A cadmium-resistant gene abycf1 derived from agaricus blazei and application thereof

Abstract

The present application relates to a cadmium-tolerant gene derived from Agaricus blazei AbYCF1 and its application. The ORF full-length sequence of the cadmium-tolerant gene AbYCF1 is shown as SEQ ID NO. 1, which is cloned from Agaricus blazei strain J77 (low Cd accumulation strain). The ORF full-length sequence of the cadmium-tolerant gene AbYCF1 is constructed into an expression vector and transformed into yeast cells, and it is found that the gene can significantly improve the cadmium tolerance of the yeast. The present application has important significance for the study of fungal cadmium tolerance and the breeding of new varieties of cadmium-tolerant Agaricus blazei.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biotechnology and environmental protection, and particularly relates to a cadmium-tolerant gene AbYCF1 from Agaricus blazei and application thereof. BACKGROUND

[0002] Agaricus blazei, also known as Agaricus blazei Murrill, is a fungus for food and medicine, which is originally from Brazil. The polysaccharide of the fungus has many benefits for human body. However, the mycelium of the fungus has strong ability to enrich cadmium, which can easily lead to excessive accumulation of cadmium in fruiting bodies, affecting food safety and export exchange. At present, due to the introduction of new "three products and one standard", the control of the state on the environment and food safety is becoming more and more strict. Therefore, it is necessary and significant to explore a gene with the function of regulating cadmium migration.

[0003] The application finds the key candidate gene of cadmium tolerance difference by comparing the transcriptome data of cadmium-tolerant difference strains J77 and J1 of Agaricus blazei, and the full length of the gene is cloned by bioinformatics analysis, and the expression vector is constructed and transformed into yeast cells, and it is found that the gene can significantly improve the cadmium tolerance of yeast. The application has important significance for the research of cadmium tolerance of fungi and breeding of new varieties of cadmium-tolerant Agaricus blazei. SUMMARY

[0004] The application aims to provide a cadmium-tolerant gene AbYCF1 from Agaricus blazei and application thereof.

[0005] To achieve the above-mentioned purpose, the application adopts the following technical solutions.

[0006] The cadmium-tolerant gene AbYCF1 from Agaricus blazei, the ORF full-length sequence of the cadmium-tolerant gene AbYCF1 is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

[0007] An expression vector containing the cadmium-tolerant gene AbYCF1.

[0008] A primer for amplifying the cadmium-tolerant gene AbYCF1, the nucleotide sequence of the primer is shown as SEQ ID NO. 3-4.

[0009] Application of the cadmium-tolerant gene AbYCF1 in improving the cadmium tolerance of fungi, the fungi being yeast and Agaricus blazei.

[0010] Application of the cadmium-tolerant gene AbYCF1 in breeding of new varieties of cadmium-tolerant Agaricus blazei.

[0011] The application has the following advantages.

[0012] The AbYCF1 gene of the application can significantly improve the cadmium tolerance of fungi, provides a basis for artificially controlling the expression of cadmium tolerance related genes, and plays an important role in the cultivation of fungi with enhanced cadmium tolerance. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 : Differential expression of AbYCF1 gene in Agaricus blazei strain J77 and Agaricus blazei strain J1.

[0014] Figure 2 : Agaricus blazei RNA (A) and AbYCF1 gene PCR (B) electrophoresis map.

[0015] Figure 3 : Comparison of cadmium tolerance of transgenic strains and original strains. DETAILED DESCRIPTION

[0016] In order to make the content of the application more convenient to understand, the technical solutions of the application will be further described below in combination with specific embodiments, but the application is not limited thereto.

[0017] Example 1

[0018] The applicant of the present application found in the previous research that Agaricus blazei strain J77 is a low Cd accumulation strain, and Agaricus blazei strain J1 is a high Cd accumulation strain, and the expression amount of AbYCF1 gene in J77 and J1 is significantly different under cadmium stress Figure 1 ), it is speculated that AbYCF1 gene is related to cadmium accumulation and tolerance of Agaricus blazei; wherein the ORF full-length sequence of AbYCF1 gene of Agaricus blazei strain J77 is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

[0019] 1. Cloning of AbYCF1 gene

[0020] 1.1 Mycelium culture

[0021] (1) The formula of PDA rich culture medium is: potato 230 g, sucrose 20 g, potassium dihydrogen phosphate 2 g, magnesium sulfate 0.5 g, vitamin B1 10 mg, add distilled water to 1 L, put into a high-pressure sterilization pot, sterilize at 120℃ for 20 min, cool to room temperature, and then reserve.

