CN112795567B - Deep-sea fungus FS 140O-methyltransferase gene GliM promoter and application thereof - Google Patents

Deep-sea fungus FS 140O-methyltransferase gene GliM promoter and application thereof Download PDF

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CN112795567B
CN112795567B CN202011550290.9A CN202011550290A CN112795567B CN 112795567 B CN112795567 B CN 112795567B CN 202011550290 A CN202011550290 A CN 202011550290A CN 112795567 B CN112795567 B CN 112795567B
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李赛妮
章卫民
叶伟
许丽琼
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Institute of Microbiology of Guangdong Academy of Sciences
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Abstract

The invention discloses a GliM promoter of a deep-sea fungus FS 140O-methyltransferase gene and application thereof. The promoter has a nucleotide sequence shown as SEQ ID NO.1, the promoter adopts a Genome Walking kit, utilizes a general primer and a reverse specificity primer, utilizes a TAIL-PCR principle to obtain an upstream promoter sequence of a deep sea fungus FS 140O-methyltransferase gene GliM, predicts a promoter core region to obtain a promoter key core region and performs functional verification, thereby laying a molecular biological foundation for improving the expression level of gliotoxin and obtaining novel gliotoxin through transcription regulation and heterologous expression at the later stage.

Description

Deep-sea fungus FS 140O-methyltransferase gene GliM promoter and application thereof
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a GliM promoter from a GliM promoter of a deep-sea fungus Geosmithia pallida FS 140O-methyltransferase gene0And applications thereof.
Background
Gliotoxin (GT) is an important hydrophobic fungal metabolite belonging to the epithiopiperazinone class of compounds (ETPs). ETPs are active secondary metabolites mainly produced by fungi, and structurally characterized by a Diketopiperazine (DKP) core containing disulfide bonds, and have a wide range of biological activities, including antiproliferative, cytotoxic, immunosuppressive, antiviral, and antibacterial biological activities. The deep sea fungus Geosmithia pallida FS140 is a fungus separated from the sediment of the south sea, 12 gliotoxins and derivatives thereof are separated from the fermentation liquor of the fungus, the gliotoxin and the derivatives thereof comprise gliotoxin dimer derivatives with rare structures, and activity tests show that part of the compounds have strong antitumor activity.
Eukaryotic gene promoters are DNA sequences which are located at the 5' upstream of the transcription start point of genes and are identified by RNA polymerase, combined with the transcription start point and start transcription, can be specifically combined with RNA polymerase and other trans-acting factors and start the expression of the genes to carry out fine regulation, and firstly, the expression regulation of the promoters and transcription factors on the transcription level is carried out, so the action of the promoters is very critical. In recent years, as filamentous fungi have rapidly progressed in the discovery of novel, highly active secondary metabolites and the research and development of active enzymes, and promoters in filamentous fungi have high transcriptional activity on endogenous genes thereof, promoters of many different species of filamentous fungi have been discovered in succession, such as: cbh1 of Trichoderma reesei (Trichoderma reesei), tef1 (translational elongation factor gene), the gpdA (glyceraldehyde triphosphate dehydrogenase gene) promoter of Aspergillus nidulans, the pki1 promoter of Trichoderma (Trichoderma sp.), the glaA promoter of Aspergillus, and the like. However, few studies on promoters of genes for biosynthesis of secondary metabolites of deep-sea fungi have been reported. Although genomic sequencing results show that GliM is an O-methyltransferase, the specific function in the GT biosynthetic pathway is not clear.
Disclosure of Invention
The first purpose of the invention is to provide a GliM promoter Gl of a deep-sea fungus Geosmithia pallida FS 140O-methyltransferase geneiM0The GliM promoter GliM of the deep-sea fungus FS 140O-methyltransferase gene0The nucleotide sequence is shown in SEQ ID NO. 1.
