium. This black fungus belongs to Auricularia auricula-judae.

[0039] Example 2:

[0040] This embodiment applies the chlorogenic acid synthesis-related genes from *Auricularia auricula-judae* (black fungus) in Example 1 to chlorogenic acid production. This application uses a cultivation substrate to regulate the expression level of these genes, thereby increasing the chlorogenic acid yield of *Auricularia auricula-judae*. The cultivation substrate consists of the following ingredients: sawdust, wheat bran, soybean flour, calcium carbonate, sucrose, superphosphate, potassium chloride, and water.

[0041] In this embodiment, experiments were conducted to determine the component content of each raw material. The specific process of the experiment and data processing is as follows: Different components and ratios of cultivation substrate were prepared, as shown in Table 1. Then, *Auricularia auricula-judae* strains were inoculated onto cultivation substrates with different components and ratios and cultured at 24℃ in the dark for 15 days. Mycelial samples were collected, pulverized with liquid nitrogen, and then subjected to real-time quantitative PCR. The primers for real-time quantitative PCR are shown in Table 2. After the PCR was completed, the 2'ΔΔCT value was calculated. Then, using the 2'ΔΔCT value of the control group (numbered K) as 1, all experimental group data were normalized, and a line graph was plotted. The final results are shown in Table 2. Figure 1 , Figure 2 and Figure 3 As shown.

[0042] Depend on Figures 1 to 3 It can be seen that sucrose can affect the gene expression of AaHCT and AaHQT in the chlorogenic acid synthesis pathway of black fungus; superphosphate can affect the gene expression of AaHCT; and potassium chloride can affect the gene expression of AaHCT, AaHQT and AaC3H.

[0043] Table 1. Cultivation materials with different components and ratios

[0044]

[0045] Table 2. Primers for Real-Time Quantitative PCR

[0046] AaHCT-PF tctggacgtaacgcttggac AaHCT-PR tcgaagggtcggttgtgatg AaHQT-PF ttgcgtactacttgcccgtt AaHQT-PR caggatcgcgctccaaaatg AaC3H-PF catgaacctatgggccgtca AaC3H-PR gctgtccgagtcgtaacaca

[0047] Based on the analysis of the qRT-PCR test results, the appropriate mass ratio of the cultivation material was determined as follows: sawdust 860g / Kg, wheat bran 100g / Kg, soybean meal 20g / Kg, calcium carbonate 20g / Kg, sucrose 2.5g / Kg, superphosphate 37.5g / Kg, potassium chloride 2.5g / Kg, and water 600g / Kg.

[0048] In this embodiment, the method of the application specifically includes the following steps:

[0049] Step 1: Prepare the cultivation substrate:

[0050] The solid components are mixed, and then water is added and stirred until evenly mixed to obtain the cultivation material.

[0051] Step two: Packaging, sterilization, and heat preservation:

[0052] Pack the prepared cultivation material from step one into sterilization bags, and then sterilize the bagged cultivation material at 120-125℃ for 2 hours under high temperature and pressure. After sterilization, when the temperature drops to 100℃, maintain it for 10-12 hours.

[0053] Step 3, vaccination:

[0054] After the bagged cultivation material, which has been sterilized and kept warm in step three, has cooled down, black fungus is inoculated onto the bagged cultivation material under aseptic conditions.

[0055] Step 4, Management during the mycelium growth period:

[0056] The mushroom bags were cultivated entirely in darkness, and the humidity in the cultivation room was always controlled at 65%.

[0057] The period of 5 to 7 days during which the mushroom bags are cultured in the culture room is the germination and colonization period. During this period, the temperature in the culture room is controlled at 26℃ to 28℃, and ventilation is carried out for 1 hour every noon.

[0058] The period of 8 to 15 days of cultivation in the culture room is the late colonization period. During this period, the temperature in the culture room should be controlled between 23°C and 25°C.

[0059] The period from 16 to 35 days of cultivation in the culture room is the robust period. During this period, the temperature in the culture room is controlled at 23℃ to 24℃, and ventilation is carried out once each morning, noon and evening, each lasting for 1 hour.