[0022] (2) Mycelium sample: Agaricus blazei strain J77 test tube seed was inoculated in PDA rich liquid culture medium, and the mycelium was collected after 20 d of static culture at 25℃, and the mycelium was rapidly frozen with liquid nitrogen and stored in a-80℃ refrigerator for standby.

[0023] 1.2 RNA extraction and first strand cDNA synthesis

[0024] The extraction of mycelium RNA was performed by using RNA-easy Isolation Reagent R701 kit of Novagen, and the specific steps were performed according to the operation instruction of the kit, and the method used in the present study was slightly modified.

[0025] (1) The mortar and pestle were pre-cooled with liquid nitrogen, and the mycelium stored in liquid nitrogen was taken out and ground into powder.

[0026] (2) The ground sample was moved to a sterile centrifuge tube, 500 μL of RNA-easy (RNA-easy Isolation Reagent) was added to each 25 mg of mycelium powder, and the sample was uniformly blown and dissolved by using a pipette.

[0027] (3) 200 μL of RNase-free ddH2O was added to the centrifuge tube, and the mixture was uniformly mixed by inverting, and was left to stand at room temperature for 10 min, and was centrifuged at 12000 x g at room temperature for 15 min.

[0028] (4) The centrifuge tube was taken out from the centrifuge, at this time, the liquid in the tube was divided into two layers, the upper water phase (containing RNA) and the lower precipitate (containing protein, DNA, polysaccharide and other impurities), the lower precipitate was discarded, and the upper water phase was carefully taken and moved to a new centrifuge tube (the lower precipitate should not be taken, which would easily contaminate the impurities).

[0029] (5) An equal volume of isopropanol was added to the centrifuge tube, and the mixture was uniformly mixed by inverting, and was left to stand at room temperature for 20 min, and was centrifuged at 12000 x g at room temperature for 10 min, and a small amount of white precipitate was observed, and the supernatant was carefully discarded.

[0030] (6) 500 μL of 75% ethanol (prepared by using RNase-free ddH2O) was added to the precipitate, the centrifuge tube wall was blown and mixed by using a pipette, the precipitate was suspended, and was inverted several times, and was centrifuged at 8000 x g at room temperature for 3 min, and all the supernatant was discarded.

[0031] (7) After being left to stand at room temperature for 3 min, 40 μL of RNase-free ddH2O was added to quickly dissolve the precipitate, and the RNA precipitate was fully dissolved by repeatedly blowing and mixing with a pipette. The extracted RNA product was divided and stored at -85 to -65 °C for a long time.

[0032] cDNA synthesis: each component was spun before use. The following reaction system and reaction conditions were established in an RNase-free PCR tube:

[0033] Table 1 cDNA synthesis reaction system

[0034]

[0035] Table 2 cDNA synthesis reaction temperature

[0036]

[0037] 1.3AbYCF1 gene ORF sequence full-length amplification

[0038] (1) According to the AbYCF1 gene sequence obtained by transcriptome sequencing, two specific primers were designed by Primer Premier 5.0 software (see Table 3), and the primer synthesis was completed by Fuzhou Bosang Biotechnology Co., Ltd. The AbYCF1 gene ORF sequence of the strain J77 of Agaricus brasiliensis was amplified by using two specific primers.

[0039] Table 3 AbYCF1 gene amplification primers

[0040]

[0041] (2) The target gene was amplified by PCR, and the reaction system and reaction conditions are shown in the table:

[0042] Table 4 PCR reaction system

[0043]

[0044]

[0045] Table 5 PCR reaction conditions

[0046]

[0047] (3) The PCR product was recovered, and the PCR product obtained after the reaction was subjected to agarose gel electrophoresis (GelGreen staining), and if the band was specific and the size was consistent with the fragment size, the gel recovery and purification could be performed. The specific steps were carried out according to the Biospin gel recovery and PCR product purification kit instructions. Figure 2

[0048] (4) The purified PCR product was connected with the cloning vector according to the system in the following table at 37°C for 30 min.