The deep sea fungus FS 140O-methyltransferase gene GliM promoter GliM of the invention0Is obtained by the following method: the gliotoxin biosynthesis gene GliM (the nucleotide sequence of which is shown in SEQ ID NO. 2) for coding O-methyltransferase is obtained by transcriptome sequencing. Specific reverse primers sp1, sp2 and sp3 are designed in the upstream sequence, and three rounds of nested PCR amplification are carried out by adopting a universal forward primer AP3 of a Genome walking kit, and the amplified product of each round is diluted to be used as a template for the next round of amplification. TA cloning is carried out on the amplification product of the last round, the amplification product is transformed into an escherichia coli competent cell, coating on an ampicillin resistance plate to screen out positive clone, PCR verification and sequencing are carried out on the positive clone by bacterial liquid to obtain a GliM upstream promoter sequence of the target gene, and the upstream promoter sequence is analyzed by utilizing promoter prediction software to obtain the GliM promoter core region GliM0(i.e., GliM promoter of GliM gene of deep sea fungus FS 140O-methyltransferase0The nucleotide sequence is shown as SEQ ID NO. 1); designing a primer and adopting a point mutation method to combine the GliM of a promoter core region0The critical region of (a) was mutated (fig. 5).
The invention utilizes a homologous recombination method to combine GliM promoter core region GliM0The TEF1 promoter element replacing the yeast vector YEp352-TEF 1-HYRB. First, GliM is designed aiming at the core region of GliM promoter0The primer sequence of the upstream and downstream primers for amplification is GliM0-F：
5'-AATTCGAGCTCGGTACCCGGGGATCCTATTTTATTTTTATGGGGGTACGCGAGCG-3'；
GliM0-R：
5'-GACGTCGCGGTGAGTTCAGGCATTCTAGAGGTGAGTTGATCTGGCAAAAGTGT-3', obtaining the product by PCR amplification and purifying the recovered fragment. The constructed YEp352-TEF1-HYRB vector was digested with BamHI and Xba I, the fragment and the digested vector were then recombinantly ligated using Clon express II One Step Cloning Kit C112(Vazyme) and transformed into E.coli competent cells, which were plated on ampicillin resistant plates and screenedAnd (4) positive cloning. After the round of molecular cloning, the target gene GliM promoter core region GliM0(the nucleotide sequence is shown as SEQ ID NO.1) replaces TEF1 promoter to construct YEp352-GliM0HYRB vector, electrotransformed into Saccharomyces cerevisiae BJ5464 cells, screened and validated using YPD plates containing 50. mu.g/mL hygromycin resistance.
Then aiming at positive clone YEp352-GliM0-HYRB engineered promoter core region GliM0Upstream and downstream primers with key core region mutation, and primer sequences thereof are GliM0-tu-F：5'-GTTAAAGAGAACGCTCAAGACATATCGTCCCTAGC-3'；GliM0-tu-R: 5'-TCTTGAGCGTTCTCTTTAACAATCGTGATGGCAAAAGTGGAGC-3', YEp352-GliM Positive clone0Using HYRB vector as template, obtaining product by PCR amplification, using DpnI to eliminate template, directly transforming into Escherichia coli competent cell, coating ampicillin resistant plate to pick out transformant, using primer GliM0-F and GliM0-R for sequencing verification. Sequencing results show that the vector YEp352-GliM0-HYRB core region GliM0The key region of (2) was successfully mutated (FIG. 5), and the construction yielded YEp352-GliM0-tu-HYRB vector, electroporated into Saccharomyces cerevisiae BJ5464 cells, screened and validated using YPD plates containing 50. mu.g/mL hygromycin resistance.
Compared with the Saccharomyces cerevisiae BJ5464 transferred with YEp352-TEF1-HYRB plasmid (positive control), the Saccharomyces cerevisiae containing the recombinant vector YEp352-GliM0-HYRB has consistent growth speed and similar number of positive colonies. Demonstration of the core region of the GliM promoter0Can promote the expression of the hygromycin resistance gene hph, and the promotion efficiency is similar to that of a constitutive promoter TEF 1. And contains the recombinant vector YEp352-GliM0The growth speed of the saccharomyces cerevisiae of-tu-HYRB is consistent with the number of negative colonies, the growth is obviously hindered, the growth of sterile colonies is proved, and the mutant core region GliM is proved050bp in (A) is the key core region of the promoter.
