CN112725337B - GliT promoter of deep-sea fungus FS140 oxidoreductase gene and application thereof - Google Patents
GliT promoter of deep-sea fungus FS140 oxidoreductase gene and application thereof Download PDFInfo
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Abstract
The invention discloses a GliT promoter of a deep-sea fungus FS140 oxidoreductase gene and application thereof. The nucleotide sequence of the promoter is shown as SEQ ID NO. 1. The invention obtains the upstream promoter sequence of the GliT gene by a chromosome walking technology and predicts the core region of the promoter to obtain the core region GliT of the GliT promoter of the deep-sea fungus FS140 oxidoreductase gene 0 And successfully verifies the key core region of the GliT promoter in prokaryotic and eukaryotic expression systems through point mutation. The promoter disclosed by the invention can efficiently start the expression of the hygromycin resistance gene hph, and the starting efficiency is similar to that of a constitutive promoter TEF 1; can also effectively start the expression of ampicillin Amp, which lays a molecular biology foundation for improving the output of gliotoxin and obtaining more novel gliotoxin with high activity through transcription regulation and heterologous expression in the later period.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to a GliT promoter of a deep-sea fungus Geosmithia pallida FS140 oxidoreductase gene and application 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, tef1 (translational elongation factor gene), gpdA (glyceraldehyde triphosphate dehydrogenase gene) promoter of Aspergillus nidulans, pki1 promoter of Trichoderma (Trichoderma sp.), glaA promoter of Aspergillus, and the like of Trichoderma, but there are few reports on related studies on promoters of secondary metabolite biosynthesis genes of deep sea fungi. No research report about GliT promoter of deep-sea fungus Geosmithia pallida FS140 oxidoreductase is available at present.
Disclosure of Invention
The first purpose of the invention is to provide a GliT promoter of a deep-sea fungus Geosmithia pallida FS140 oxidoreductase gene GliT 0 The GliT promoter GliT of the deep sea fungus FS140 oxidoreductase gene 0 The nucleotide sequence is shown in SEQ ID NO. 1.
The GliT promoter GliT of the deep sea fungus FS140 oxidoreductase gene 0 Is obtained by the following method: the gliotoxin biosynthesis gene GliT (the nucleotide sequence of which is shown in SEQ ID NO. 2) for coding oxidoreductase is obtained by transcriptome sequencing. Specific reverse primers sp1, sp2 and sp3 are designed in the upstream sequence of the primer, and a general forward primer AP of a Genome walking kit is adopted3, three rounds of nested PCR amplification are carried out, 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 GliT upstream promoter sequence of the target gene, and the upstream promoter sequence is analyzed by utilizing promoter prediction software to obtain the GliT promoter core region GliT 0 (i.e. a GliT promoter of a deep-sea fungus FS140 oxidoreductase gene, the nucleotide sequence of which is shown in SEQ ID NO. 1); designing a primer to use a point mutation method to convert a promoter core region GliT 0 And GliT 1 The critical region of (a) was mutated (fig. 2).
The second purpose of the invention is to provide an expression vector containing the deep-sea fungus FS140 oxidoreductase gene GIlT promoter GliT 0 。
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 BJ5464 and Escherichia coli.
The fourth purpose of the invention is to provide the GliT promoter GliT of the deep sea fungus FS140 oxidation-reduction enzyme gene GliT 0 The 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, Saccharomyces cerevisiae BJ5464 and Escherichia coli.
The downstream gene is preferably an oxidoreductase gene GliT, a hygromycin resistance gene hph or an ampicillin gene Am p.
