CN112608931B - Deep-sea fungus FS140 anti-gliotoxin self-protection gene GliM and application thereof - Google Patents
Deep-sea fungus FS140 anti-gliotoxin self-protection gene GliM and application thereof Download PDFInfo
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Abstract
The invention discloses a deep-sea fungus FS140 anti-gliotoxin self-protection gene GliM and application thereof. The nucleotide sequence of the gliotoxin self-protection gene GliM is shown in SEQ ID NO. 1. Therefore, the invention obtains the GliM sequence of the gliotoxin self-protection gene from the cDNA library of the deep-sea fungus FS140, and successfully introduces the GliM sequence into the saccharomyces cerevisiae S.cerevisiae BJ5464 for anti-toxin function verification, thereby laying a molecular biological foundation for improving the gliotoxin resistance of the saccharomyces cerevisiae in the later period, improving the heterologous expression level of the gliotoxin and obtaining the novel gliotoxin.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a gliotoxin self-protection gene GliM for deep-sea fungi Geosmithia pallida FS140 and application thereof.
Background
More than 20 gliotoxin compounds are separated from deep sea fungus Geosmithia pallida FS140 in the previous subject group, including gliotoxin dimer compounds with rare structures, wherein most gliotoxin compounds have obvious antitumor activity.
Gliotoxin (GT) is a diketopiperazine compound (ETP) which is an important virulence factor, produces specific toxicity to various cells through different pathways, and plays an important synergistic role in invasive aspergillosis. ETP exerts toxic and side effects mainly through disulfide bonds and crosslinking of sulfydryl and target protein to further inactivate the activity of the protein, and can generate toxic Reactive Oxygen Species (ROS) through redox circulation, and the ROS generation mechanism is considered to be a mechanism of GT for generating cytotoxicity. The current studies on GT are limited to being one of the ETP family members or the metabolites of fumonisins, and therefore many toxicity (e.g. immunosuppressive toxicity) studies on gliotoxin have focused only on exerting toxicity as a primary and secondary metabolite, while their own toxicity, such as cytotoxicity, DNA damage and other toxic mechanisms to the host, are not known and remain to be studied further.
The toxicity of gliotoxin to the host mainly includes: inducing apoptosis; leading to an imbalance in redox reactions; inhibiting proteasome activity results in inhibition of NF- κ B activity, decreased immunological activity, etc. It is toxic not only to animal and plant cells, but also to host bacteria (Kamei K, Watanabe A. Aspergillus mycotoxin and the effect on the host. medical Mycology,2005,43: S95-S99.).
Disclosure of Invention
The first purpose of the invention is to provide a deep sea fungus Geosmithia pallida FS140 gliotoxin self-protection gene GliM, the nucleotide sequence of which is shown in SEQ ID NO. 1.
The deep-sea fungus Geosmithia pallida FS140 gliotoxin self-protection gene GliM is obtained by the following method: predicting a sequence of a GliM (gliotoxin self-protection gene) for coding the anti-gliotoxin through a transcriptome sequencing result, and designing a specific primer at the upstream and the downstream of the sequence, wherein the primer sequence is GliM-F: 5'-ATGGAAGCCAACAACACCGAC-3'; 5'-CTACTTCTTCAGCCGTAATTCCAA-3', using cDNA library reverse transcription from FS140 transcriptome of deep-sea fungus as template, obtaining product by PCR amplification and purifying and recovering fragment to obtain gliotoxin self-protective gene GliM with nucleotide sequence shown in SEQ ID NO. 1.