The second purpose of the invention is to provide an expression vector containing the GliM promoter GliM of the deep sea fungus FS 140O-methyltransferase gene GliM0。
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 GliM promoter GliM of the deep sea fungus FS 140O-methyltransferase gene GliM0The use of a promoter to promote expression of a downstream gene in a host cell.
The host cell is preferably a deep-sea fungus Geosmithia pallida FS140 or Saccharomyces cerevisiae cer evisiae BJ 5464.
The downstream gene is preferably O-methyltransferase gene GliM or hygromycin resistance gene hph.
The fifth object of the present invention is to provide an expression cassette comprising the GliM promoter of the above deep sea fungus FS 140O-methyltransferase gene GliM0。
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. As the promoter of deep sea fungus Geosmithia pallida FS140 is not reported at present, transcriptome sequencing and literature research results also show that the expression level of a biosynthesis gene GliM for encoding gliotoxin is higher, which indicates that the promoter of the gene has higher transcription activity. Therefore, the invention adopts a Genome Walking kit, utilizes a universal primer and a reverse specific primer, utilizes the TAIL-PCR principle to obtain an upstream promoter sequence of deep-sea fungus FS 140O-methyltransferase gene GliM, predicts a promoter core region to obtain a promoter key core region and performs functional verification, thereby laying a molecular biology foundation for improving the expression level of the gliotoxin and obtaining the novel gliotoxin through transcription regulation and heterologous expression in the later stage.
The deep sea fungus Geosmithia pallida FS140 of the invention is disclosed in the literature: 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 Thiodikepiprowazine with inhibition Activity against Enzyme antigen-Converting Enzyme from a Deep-Sea-Derived 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.
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FIG. 1 shows the obtaining of the promoter sequence of the deep sea fungus FS140 GliM; wherein A is an electrophoretogram of chromosome walking amplification products of a GliM promoter, G1 is a first nested PCR amplification product, G2 is a second nested PCR amplification product, and G3 is a third nested PCR amplification product; b is GliM promoter core region GliM0Electrophoretogram of the amplified product;
FIG. 2 shows a recombinant vector YEp352-GliM0-construction of HYRB; wherein A is YEp352-TEF1-HYRB vector map; b is YEp352-GliM0-a HYRB vector map; c is YEp352-GliM0-tu-HYRB vector map;
FIG. 3 shows pairs of hygromycin-resistant YPD plates containing No promoter (No Pro), YEp352-TEF1-HYRB plasmid (TEF1), YEp352-GliM, and 50. mu.g/mL0-HYRB plasmid (GliM)0) And YEp352-GliM0-tu-HYRB plasmid (GliM)0-tu) selection of saccharomyces cerevisiae;
FIG. 4 shows the core region GliM of the GliM promoter0The electrophoretogram of the PCR amplification product of the saccharomyces cerevisiae colony.
FIG. 5 is a sequence alignment chart of the results of successful mutation sequencing of the key core region of the GliM promoter.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
In this example, the Genome walking kit and QuickCut (DPnI) enzyme were purchased from Takara bioengineering, Inc. (Dalian, China); the TA cloning kit was purchased from novispan biotechnology, inc (Nanjing, China).
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, 30g of agar powder and the balance of distilled water, and the preparation method comprises the following steps: mixing the components of the culture medium, stirring for dissolving, and sterilizing.