The fifth object of the present invention is to provide an expression cassette comprising the GliT promoter of the deep sea fungus FS140 oxidoreductase gene GliT 0 。
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. In view of the fact that no report is available on the promoter of deep-sea fungi Geosmithia pallida FS140 at present, transcriptome sequencing and literature research results also show that the expression level of a biosynthesis gene GliT for encoding gliotoxin is high, which indicates that the promoter of the gene has high 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 the deep-sea fungal FS140 oxidoreductase gene GliT, predicts a promoter core region to obtain a promoter key core region, and respectively carries out functional verification in an eukaryotic expression vector and a prokaryotic expression vector, thereby laying a molecular biological foundation for improving the expression level of the gliotoxin and obtaining the novel gliotoxin through transcription regulation and heterologous expression in the later period.
The deep sea fungus Geosmithia pallida FS140 of the invention is disclosed in the literature: Zhang-Hua Sun, Jiangyon g Gu, Wei Ye, Liang-Xi Wen, Qi-Bin Lin, Sai-Ni Li, Yu-Chan Chen, Hao-Hua Li, Wei-Min Zhang.Geospallins A-C New Thiodikepiprowazines 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 that the strain is provided to the public within 20 years from the filing date of the invention.
Drawings
FIG. 1 shows the obtaining of the GliT promoter sequence of deep-sea fungus FS 140; wherein A is an electrophoretogram of chromosome walking amplification products of a GliT 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 and C are electrophoresis images of amplification products of the core region of the GliT promoter, and B is pET30a-Amp-GliT 0 An electrophoretogram; c is YEp352-GliT 0 -an electropherogram of HYRB amplification products;
FIG. 2 promoter prediction website (http:// www.fruitfly.org/seq _ tools/promoter. html) predicts key core regions of promoters;
FIG. 3 shows hygromycin at 100. mu.g/mLThe pair of YPD plates resistant to the biotin contained no promoter (A), YEp352-TEF1-H YRB plasmid (B), YEp352-GliT 0-tu HYRB plasmid (C) and YEp352-GliT 0 -screening of the HYRB plasmid (D) saccharomyces cerevisiae;
FIG. 4 shows the selection of a non-resistant LB plate (A) and an LB plate (B) containing 100. mu.g/mL ampicillin resistance, in which ACP stands for pET30 a-ACP-Amp; pEASY-T1 represents pEASY-T1; Amp-T 0 Representing pET30a-A mp-GliT 0 ；Amp-T 1 -Tu stands for pET30a-Amp-GliT 1-tu ；
FIG. 5 shows the core region GliT of the GliT promoter 0 The electrophoresis of the amplified product, wherein A is an electrophoretogram of a PCR amplified product of a saccharomyces cerevisiae colony; b is the electrophoretogram of colony PCR amplification product.
FIG. 6 is a sequence alignment chart of the results of successful mutation sequencing of core region of GliT promoter, wherein A is eukaryotic expression vector YEp352-GliT 0-tu -a HYRB contrast map; b is prokaryotic expression vector pET30a-Amp-GliT 1-tu Compare the figures.