[0049] Table 6 Cloning vector ligation system

[0050]

[0051] ​(5) Take the E. coli Trans1-T1 competent cells to place in the ice box to melt, mix the ligation product with 50 μL of the melted Trans1-T1 competent cells gently, and ice bath for 30 min; 50 μL of the melted Trans1-T1 competent cells are added with the gel recovery liquid as a negative control; after the ice bath, 42°C water bath heat shock for 30 s, and then immediately placed in the ice box to stand for 2 min; 250 μL of the normal LB liquid medium is added, and cultured at 37°C, 200 r·min -1 under the condition for 1 h; after the culture, centrifuged at 1500 x g for 1 min, the supernatant is discarded, 100-150 μL is reserved, and the remaining culture liquid is taken to the LB plate containing 100 mg·L -1 Amp with a sterile glass coating rod; after the coating, cultured at 37°C for 12 h; after the culture, a single colony is picked up with a sterile inoculation needle in the LB liquid medium containing 100 mg·L -1 Amp, and then expanded in the LB liquid medium at 37°C, 200 r·min -1 under the condition for 7 h.

[0052] (6) The bacterial liquid PCR verification is carried out according to the ORF amplification system and condition, and finally the positive clone is sent to ShangHai ShengGong Bioengineering Co., Ltd. for sequencing. The AbYCF1 gene ORF full-length sequence of the Brazilian mushroom strain J77 is shown as SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2.

[0053] 2. Transformation of AbYCF1 gene into yeast and analysis of cadmium tolerance

[0054] 2.1 Culture medium

[0055] The formula of the yeast complete culture medium (YPDA) is: 1 wt% yeast extract, 2 wt% tryptone, 2 wt% glucose, and 0.02 wt% adenine sulfate.

[0056] The formula of the yeast deficient screening culture medium is: SD-URA (SD-U) and SG-URA (SG-U)

[0057] The formula of the SD-U culture medium (mg / L) is: yeast nitrogen base (YNB), 6700; glucose, 20000; adenine sulfate, 40; L-arginine hydrochloride, 20; L-aspartic acid, 100; L-glutamic acid, 100; L-histidine, 20; L-leucine 60; L-lysine, 30; L-methionine, 20; L-phenylalanine, 50; L-serine, 375; L-threonine, 200; L-tryptophan, 40; L-tyrosine, 30; L-valine, 150.

[0058] SG-U medium formulation (mg / L): Yeast nitrogen basal (YNB), 6700; D-galactose, 20000; adenine sulfate, 40; L-arginine hydrochloride, 20; L-aspartic acid, 100; L-glutamic acid, 100; L-histidine, 20; L-leucine, 60; L-lysine, 30; L-methionine, 20; L-phenylalanine, 50; L-serine, 375; L-threonine, 200; L-tryptophan, 40; L-tyrosine, 30; L-valine, 150.

[0059] 2.2 Stock solution:

[0060] 10×TE: 0.1M Tris-HCl (pH 7.5), 10mM EDTA, autoclaved;

[0061] 10×LiAc: 1M LiAc (pH 7.5), autoclaved;

[0062] Cd 2+ Mother liquor: Dissolve 2.0315g of CdCl2·2.5H2O in 1L of distilled water to prepare a solution with a concentration of 1g·L⁻¹. -1 Cd 2+ After preparing the stock solution and SG-U solid medium (SG-U medium with 2% agar), add Cd. 2+ The mother liquor was used to make the cadmium concentration in the culture medium 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 μM, respectively.

[0063] 2.3 Construction of expression vector

[0064] The homologous recombination primer pYES2-AbYCF1-F / R was designed. Using the cDNA of Agaricus blazei strain J77 as a template, the full-length ORF sequence of the AbYCF1 gene with BglII and EcoRI restriction sites was amplified by PCR. The ORF sequence was then ligated with the pYES2 vector that had been digested with BglII and EcoRI to construct the recombinant expression vector, named pYES2-AbYCF1.

[0065] Table 7 Primers for Vector Construction

[0066]

[0067] 2.4 Transformation of yeast strain ycf1 with expression vector

[0068] The recombinant plasmid pYES2-AbYCF1 was transformed into yeast strain ycf1 using a high-efficiency LiAc / ssDNA / PEG transformation method. The specific steps of the yeast transformation method are as follows:

[0069] (1) Prepare YPDA liquid culture medium and dispense 4 mL into sterile centrifuge tubes. Then, pick a single colony of ycf1 from the YPDA plate and inoculate it into the centrifuge tube. Incubate at 30°C and 225 rpm. -1 Under these conditions, culture with shaking for 18-20 hours overnight until OD600 > 1.5.

[0070] (2) After the transfer requirements are met, the bacteria are transferred to YPDA liquid medium with a culture volume of 2 × 50 mL. When the initial OD600 = 0.2, the culture is carried out at 30℃ and 225 r·min. -1 Under the specified conditions, culture with shaking for 4-5 hours until OD600 = 0.6.

[0071] (3) At room temperature, 4000 r·min -1 Under the specified conditions, centrifuge for 5 minutes to collect the bacteria.