Example 1 obtaining of promoter sequence of GliM Gene of deep sea fungus Geosmithia pallida FS 140O-methyltransferase
GliM promoter GliM0Amplification of (3): deep sea fungus Geosmithia pallida FS140 is inoculated on a YPD medium plate, cultured for 72 hours at 28 ℃, fresh mycelium is picked, and genome DNA is extracted by using a fungus DNA extraction kit. Adopting a Genome walking kit, amplifying by TAIL-PCR technology to obtain an upstream sequence of deep sea fungus FS 140O-methyltransferase gene GliM (the GliM gene sequence is shown in SEQ ID NO. 2), obtaining a nucleotide sequence of the upstream sequence by TA cloning and sequencing, and obtaining a core region GliM of the promoter by using promoter analysis software0And its critical core area. Designing specific reverse primers sp1, sp2 and sp3 (table 1) in the GliM gene sequence, performing nested PCR amplification by using a general forward primer AP3 of a Genome walking kit, performing Genome amplification on FS140 through 3 times of nested PCR reaction to obtain corresponding amplification products G-1, G-2 and G-3, performing TA cloning on a target band obtained by the last nested PCR amplification product G-3 by using a pEASY-T1 kit, transforming the target band to an escherichia coli competent cell, coating the escherichia coli competent cell on an ampicillin plate to screen out a positive clone, performing bacterial liquid PCR verification on the positive clone by using M13-F (5'-GTAAAACGACGGCCAGT-3') and M13-R (5'-CAGGAAACAGCTATGAC-3') primers, and sequencing to obtain the GliM upstream promoter sequence of the target gene (the nucleotide sequence of which is shown as SEQ ID NO. 3), and analyzing the sequence of the upstream promoter by using promoter prediction software (http:// www.fruitfly.org/seq _ tools/promoter. html) to obtain the core region GliM of the GliM promoter0670bp in size
(tattttatttttatgggggtacgcgagcgggtttcgcgggaatatggacgactatttcgacaggcagctgcagttcacgccgagattctagagactgtgatttgtgaaggggtttattctggattgaagctgacttggagctgaattgatatttccactggacactcatagtaattcccatgtaccccgtagttacacagccataattggattattcttttctttttttttaatcacttatactcctgcttaccatgtcagcaatgacttcaataataatcaagacgagttacacagccgatgctgactgtaaagtggcgtcagctgcaatatacgcggatgtcacctcttgggtcattgtgcatcaaccaccgccagccatcctgcttggaggatagaactgcttagctgaatgatgagtgtccctggcaggatccattatttggtagcccaattaatggtctcaagatgactgctctgactatacctgcaggtcttggactcctggccggctccacttttgccatcacgattgttaaagagagggtttttgatataagttcggcagttacagttctggaatttctggagtaaacgctcaagacatatcgtccctagcatctatagtttctatacagacagacagacacttttgccagatcaactcacc, the nucleotide sequence of which is shown in SEQ ID NO.1), wherein the key core region is: 5'-GGGTTTTTGATATAAGTTCGGCAGTTACAGTTCTGGAATTTCTGGAGTAA-3', size 50 bp.
TABLE 1 GliM-specific reverse primer sequences of target genes
Figure BDA0002857703410000071
Example 2 GliM promoter core region GliM0Function verification of
First, a hygromycin-resistant gene hygromycin-B (GenBank Accession: XM-003071606) was inserted between the Xba I and Sal I cleavage sites of yeast vector YEp352-TEF1-CYC1 (YEp352-TEF1-CYC1 was an early construction plasmid carrying constitutive promoter TEF1 and terminator CYC1, and a control plasmid YEp 352-TEF-41 was constructed as a known product in the art: Xiaodan Ouyan, Yaping Cha, Wen Li, Chaoyi Zhu, Muzi Zhu, Shuang Li, Min Zhuo, Shaobin Huang and Jianjun Li.Stepwi engineering of Saccharomyces cerevisiae to genes and related (+) -modified plasmids, see FIG. Stepwi gene engineering of Saccharomyces cerevisiae to genes RB Adv, RSC 2019, HY-C-36352).