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 GliT promoter sequence of deep sea fungus Geosmithia pallida FS140 oxidoreductase gene
GliT promoter GliT 0 Amplification 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. Obtaining deep-sea fungi by amplification of TAIL-PCR technology by adopting Genome walking kitAn upstream sequence of FS140 oxidoreductase gene GliT gene (the sequence of the GliT gene is shown in SEQ ID NO. 2), a nucleotide sequence of the upstream sequence is obtained by TA clone sequencing, and a core region GliT of the promoter is obtained by utilizing promoter analysis software 0 And its critical core area. Designing specific reverse primers sp1, sp2 and sp3 (table 1) in a GliT gene sequence, performing nested PCR amplification by using a general forward primer AP3 of a Genome Walkin G kit, performing Genome amplification on FS140 through 3 times of nested PCR reactions to obtain corresponding amplification products G-1, G-2 and G-3, performing TA cloning on a target band of the final nested PCR amplification product G-3 by using a pEASY-T1 kit, transforming the target band into an Escherichia coli competent cell, coating the Escherichia coli competent cell on a penicillin ampicillin resistance plate to screen out positive clones, performing PCR bacterial liquid verification positive clones by using M13-F (5'-GTAAAACGACGGCCAGT-3') and M13-R (5'-CAGGAAACAGCTATGAC-3') primers, and sequencing to obtain a GliT 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 GliT of the GliT promoter 0 The size of the gene is 981bp (the nucleotide sequence is shown as SEQ ID NO.1, ctaggtgtagttcaacgtgggctggtttccatgtcctctgtcgcgactgacgaggggaggccttacaaagcgcctgtactgaataggaatgatggcggtcgtcgtgcatgatggatgcgcggtcttgatctcgagtccattctgtgggttgaagctgaagcacccgaggacatggcgtagtcgatttcagctggtgaacgagaccaacacggcagggttgttgcggacgagaagatatctcatggcatcatcgaattcatcagccttggtgcccatgtgcgagtactcgcctaggaattggttgtaatgttgctttatatagatgattagcggccagttatacagtaaaagaaatgacctacttgatagacagctgagactgagggagcgtctggacggtatcgatttcggacacaggaagaacgaccacctgctgtccggggacacccagcgcaaagggagtgtctggatactttgtatatatagcatcatctcatacaaaaagacataggaagagtacccgggcgtgtcccccctccaaggttttcacaatatcttcatcgtcacactgaagaacaggcaagtccagcgctttgagactcttggtggcagcaaaaacagtcgatttatccagtacaacaggccaatcccgacagagacaaagaccagaatctgcaggggttggtgggaaagatatagatagtccatggtatcctgttgccactcactgcccttgccaatccttcgcaggtatgggtccagttcagaaaacaaagcctggagcatctcattaatagagcagaaggattatccatttgtataaatgatcattccagattctca)atgttgccacggacccctttttcggctgccgagggggaaagtccccgagtggttcttaataaatagatagtttgctggtctatcttaacaaagtaaatattcctttcgttcttccactcttgttagaatttcccaaac) in which the key core region sequences (fig. 2).
TABLE 1 GliT-specific reverse primer sequences for target genes
Example 2 GliT promoter core region GliT 0 Eukaryotic expression vector function verification
First, the hygromycin-resistant gene hygromycin-B (GenBank Accession: XM-003071606) was inserted between the Xba I and Sal I cleavage sites of the yeast vector YEp352-TEF1-CYC1 (YEp352-TEF1-CYC1 is an early construction plasmid carrying the constitutive promoter TEF1 and the terminator CYC1, and is constructed as a positive plasmid of Xiaodan Ouyang, Yaping Cha, Wen Li, Chaoyi Zhu, Muzi Zhu, Shuang Li, Min Zhu o, Shaobin Huang Li.Stepwis engineering of Saccharomyces cerevisiae to positive 35urea and related bits (+) -related sequences, RSC Adv.2019, RSC 359, 76I 3532-T35 RB, HYGR I3526-T35F 3526/32 by enzyme ligation), and a control.
Secondly, the core region GliT of the GliT promoter is converted into GliT by homologous recombination 0 (the nucleotide sequence of which is shown in SEQ ID NO.1) is inserted into the yeast vector YEp352-TEF1-HYRB by means of the replacement element TEF 1. First, design the core region GliT of the GliT promoter 0 (SEQ ID NO.1) amplified upstream and downstream primers HYRB-GliT 0 -F: 5'-ATTCGAGCTCGGTACCCGGGGATCCCTAGGTGTAGTTCAACGTGGGCT-3' and HYRB-GliT 0 -R: 5'-CGCGGTGAGTTCAGGCATTCTAGAGTTTGGGAAATTCTAACAAGAGTGGAAGAAC-3', obtaining the product by PCR amplification. The TEF1 promoter was excised by double digestion with BamH I and Xba I and the product recovered for vector YEp352-TEF1-HYRB, and the two products were then recombinantly ligated and transformed into E.coli DH 5. alpha. competent cells using Clonexpress II One Step Cloning Kit C112(Vazyme) to screen for positive clones. With primer GliT 0 -F:5'-CTAGGTGTAGTTCAACGTGGGCT-3'；GliT 0 5'-GTTTGGGAAATTCTAACAAGAGTGGAAGAAC-3' shows that the results of colony PCR verification indicate GliT 0 Successful insertion into the YEp352-HYRB vector and confirmation by sequencing gave YEp352-GliT 0 -a HYRB carrier.