[0072] (4) Resuspend the bacterial cells in 20 mL of sterile water, mix well by pipetting, and incubate at room temperature at 4000 rpm. -1 Under the specified conditions, centrifuge for 5 minutes, discard the supernatant, and collect the bacteria.

[0073] (5) Resuspend the bacterial cells in 5 mL of 0.1 M LiAc, mix by pipetting, and incubate at room temperature at 4000 r·min. -1 Under the specified conditions, centrifuge for 5 minutes, discard the supernatant, and collect the bacteria.

[0074] (6) Next, resuspend the bacterial cells in 500uL of 0.1M LiAc, mix them by pipetting, and dispense 50uL into 1.5mL sterile centrifuge tubes for later use.

[0075] (7) Add the following reagents to the 1.5 mL centrifuge tubes in sequence, mix them by pipetting or by shaking with a micro shaker for about 1 minute until they are completely mixed.

[0076] Table 8. Yeast Transformation System Using Expression Vectors

[0077]

[0078] (8) Incubate in a water bath at 30°C for 30 minutes, then remove.

[0079] (9) Heat shock for 25 minutes in a water bath at 42°C, then remove.

[0080] (10) Under the condition of 30°C in a water bath, revive for 30 minutes and then remove.

[0081] (11) Centrifuge at room temperature and 4000 r·min-1 for 5 min, discard the supernatant and collect the bacteria.

[0082] (12) Centrifuge the bacteria from the previous step, suspend the bacteria with 200uL sterile water, mix gently, and spread on the corresponding deficient screening plate.

[0083] (13) Incubate at 30℃ for 4 days, and take pictures.

[0084] Screening of positive transformants:

[0085] Spread the transformed strain (ycf1-pYES2-AbYCF1) and the empty vector control strain (ycf1-pYES2-NTB) on SD-U plates and incubate for 4 days. Randomly pick several points from the transformed plates, streak for preservation, and perform PCR verification using the Forward Primer (5'-TAATACGACTCACTATAGG-3') and the Reverse Primer (5'-AGGGTTAGGGATAGGCTTACCT-3'). The PCR system is as follows:

[0086] Table 9 PCR reaction system

[0087]

[0088]

[0089] 2.5 Cadmium tolerance of transgenic strains

[0090] 2.5.1 Expansion culture of positive transformants

[0091] From the successfully verified yeast single colonies, randomly pick one single colony and inoculate it in 4mL SD-Ura liquid medium, incubate at 30℃ with 225r·min-1 shaking for about 24h until the OD600 is greater than 1.0 (the negative control is also shaken in the same way).

[0092] 2.5.2 Determination of bacterial liquid concentration

[0093] Adjust the initial OD600 concentration of the bacterial liquid to 0.8-1.0, and then dilute it by 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 .

[0094] 2.5.3 Plating and incubation

[0095] Diluted bacterial liquid is spotted on SG-Ura plates containing 0, 10, 20, 30, 40, 50, 60, 70, 80, 90μM CdCl2, and placed in a 30℃ incubator for incubation. Observe the plates and take pictures.

[0096] Cd stress tests were performed on expanded cultures of ycf1-pYES2-AbYCF1 and ycf1-pYES2-NTB colonies to reveal ( Figure 3 ), under stress for 12 hours, at low concentrations of Cd 2+ Under (0–20 μM) stress, the AbYCF1 gene-transformed strain (ycf1-pYES2-AbYCF1) did not show Cd tolerance advantage; at medium concentrations of Cd… 2+ Under (30–50 μM) stress, the growth of strain ycf1-pYES2-AbYCF1 was stronger than that of strain ycf1-pYES2-NTB transfected with an empty vector; under high concentrations of Cd 2+ Under (60–90 μM) stress, the ycf1-pYES2-AbYCF1 strain exhibited a strong Cd tolerance advantage.

[0097] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. AbYCF1 The use of a gene to increase the cadmium tolerance of a fungus, characterized in that: PCR amplification with BglII and EcoRΙ Enzyme cleavage sites AbYCF1 The full-length sequence of the gene's ORF, and the process BglII and EcoRΙ The pYES2 vector, after being digested with enzymes, was ligated to construct the recombinant vector pYES2-AbYCF1; the recombinant vector pYES2-AbYCF1 was then transferred into fungi to improve the fungal cadmium resistance. The gene ORF full-length sequence is shown as SEQ ID NO.

1. AbYCF1 The gene ORF full-length sequence is shown as SEQ ID NO.

1. wherein the fungus is a yeast.

Images