[0060] The period from when the mushroom bags are cultured in the culture room for 35 days until the end of the culture is called the maturation period. During the maturation period, the temperature in the culture room is controlled at 18℃-22℃, and ventilation is carried out once each morning, noon and evening, each time for 1 hour.

[0061] Step 5, Ear Management:

[0062] During the primordium formation period, maintain the humidity in the culture room at 80-85%, control the temperature at around 20℃, provide some diffused light, spray water on the straw mat every morning and evening to keep it moist, and open both ends of the straw mat for ventilation at night; after about 7-15 days, dark brown primordia will be visible, at which point the incision line can be sealed.

[0063] During the fruiting body differentiation period, maintain the humidity in the culture room at 85-90%, control the temperature at 15-25℃, provide some diffused light, and appropriately increase the ventilation. After about 15 days, the primordia will differentiate into irregular 1-2cm auricles.

[0064] During the growth and development of the fruiting bodies, maintain the humidity in the culture room at 90-95%. Water can be sprayed directly when the earlobes are 1cm in size. Frequent, light, and fine spraying is necessary to keep the earlobes moist and prevent them from curling. In the later stages, the amount of water should be increased appropriately, and alternating between dry and wet conditions is required (specifically, dry-wet-dry).

[0065] Before the earlobes mature, reduce the amount of water sprayed, reduce the air humidity to 85-90%, control the temperature at 15-25℃, adopt open (full light) cultivation, water thoroughly only in the evening and morning, ventilate 2-3 times a day for 30-40 minutes each time, and maintain sufficient fresh air.

[0066] The fruiting bodies require strong diffused light for growth and development. When the temperature is suitable, the covering should be removed to allow the ear lobes to grow and develop under sufficient light.

[0067] In this embodiment, after the culture was completed, mature fruiting bodies were collected, and the chlorogenic acid content in black fungus was determined by high performance liquid chromatography. The chlorogenic acid content in black fungus was 0.008 mg / g.

[0068] Comparative Example 1:

[0069] This comparative example applies the gene related to chlorogenic acid synthesis from black fungus in Example 1 to the production of chlorogenic acid. The method is basically the same as that in Example 2, except that the cultivation material is different.

[0070] The cultivation substrate in this comparative example consists of the following raw materials by mass: 860 g / kg sawdust, 100 g / kg wheat bran, 20 g / kg soybean flour, 20 g / kg calcium carbonate, and 600 g / kg water.

[0071] In this comparative example, after the culture was completed, mature fruiting bodies were collected, and the chlorogenic acid content in black fungus was determined by high performance liquid chromatography. The chlorogenic acid content in black fungus was 0.005 mg / g.

[0072] As can be seen from Example 2 and Comparative Example 1 above, the chlorogenic acid content of Example 2 increased by 60% compared with Comparative Example 1, indicating that the cultivation material with specific components and ratios can affect the expression level of genes related to chlorogenic acid synthesis in black fungus, thereby increasing the yield of chlorogenic acid.

Claims

1. Application of Auricularia auricular-j chlorogenic acid synthesis related genes in chlorogenic acid production, characterized in that, The application regulates the expression amount of chlorogenic acid synthesis related genes of Auricularia auricula by using cultivation material, so as to improve the yield of chlorogenic acid of the fungus The fungus is Auricularia auricula The cultivation material is composed of the following raw materials in mass fraction: 800-900 parts of sawdust, 50-200 parts of bran, 10-30 parts of bean powder, 10-30 parts of calcium carbonate, 0.5-5 parts of sucrose, 10-50 parts of superphosphate, 0.5-5 parts of potassium chloride and 400-800 parts of water The auricularia auricular black green acid synthesis related gene is AaHQT gene, AaHCT gene and AaC3H gene; The AaHQT The AaHCT The AaC3H 2. Use according to claim 1, wherein The cultivation material is composed of the following raw materials in mass fraction: 860 parts of sawdust, 100 parts of bran, 20 parts of bean powder, 20 parts of calcium carbonate, 2.5 parts of sucrose, 37.5 parts of superphosphate, 2.5 parts of potassium chloride and 600 parts of water.

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