Then aiming at positive clone YEp352-GliT 0 HYRB is designed to the GliT promoter core region GliT 0 The primer sequence of the mutated upstream and downstream primers is HYRB-GliT 0-tu -F：5'-GAGCAGAAGGATCCTTTTTCGGCTGCCGAGGGGGAAAGTCC-3'；HYRB-GliT 0-tu -R: 5'-CCGAAAAAGGATCCTTCTGCTCTATTAATGAGATGCTCCAGGCTTTGTTTTCT-3', YEp352-GliT Positive clone 0 Using 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 to pick out a transformant, and using a primer GliT 0 -F and GliT 0 -R for sequencing verification. Sequencing results show that YEp352-GliT 0 GliT in HYRB vector 0 The key core region of the fragment is successfully mutated to construct YEp352-GliT 0-tu HYRB vector (FIG. 6A), which was electroporated into Saccharomyces cerevisiae BJ5464 cells, screened and validated using YPD plates containing 100. mu.g/mL hygromycin resistance.
Preparing competent cells of Saccharomyces cerevisiae BJ5464, and culturing YEp352-TEF1-HYRB plasmid vector (positive control) and YEp352-GliT 0 -HYRB and YEp352-GliT 0-tu The HYRB plasmid vectors are respectively electroporated into Saccharomyces cerevisiae BJ5464 cells (1500V, 5ms), evenly coated on YPD plates containing 100 mu g/mL hygromycin resistance, cultured for 2d at 30 ℃, screened for positive clones by colony PCR, and further sequenced and verified. No colonies were generated on the negative control plate without promoter (without addition of plasmid vector) (FIG. 3A), indicating that a hygromycin resistance concentration of 100. mu.g/mL could be used to screen for Saccharomyces cerevisiae BJ 5464. Containing YEp352-GliT, in contrast to the positive control (containing YEp352-TEF1-HYRB plasmid vector, FIG. 3B) 0 The consistent growth rate of Saccharomyces cerevisiae with similar colony numbers of HYRB plasmid vectors (FIG. 3D) demonstrated that the core region of the GliT promoter, GliT 0 Can effectively start the expression of hygromycin resistance gene hph, and the starting efficiency is the same as that of a constitutive promoter TEF1And (4) approaching. And contains the recombinant vector YEp352-GliT 0-tu Saccharomyces cerevisiae growth rate of HYRB was consistent with the number of negative colonies, significantly hindered growth, and colony-free growth (FIG. 3C), demonstrating the mutated GliT 0-tu The 50bp of the fragment is the key core region of the promoter.
Picking the vector containing the recombinant plasmid YEp352-GliT 0 Colony PCR of Saccharomyces cerevisiae of HYRB, with HYRB-GliT as primer 0 -F and HYRB-GliT 0 -R, obtaining the GliT promoter core region GliT by amplification 0 Screening to obtain positive clones (FIG. 5), and verifying by sequencing; picking the vector containing the recombinant plasmid YEp352-GliT 0-tu Colony PCR of Saccharomyces cerevisiae of HYRB with primer GliT 0 -F and GliT 0 Sequencing verification is carried out on the-R, and the result shows that the amplification obtains the GliT promoter fragment GliT 0 The core region in (A) has been successfully mutated to YEp352-GliT 0-tu HYRB (FIG. 6A).