The invention utilizes a homologous recombination method to insert GliM gene into an expression cassette of a yeast vector YEp352-TEF1-CYC 1. First, the upstream and downstream primers of the GliM gene containing homology arms are designed, and the primer sequence is YEp352-GliM-F: 5-A TAGCAATCTAATCTAAGTCTAGAATGGAAGCCAACAACACCGAC-3'；YEp352-GliM-R:5'-TACATGATGCG GCCCGTCGACCTACTTCTTCAGCCGTAATTCCAA-3' (the underlined sequence is a homologous arm fragment), the product was obtained by PCR amplification and the fragment was purified and recovered. The constructed YEp352-TEF1-CYC1 vector was digested with Sal I and Xba I, and the fragment and the digested vector were ligated by recombination using Cloneexpress II One Step Cloning Kit C112(Vazyme), transformed into E.coli competent cells, plated on ammonia cells, and ligatedPositive clones were selected on the benzyl penicillin resistant plates. Through the molecular cloning, a target gene GliM (the nucleotide sequence of which is shown in SEQ ID NO.1) is inserted between a promoter TEF1 and a terminator CYC1 to construct a YEp352-TEF1-GliM vector, the vector is electrically transferred into a saccharomyces cerevisiae BJ5464-D cell, and screening and verification are carried out by using a uracil-deficient SD culture medium plate. Compared with Saccharomyces cerevisiae BJ5464-D transferred with YEp352-TEF1-CYC1 plasmid (negative control), the growth speed of the Saccharomyces cerevisiae containing the recombinant vector YEp352-TEF1-GliM is obviously accelerated, the colony density is higher in the same culture time, and the functional gene GliM is proved to be capable of effectively assisting the Saccharomyces cerevisiae to resist exogenous gliotoxin and lay a foundation for reconstructing a gliotoxin biosynthesis pathway in the Saccharomyces cerevisiae.
The second objective of the invention is to provide an expression vector containing the self-protective gene GliM against gliotoxin of the deep-sea fungus Geosmithia pallida FS140.
The third object of the present invention is to provide a host cell containing the above-mentioned expression vector.
The host cell is preferably Saccharomyces cerevisiae BJ 5464.
The fourth purpose of the invention is to provide the application of the self-protective gene GliM of the deep-sea fungus Geosmithia pallida FS140 against gliotoxin in assisting host cells in resisting the gliotoxin.
The host cell is preferably deep sea fungus Geosmithia pallida FS140 anti-gliotoxin or Saccharomyces cerevisiae BJ 5464.
The fifth object of the present invention is to provide an expression cassette comprising the self-protective gene GliM against gliotoxin of the deep-sea fungus Geosmithia pallida FS140 as described above.
Compared with the prior art, the invention has the following beneficial effects:
the deep sea fungus Geosmithia pallida FS140 related by the invention is separated from the sediment of the south sea, and transcriptome sequencing is carried out on the strain and related genes for biosynthesis of gliotoxin are annotated at the early stage of the subject group. Given the current few studies on the deep-sea fungal Geosmithia pallida FS140 anti-gliotoxin self-protective gene. Therefore, the invention obtains the GliM sequence of the gliotoxin self-protection gene from the cDNA library of the deep-sea fungus FS140, and successfully introduces the GliM sequence into the saccharomyces cerevisiae S.cerevisiae BJ5464 for anti-toxin function verification, thereby laying a molecular biological foundation for improving the gliotoxin resistance of the saccharomyces cerevisiae in the later period, improving the heterologous expression level of the gliotoxin and obtaining the novel gliotoxin.
The deep sea fungus Geosmithia pallida FS140 disclosed by the invention is prepared from the following documents: Zhuang-Hua Sun, Jiangyong Gu, Wei Ye, Liang-Xi Wen, Qi-Bin Lin, Sai-Ni Li, Yu-Chan Chen, Hao-Hua Li, Wei-Min Zhuang. Geospallins A-C New Thiodikepiprazine with inhibition Activity against Enzyme antigen-Converting Enzyme from a Deep-Sea-Derived fungi drug FS140.Marine Drugs,2018,16(12),464.https:// doi. org/10.3390/md 16120464. The applicant also holds the strain and guarantees that it will be provided to the public within 20 years from the date of filing.
Drawings
FIG. 1 shows the structural formula of 7-deoxy-6,7-didehydrogliotoxin (FS140-12-2) used in the experiment.
FIG. 2 shows the sequence of the deep-sea fungus FS140 GliM gene: electrophoresis chart of gene GliM amplification product with FS140 cDNA library as template;
FIG. 3 shows the construction of recombinant vector YEp352-TEF 1-GliM; wherein A is YEp352-TEF1-CYC1 vector map; b is YEp352-TEF1-GliM vector map; c is an electrophoretogram of colony PCR amplification products of the gene GliM;
FIG. 4 is a graph showing the effect of three species of Saccharomyces cerevisiae cultured on YPD plates and YPD-FS140-12-2 plates (2.5. mu.M) for 30 hours. A. Saccharomyces cerevisiae BJ5464-D (YEp352-TEF1-CYC 1); B. saccharomyces cerevisiae BJ 5464; c, Saccharomyces cerevisiae BJ5464-D (YEp352-TEF 1-GliM). 10 -2 、10 -3 、10 -4 Each represents OD 600 About 0.01, 0.001, and 0.0001. mu.L of the bacterial suspension sample.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The formulation of the SD solid medium used in this example was: the nutrient solution contains 20g of glucose, 0.62g of Do supplement (-Leu/-Trp/-Ura, Clontech), 6.7g of nitrogen source YNB (Puboxin), 0.06g of leucine, 0.04g of tryptophan and 20g of agar powder per liter, and the balance of distilled water. Gliocladin FS140-12-2 was isolated from the deep sea fungus Geosmithia pallida FS140 for this group of subjects.