Secondly, the core region GliM of the GliM promoter is converted into GliM by homologous recombination0(the nucleotide sequence of which is shown in SEQ ID NO.1) is inserted into the yeast vector YEp352-TEF1-HYRB by way of the replacement element TEF 1. First, GliM is designed aiming at the core region of GliM promoter0(SEQ ID NO.1) upstream and downstream primers GliM for amplification0-F (5'-AATTCGAGCTCGGTACCCGGGGATCCTATTTTATTTTTATGGGGGTACGCGAGCG-3') and GliM0-R(5'-gacgtcgcggtgagttcaggcatTCTAGAGGTGAGTTGATCTGGCAAAAGTGT-3'), and the product was obtained by PCR amplification. The vector YEp352-TEF1-HYRB was used to excise TEF1 promoter with BamHI and Xba I and recover the product, which was then recombinantly ligated using Clon express II One Step Cloning Kit C112(Vazyme) and transformed into E.coli DH5 alpha competent cells to screen for positive clones. With primer GliM0-F and GliM0Colony PCR verification is carried out on-R, and the result shows that GliM0Successful insertion into the YEp352-HYRB vector and confirmation by sequencing gave YEp352-GliM0-HYRB vector (YEp 352-GliM)0The HYRB carrier map is shown in FIG. 2B).
Then aiming at positive clone YEp352-GliM0HYRB design against the GliM promoter core region GliM0The primer sequence of the mutated upstream and downstream primers is GliM0-tu-F：5'-GTTAAAGAGAACGCTCAAGACATATCGTCCCTAGC-3'；GliM0-tu-R: 5'-TCTTGAGCGTTCTCTTTAACAATCGTGATGGCAAAAGTGGAGC-3', YEp352-GliM Positive clone0Using HYRB as a template, obtaining a product through PCR amplification, directly transforming the product into escherichia coli competent cells after eliminating the template by using DpnI, coating the escherichia coli competent cells on an ampicillin resistant plate, selecting a transformant, and using a primer GliM0-F and GliM0-R for sequencing verification. Sequencing results show that YEp352-GliM0Glim in HYRB vectors0The key core region of the fragment was successfully mutated (FIG. 5), and the construction yielded YEp352-GliM0The tu-HYRB vector (vector map see FIG. 2C), which was electroporated into Saccharomyces cerevisiae BJ5464 cells, was screened and validated using YPD plates containing 50. mu.g/mL hygromycin resistance.
Preparing competent cells of Saccharomyces cerevisiae BJ5464, and culturing YEp352-TEF1-HYRB plasmid vector (positive control) and YEp352-GliM0-HYRB and YEp352-GliM0the-tu-HYRB plasmid vector is respectively transferred into saccharomyces cerevisiae BJ5464 cells (1500V, 5ms), evenly coated in YPD plates containing 50 ug/mL hygromycin resistance, cultured for 2d at 30 ℃, screened for positive clones by colony PCR, and further sequenced and verified. No colonies were produced on the negative control plate without promoter (without plasmid vector addition) (FIG. 3-No Pro), indicating 50. mu.g/mL hygromycinThe concentration of the antibiotic resistance can be used for screening saccharomyces cerevisiae BJ 5464. Containing YEp352-GliM compared to a positive control (containing YEp352-TEF1-HYRB plasmid vector, TEF1)0Saccharomyces cerevisiae (GliM) of the HYRB plasmid vector0) Consistent growth rates with similar numbers of colonies (FIG. 3), demonstrating the GliM promoter core region GliM0Can effectively promote the expression of the hygromycin resistance gene hph, and the promotion efficiency is similar to that of a constitutive promoter TEF 1. The growth rate of the saccharomyces cerevisiae (GliM0-tu) containing the recombinant vector YEp352-GliM0-tu-HYRB is consistent with the number of negative colonies, obvious growth is hindered, and aseptic colony growth (figure 3) proves that the mutated GliM0The 50bp of the fragment is the key core region of the promoter.