Example 2 GliT promoter core region GliT 0 Functional verification of prokaryotic expression vector
First, the core region GliT of the GliT promoter is modified by homologous recombination 0 Was constructed into pET30a-Amp vector. Firstly, designing a homologous arm primer aiming at a pET30a-Amp vector, carrying out PCR by using PrimeSTAR MAX Premix enzyme and using a pEASY-T1 vector as a template to obtain an ampicillin resistance gene Amp fragment, simultaneously amplifying a pET30a-ACP vector, purifying and recovering a PCR product, recombining the Amp fragment to pET30a-ACP by using Clone II One Step Cloning Kit C112(Vazyme), converting the mixture into a trans5 alpha competence, coating on a kanamycin resistance plate to screen positive clones, and obtaining pET30a-ACP-Amp no-load with ampicillin resistance through colony PCR and sequencing verification of an upgraded particle. Design against pET30a-Amp-GliT 0 The primer sequence of the homologous arm primer of (1) is Amp-GliT 0 -F：5'-TTAGCAGAATGAATCACCGATACGCTAGGTGTAGTTCAACGTGGGCT-3'；Amp-GliT 0 -R: 5'-CACGGAAATGTTGAATACTCATGTTTGGGAAATTCTAACAAGAGTGGAAG-3', obtaining the product by PCR amplification and purifying the recovered fragment. Amplifying to obtain GliT 0 The promoter fragment was used to double-load pET30a-ACP-Amp with restriction enzymes NdeI and Xho IAnd (4) enzyme digestion, identifying through agarose gel electrophoresis, cutting the gel, recovering the PCR product and the enzyme digestion product, and determining the concentration of the PCR product and the enzyme digestion product. GliT Using homologous recombination kit 0 Homologous recombination is carried out on the promoter fragment and the empty vector pET30a-ACP-Amp, the promoter fragment is transformed into trans5 alpha competence, the promoter fragment is coated on a kanamycin-resistant plate to screen positive clones, and pET30a-Amp-GliT is obtained through colony PCR and sequencing verification 0 And (3) a carrier.
Thirdly, positive clone pET30a-Amp-GliT is used 0 As template, the mutant primer Amp-GliT was used 1-tu -F:5'-TCTGCAGGGGCCTTGCCAATCCTTCGCAGGTATGGGTCCAGTTCAG-3'
Amp-GliT 1-tu 5'-ATTGGCAAGGCCCCTGCAGATTCTGGTCTTTGTCTCTGTCGGGATTG-3' construction of pET30a-Amp-GliT 1-tu Vector (FIG. 6B). pET30a-ACP-Amp (negative control) was spread on LB plates containing 100. mu.g/mL ampicillin resistance, and cultured at 30 ℃ for 24 hours, and no colonies were produced on the negative control plates without promoter (without addition of plasmid vector), indicating that a concentration of 100. mu.g/mL ampicillin resistance could be used for selection.
Fourthly, pET30a-Amp-GliT 0 、pET30a-Amp-GliT 1-tu Positive transformants, pEASY-T1 (positive control) and pET30a-ACP-Amp were inoculated in LB broth medium containing kanamycin (negative control), shake-cultured at 37 ℃ and OD of each bacterial solution 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 result of culturing the dilutions of (A) on LB plates with an ampicillin concentration of 0 and 100. mu.g/mL, respectively, at 37 ℃ for 12 hours is shown (see FIG. 4).
Compared with pEASY-T1 plasmid (positive control) Escherichia coli, the recombinant vector pET30a-Amp-GliT 0 The growth speed of the escherichia coli is consistent, and the number of positive colonies is similar. Demonstration of the core region GliT of the GliT promoter 0 Can regulate and control the expression of ampicillin resistance gene Amp and start effectThe rate is similar to that of positive pEASY-T1. And contains a recombinant vector pET30a-Amp-GliT 1-tu The growth speed of the escherichia coli is consistent with the number of negative colonies, obvious growth is blocked, the growth of the bacteria-free colonies is proved, and the mutant core region GliT 1-tu 50bp in the expression vector is a key core region of the promoter in a prokaryotic expression vector.