The YPD solid medium used in this example was formulated as follows: each liter contains 10g of yeast powder, 20g of peptone, 20g of glucose, 20g of agar powder and the balance of distilled water, and the preparation method comprises the steps of uniformly mixing the components and sterilizing.
Example 1 obtaining of self-protective Gene sequence against Gliocladium toxin from deep-sea fungus Geosmithia pallida FS140
Amplification of the gene GliM: deep sea fungus Geosmithia pallida FS140 is inoculated on a YPD medium plate, the YPD medium plate is cultured for 72 hours at 37 ℃, fresh mycelium is picked, RNA is extracted by a fungus RNA extraction Kit, and then All-in-one RT Master Kit is used for reverse transcription to obtain cDNA. Predicting a GliM sequence of the coding anti-trichothecene self-protection gene according to a transcriptome sequencing result, and designing specific primers at the upstream and downstream of the GliM sequence, wherein the primer sequence is GliM-F: 5'-ATGGAAGCCAACAACACCGAC-3'; GliM-R:5'-CTACTTCTTCAGCCGTAATTCCAA-3', and PCR products were obtained by amplification using cDNA library as a template (FIG. 1). Recovering the product, performing TA cloning by using a pEASY-T1 kit, transforming the product into escherichia coli competent cells, coating the escherichia coli competent cells on an ampicillin resistance plate to screen out positive clones, performing bacterial liquid PCR (polymerase chain reaction) verification on the positive clones by using universal primers M13-F (5'-GTAAAACGACGGCCAGT-3') and M13-R (5'-CAGGAAACAGCTATGAC-3') and sequencing (figure 2) to obtain a target gene GliM sequence (the nucleotide sequence of the target gene GliM sequence is shown as SEQ ID NO.1 and atggaagccaacaacaccgaccaaatccccaagcgcagacacaccgatgacctgtacgagctctcggttaatatctccagcgcggtcgagaccttcctcggaaggctggacgctgtaggagccccacgacccaccctggataacccgttcccggagcttatccacgacgaaggggcccagattgcccggatgaagattctccgcctatgcgagaggctcatggcgctggtgcagggccccgtccagtggctcatgtttcagaatatgtgcttcgtcgaaccagcttgtattggggcgatggcggaaatggggatccatgagattgtggctcctggtccggaaccgacgtctctagaccagattgtggaggctaccggtgcctctaaggatattctgaagcgggttatgcgagtctgtacccagagactggtctttgatgagattgcgccggagcaattcatccacaatggtgtctccttgcaatttcttgctcctcctgtacaggcgctgattagccatgcctgcgacgatggactgcgcattgcgtcccgtttctccgactctctgaagaaaaccaacttcaaaggcagcgataaaccagaagaaacggccttcagtctcgcctttggaacggataagggcctcttcgactacttctacagcgatgacatcgctcgcggacaacgcttcgccctcggtatggcaggtaatgatatcgtcagatcccggaccgaggatatgttcccattcgacaccctcccacagggcgcgaagttggtggacgttggaggcggtcgaggccatgtcgacgtgcgcattgccgagagggtccccggcttgaactttgtcgtgcaggacgacgtatccgttctcgaagccggacaggcagagggtgttcctgcggccgttaagggtcggatcgagttcatgccgcatgacttttttaaggagcagcctgtcaagggggcggatgcgtatctcctgcgattcattatgcatgatcacccggatagtgtctgtgcaaagatcctgtcccatattgtggacgcgatggaccccgagaagtctcggatcctgattgacgatgcggtcgtccccagcttcttgggtccggagagttcgcgattcttcaatttcctggatctgtatatgctgttctcactgaatgggaaggagaggactctggagatgtggaatcatctgttccagatggtcagtccgaacttggtgttggagaagatttggaaaatgcctggcagtgggcctgagtcggggaatgtgttggaattacggctgaagaagtag).