Picking the vector containing the recombinant plasmid YEp352-GliM0Colony PCR of Saccharomyces cerevisiae of HYRB with primer GliM0-F and GliM0-R, amplification to obtain GliM promoter core region GliM0Positive clones were screened and verified by sequencing (FIG. 4); picking the vector containing the recombinant plasmid YEp352-GliM0Colony PCR of Saccharomyces cerevisiae of-tu-HYRB with primer GliM0-F and GliM0Sequencing verification is carried out on the-R, and the result shows that the amplification obtains the GliM promoter fragment GliM0The core region in (A) has been successfully mutated to GliM0Tu (FIG. 5).
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 FS 140O-methyltransferase gene GliM promoter and application thereof
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tattttattt ttatgggggt acgcgagcgg gtttcgcggg aatatggacg actatttcga 60
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ggattgaagc tgacttggag ctgaattgat atttccactg gacactcata gtaattccca 180
tgtaccccgt agttacacag ccataattgg attattcttt tctttttttt taatcactta 240
tactcctgct taccatgtca gcaatgactt caataataat caagacgagt tacacagccg 300
atgctgactg taaagtggcg tcagctgcaa tatacgcgga tgtcacctct tgggtcattg 360
tgcatcaacc accgccagcc atcctgcttg gaggatagaa ctgcttagct gaatgatgag 420
tgtccctggc aggatccatt atttggtagc ccaattaatg gtctcaagat gactgctctg 480
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gagagggttt ttgatataag ttcggcagtt acagttctgg aatttctgga gtaaacgctc 600
aagacatatc gtccctagca tctatagttt ctatacagac agacagacac ttttgccaga 660
tcaactcacc 670
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atggaagcca acaacaccga ccaaatcccc aagcgcagac acaccgatga cctgtacgag 60
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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
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<400> 3
tattttattt ttatgggggt acgcgagcgg gtttcgcggg aatatggacg actatttcga 60
caggcagctg cagttcacgc cgagattcta gagactgtga tttgtgaagg ggtttattct 120
ggattgaagc tgacttggag ctgaattgat atttccactg gacactcata gtaattccca 180
tgtaccccgt agttacacag ccataattgg attattcttt tctttttttt taatcactta 240
tactcctgct taccatgtca gcaatgactt caataataat caagacgagt tacacagccg 300
atgctgactg taaagtggcg tcagctgcaa tatacgcgga tgtcacctct tgggtcattg 360
tgcatcaacc accgccagcc atcctgcttg gaggatagaa ctgcttagct gaatgatgag 420
tgtccctggc aggatccatt atttggtagc ccaattaatg gtctcaagat gactgctctg 480
actatacctg caggtcttgg actcctggcc ggctccactt ttgccatcac gattgttaaa 540
gagagggttt ttgatataag ttcggcagtt acagttctgg aatttctgga gtaaacgctc 600
aagacatatc gtccctagca tctatagttt ctatacagac agacagacac ttttgccaga 660
tcaactcacc atggaagcca acaacaccga ccaaatcccc aagcgcagac acaccgatga 720
cctgtacgag ctctcggtta atatctccag cgcggtcgag accttcctcg gaaggctgga 780
cgctgtagag cacacacctt cgattat 807

Claims (7)

1. Promoter GliM0The nucleotide sequence is shown as SEQ ID NO. 1.
2. An expression vector comprising the promoter GliM according to claim 10。
3. A host cell comprising the expression vector of claim 2.
4. The host cell of claim 3, wherein said host cell is Saccharomyces cerevisiaeSaccharomyces cerevisiae BJ5464。
5. The promoter GliM of claim 10Application of promoting expression of downstream gene in host cell, wherein the host cell is deep sea fungusGeosmithia pallidaFS140 or Saccharomyces cerevisiaeSaccharomyces cerevisiaeBJ5464。
6. The use of claim 5, wherein the downstream gene is an O-methyltransferase geneGliMOr hygromycin resistance genehph。
7. An expression cassette comprising the promoter GliM of claim 10。
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