Picking the recombinant plasmid containing pET30a-Amp-GliT 0 The Escherichia coli is subjected to colony PCR, and the adopted primer is Amp-GliT 0 -F and Amp-GliT 0 -R, amplification to obtain GliT promoter core region GliT 0 Screening to obtain positive clones, and sequencing to obtain verification; picking a plasmid containing the recombinant plasmid pET30a-Amp-GliT 1-tu The Escherichia coli (E.coli) was subjected to colony PCR using a primer GliT 0 -F and GliT 0 Sequencing verification is carried out on the-R, and the result shows that the amplification obtains the GliT promoter fragment GliT 1 The core region in (A) has been successfully mutated to pET30a-Amp-GliT 1-tu (FIG. 6B).
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 should be considered to be 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 oxidoreductase gene GliT promoter and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 981
<212> DNA
<213> fungus FS140(Geosmithia pallida)
<400> 1
ctaggtgtag ttcaacgtgg gctggtttcc atgtcctctg tcgcgactga cgaggggagg 60
ccttacaaag cgcctgtact gaataggaat gatggcggtc gtcgtgcatg atggatgcgc 120
ggtcttgatc tcgagtccat tctgtgggtt gaagctgaag cacccgagga catggcgtag 180
tcgatttcag ctggtgaacg agaccaacac ggcagggttg ttgcggacga gaagatatct 240
catggcatca tcgaattcat cagccttggt gcccatgtgc gagtactcgc ctaggaattg 300
gttgtaatgt tgctttatat agatgattag cggccagtta tacagtaaaa gaaatgacct 360
acttgataga cagctgagac tgagggagcg tctggacggt atcgatttcg gacacaggaa 420
gaacgaccac ctgctgtccg gggacaccca gcgcaaaggg agtgtctgga tactttgtat 480
atatagcatc atctcataca aaaagacata ggaagagtac ccgggcgtgt cccccctcca 540
aggttttcac aatatcttca tcgtcacact gaagaacagg caagtccagc gctttgagac 600
tcttggtggc agcaaaaaca gtcgatttat ccagtacaac aggccaatcc cgacagagac 660
aaagaccaga atctgcaggg gttggtggga aagatataga tagtccatgg tatcctgttg 720
ccactcactg cccttgccaa tccttcgcag gtatgggtcc agttcagaaa acaaagcctg 780
gagcatctca ttaatagagc agaaggatta tccatttgta taaatgatca ttccagattc 840
tcaatgttgc cacggacccc tttttcggct gccgaggggg aaagtccccg agtggttctt 900
aataaataga tagtttgctg gtctatctta acaaagtaaa tattcctttc gttcttccac 960
tcttgttaga atttcccaaa c 981
<210> 2
<211> 999
<212> DNA
<213> fungus FS140(Geosmithia pallida)
<400> 2
atgtccatcg gaaaacttct cgccaacgga gccctgttgg ttgatgtcct catcatcggt 60
gcaggcccct cgggtctgtc taccgcaacc ggactggccc gtcagcttca taccgcggtc 120
gtctttgact ccggagtgta tcgcaacgca aagacacagc acatgcacaa tgtcctaggc 180
tgggaccacc ggaatccgtc cgagctacgg gccgccggtc gagctgatct cgctgcgcgg 240
tactcgacga tccagttcca gaatgccacc gtcgagacga tcaagaggat cggggagaag 300
caactcttcg aggcgcgtga cacggacggt aagcgctggt atggtcggaa ggtcgtgttg 360
gccacgggag tccgagacat tcctctggat attgagggtt actcggaatg ctgggccaat 420
ggaatctacc actgcctgtt ctgtgacggc tatgaagaac gaggccagga gaccgtcggt 480