Example 2 functional verification of gliotoxin self-protective Gene GliM
The gene GliM was inserted into the yeast vector YEp352-TEF1-CYC1 using homologous recombination (YEp352-TEF1-CYC1 was an early construction plasmid carrying a constitutive promoter TEF1 and a terminator CYC 1. the vector map is shown in FIG. 3A, which is a product known in the art: Xiaodan Ouyang, Yaping Cha, Wen Li, Chaoyi Zhu, Muzi Zhu, Shuang Li, Min Zhuo, Shaobin Huang and Jianjun Li. Stepwise engineering of Saccharomyces cerevisiae (+) -gene and its related sesterpenes, RSC adv, 2019,9,30171, DOI:10.1039/c9ra05558 d). First, designing the upstream and downstream primers YEp352-GliM-F and YEp352-GliM-R for gene GliM (SEQ ID NO.1) amplification, wherein the primer sequences are YEp352-GliM-F:5' -ATAGCAATCTAATCTAAGTCTAGAATGGAAGCCAACAACACCGAC-3'；YEp352-GliM-R:5'-TACATGATGCGGCCCGTCGACCTACTTCTTCAGCCGTAATTCCAA-3' (underlined sequences are homologous arm fragments), and the product was obtained by PCR amplification. The vector YEp352-TEF1-CYC1 was double digested with Sal I and Xba I and the products recovered, then the two products were recombinantly ligated and transformed into DH5 alpha using Clonexpress II One Step Cloning Kit C112(Vazyme) to screen for positivesAnd (4) cloning. Colony PCR verification using primers YEp352-GliM-F and YEp352-GliM-R showed that the gene GliM was successfully inserted into YEp352-TEF1-CYC1 vector (FIG. 3C), and confirmed by sequencing to obtain YEp352-TEF1-GliM vector (vector map shown in FIG. 3B).
Preparation of competent cells of the toxin-sensitive s.cerevisiae BJ5464-D (Relentgenotype: Δ pdr5 Δ pdr10 Δ pdr15), a strain known in the art which is more sensitive to toxic compounds, Wolfgang Schweiger, Jayanand Boddu, Sanghyun Shin, Brigitte Poppenberger, Franz Bertholler, Marc Lemmens, Gary J.Muehlbauer, and Gerhard Adam.Validation of a Candidate Deoxynivalenol-Inactivatingg UDP-glucopyranosyl transfer from Barley ecology groups Expression in Yeast No, 2010, Vol.23, MPI.7, DOI 10.1094-7-0977). YEp352-TEF1-GliM plasmid vector and YEp352-TEF1-CYC1 plasmid vector (negative control) were electroporated into Saccharomyces cerevisiae BJ5464-D cells (1500V, 5ms), respectively, uniformly spread on uracil-deficient SD plates, cultured at 30 ℃ for 2D, and positive clones were selected by colony PCR to obtain Saccharomyces cerevisiae BJ5464-D cells containing YEp352-TEF1-GliM plasmid and YEp352-TEF1-CYC1 plasmid, respectively.