gtcctcgcca tgggccccat cgccaatcct ccacgagccc tacacttggc ccgaatggcc 540
catcgactct ctgaatctgt caccgtctac acccacggcg atgagcaact ggccaaggag 600
attcagcagg cggccggggg tgattcctcg tggctgaagc tggagacccg gcccatcgtg 660
cgattcgaga agggggatgt tgccaaaacc gttatcgtcc atttctccga gacgacagac 720
acgaagcaag aaggcttcct ggcctataac cccaagacgg agatcaacgg cccctttgcc 780
aaccagctct cattgcagtt gtccgaagtc ggggacatcc agacctcggc tccgttctat 840
gagaccagtg tgcccggggt attcgccgtt ggagactgtg ccaccccgtt gaaggccgtc 900
agtccggcga ttgcaatggg atcgttggct gctggaggtc tggttgctca gctgcaggcc 960
cagccagtga tggaatttgg gattgattgg gagccatag 999
<210> 3
<211> 1078
<212> DNA
<213> fungus FS140(Geosmithia pallida)
<400> 3
ctaggtgtag ttcaacgtgg gctggtttcc atgtcctctg tcgcgactga cgaggggagg 60
ccttacaaag cgcctgtact gaataggaat gatggcggtc gtcgtgcatg atggatgcgc 120
ggtcttgatc tcgagtccat tctgtgggtt gaagctgaag cacccgagga catggcgtag 180
tcgatttcag ctggtgaacg agaccaacac ggcagggttg ttgcggacga gaagatatct 240
catggcatca tcgaattcat cagccttggt gcccatgtgc gagtactcgc ctaggaattg 300
gttgtaatgt tgctttatat agatgattag cggccagtta tacagtaaaa gaaatgacct 360
acttgataga cagctgagac tgagggagcg tctggacggt atcgatttcg gacacaggaa 420
gaacgaccac ctgctgtccg gggacaccca gcgcaaaggg agtgtctgga tactttgtat 480
atatagcatc atctcataca aaaagacata ggaagagtac ccgggcgtgt cccccctcca 540
aggttttcac aatatcttca tcgtcacact gaagaacagg caagtccagc gctttgagac 600
tcttggtggc agcaaaaaca gtcgatttat ccagtacaac aggccaatcc cgacagagac 660
aaagaccaga atctgcaggg gttggtggga aagatataga tagtccatgg tatcctgttg 720
ccactcactg cccttgccaa tccttcgcag gtatgggtcc agttcagaaa acaaagcctg 780
gagcatctca ttaatagagc agaaggatta tccatttgta taaatgatca ttccagattc 840
tcaatgttgc cacggacccc tttttcggct gccgaggggg aaagtccccg agtggttctt 900
aataaataga tagtttgctg gtctatctta acaaagtaaa tattcctttc gttcttccac 960
tcttgttaga atttcccaaa catgtccatc ggaaaacttc tcgccaacgg agccctgttg 1020
gttgatgtcc tcatcatcgg tgcaggcccc tcgggtctgt ctaccgcagc cgacggcc 1078
Claims (6)
1. Promoter GliT 0 The nucleotide sequence is shown as SEQ ID NO. 1.
2. An expression vector comprising the promoter GliT according to claim 1 0 。
3. A host cell comprising the expression vector of claim 2, wherein the host cell is Saccharomyces cerevisiae BJ5464 or Escherichia coli.
4. The promoter GliT of claim 1 0 The application of the promoter in the expression of downstream genes in host cells, wherein the host cells are deep-sea fungi Geosmithia pallida FS140, Saccharomyces cerevisiae BJ5464 and Escherichia coli.
5. The use according to claim 4, wherein the downstream gene is the oxidoreductase gene GliT, the hygromycin resistance gene hph or the ampicillin gene Amp.
6. An expression cassette comprising the promoter GliT according to claim 1 0 。
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