Saccharomyces cerevisiae BJ5464-D (YEp352-TEF1-CYC1) and Saccharomyces cerevisiae BJ5464-D (YEp352-TEF1-GliM) were inoculated into the corresponding deficient SD medium and cultured at 30 ℃ for 2 days, respectively. The OD of each bacterial liquid was measured with a spectrophotometer 600 Diluting each bacterial solution to OD with sterile water 600 About 1.0 as stock solution, and diluting to 10 μ L with 100 μ L stock solution and 900 μ L sterile water -1 Diluted to 10 in the same manner -2 、10 -3 、10 -4 .10 of each 5. mu.L of different strains were taken -2 、10 -3 、10 -4 The dilutions of (A) were spotted on YPD plates and YPD-FS140-12-2 plates (containing 2.5. mu.M FS140-12-2 gliotoxin, FIG. 1), respectively, and cultured at 30 ℃ and observed in real time, using Saccharomyces cerevisiae BJ5464 as a positive control. The results of the 36-h plate culture (FIG. 4) showed that Saccharomyces cerevisiae BJ5464-D (YEp352-TEF1-CYC1) and Saccharomyces cerevisiae BJ5464-D (YEp352-TEF1-GliM) grew nearly uniformly on YPD plates without any added toxin, but on YPD plates containing 2.5. mu.MThe negative control BJ5464-D (YEp352-TEF1-CYC1) on YPD plates of FS140-12-2 gliotoxin showed significantly hindered growth and almost failed to grow. The saccharomyces cerevisiae introduced with the GliM functional gene grows well, and the density of thalli under different dilutions is equivalent to that of normal saccharomyces cerevisiae, so that the GliM functional gene derived from deep-sea fungi Geosmithia pallida FS140 partially or completely restores the tolerance of the saccharomyces cerevisiae BJ5464-D to exogenous added toxin, and effectively helps the normal growth of the saccharomyces cerevisiae in an environment containing the toxin.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
<120> deep-sea fungus FS140 anti-gliotoxin self-protection gene GliM and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1305
<212> DNA
<213> deep-sea fungus FS140(Geosmithia pallida)
<400> 1
atggaagcca acaacaccga ccaaatcccc aagcgcagac acaccgatga cctgtacgag 60
ctctcggtta atatctccag cgcggtcgag accttcctcg gaaggctgga cgctgtagga 120
gccccacgac ccaccctgga taacccgttc ccggagctta tccacgacga aggggcccag 180
attgcccgga tgaagattct ccgcctatgc gagaggctca tggcgctggt gcagggcccc 240
gtccagtggc tcatgtttca gaatatgtgc ttcgtcgaac cagcttgtat tggggcgatg 300
gcggaaatgg ggatccatga gattgtggct cctggtccgg aaccgacgtc tctagaccag 360
attgtggagg ctaccggtgc ctctaaggat attctgaagc gggttatgcg agtctgtacc 420
cagagactgg tctttgatga gattgcgccg gagcaattca tccacaatgg tgtctccttg 480
caatttcttg ctcctcctgt acaggcgctg attagccatg cctgcgacga tggactgcgc 540
attgcgtccc gtttctccga ctctctgaag aaaaccaact tcaaaggcag cgataaacca 600
gaagaaacgg ccttcagtct cgcctttgga acggataagg gcctcttcga ctacttctac 660
agcgatgaca tcgctcgcgg acaacgcttc gccctcggta tggcaggtaa tgatatcgtc 720
agatcccgga ccgaggatat gttcccattc gacaccctcc cacagggcgc gaagttggtg 780
gacgttggag gcggtcgagg ccatgtcgac gtgcgcattg ccgagagggt ccccggcttg 840
aactttgtcg tgcaggacga cgtatccgtt ctcgaagccg gacaggcaga gggtgttcct 900
gcggccgtta agggtcggat cgagttcatg ccgcatgact tttttaagga gcagcctgtc 960
aagggggcgg atgcgtatct cctgcgattc attatgcatg atcacccgga tagtgtctgt 1020
gcaaagatcc tgtcccatat tgtggacgcg atggaccccg agaagtctcg gatcctgatt 1080
gacgatgcgg tcgtccccag cttcttgggt ccggagagtt cgcgattctt caatttcctg 1140
gatctgtata tgctgttctc actgaatggg aaggagagga ctctggagat gtggaatcat 1200
ctgttccaga tggtcagtcc gaacttggtg ttggagaaga tttggaaaat gcctggcagt 1260
gggcctgagt cggggaatgt gttggaatta cggctgaaga agtag 1305
Claims (5)
1. An anti-gliotoxin self-protection gene GliM is characterized in that the nucleotide sequence is shown as SEQ ID NO. 1.
2. An expression vector comprising the gliotoxin self-protective gene GliM of claim 1.
3. A host cell comprising the expression vector of claim 2, wherein the host cell is Saccharomyces cerevisiae BJ 5464.
4. The use of the gliotoxin self-protective gene GliM of claim 1 to help host cells against gliotoxin, said host cells being the deep sea fungus Geosmithia pallid FS140 or the Saccharomyces cerevisiae BJ 5464.
5. An expression cassette comprising gliotoxin self-protecting gene GliM according to claim 1.
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| CN110950940A (en) * | 2019-10-29 | 2020-04-03 | 广东省微生物研究所（广东省微生物分析检测中心） | Novel transcription regulation factor DcGliZ from deep-sea fungi and application thereof |
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