CN115772519A - Strong promoter from cordyceps militaris and application thereof - Google Patents
Strong promoter from cordyceps militaris and application thereof Download PDFInfo
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Abstract
The invention discloses a strong promoter, the base sequence of which is shown as (a) or (b): (a) as shown in SEQ ID NO: 1; (b) Hybridizing the nucleotide sequence defined in (a) under strict hybridization conditions and coding the nucleotide sequence with the function of a promoter. The invention also discloses a recombinant vector containing the strong promoter. The invention also discloses a host cell containing the strong promoter or the recombinant vector. The invention also discloses a construction method of the recombinant vector. The invention also discloses application of the strong promoter, the recombinant vector or the host cell in expression of exogenous genes or positioning of homologous genes in expression positions of fungal cells. The strong promoter provided by the invention is applied to strong expression of control protein in cordyceps militaris in vivo, so as to meet the requirement of homologous/heterologous gene expression of edible fungi and realize large-scale production of cordyceps militaris.
Description
Technical Field
The invention belongs to the technical field of large fungus strain modification, and relates to a strong promoter derived from cordyceps militaris and application thereof.
Background
Cordyceps militaris, also known as Cordyceps militaris, etc., belongs to Ascomycota (Ascomycota), pyrenomycetes (Pyrenomycetes), sphaeriales (Sphaeriales), clavicipitaceae (Sordariomycetes), cordyceps (Cordyceps), and is an important edible and medicinal fungus. Cordyceps militaris contains various active ingredients, mainly including cordycepin, cordycepic acid, cordyceps polysaccharide, novel carotenoid and the like, and the active ingredients have various pharmacological effects of resisting fatigue, inflammation, tumor, oxidation and the like. At present, cordyceps militaris has become a wide range of health-care products and medicinal fungi in China, and gradually replaces cordyceps sinensis to carry out deep processing and utilization.
The natural Cordyceps militaris is mainly prepared by infecting larva and pupa of lepidoptera insect with Cordyceps militaris fungus, growing in the larva to destroy larva tissue and complete proliferation, and gradually maturing mycelium to obtain fruiting body after vegetative growth. China already realizes large-scale artificial cultivation of cordyceps militaris, and researches prove that the artificially cultivated cordyceps militaris and wild cordyceps militaris have no obvious difference in terms of bioactive components. On the other hand, the artificial cultivation of the cordyceps militaris requires proper temperature, humidity and illumination, the growth period is relatively long, and the large-scale utilization of the cordyceps militaris is also limited. Compared with the fruiting body culture, the mycelium can be cultured in a large amount by means of liquid fermentation and the like, and the culture condition requirement is relatively simple. Therefore, the mycelium is used as a carrier, the overexpression of a target product is realized by using a genetic engineering means, the requirement of cordyceps militaris as a medical health-care product can be met, and the large-scale production is realized.
Most of the researches on cordyceps militaris at home and abroad are focused on the aspects of active ingredients, cultivation condition optimization and the like, and relatively few researches on the aspect of molecular biology. The cordyceps militaris complete genome database is published in 2011, which provides a certain foundation for the relevant research of the molecular biology of cordyceps militaris. In the gene expression process, the most important work is the first stage of gene expression, namely the transcription process, the transcription 'switch' promoter determines the transcription initiation site, the degree of gene expression, and the finding of a strong promoter capable of high-strength and stable expression is particularly important. One of the bottleneck problems in the development of cordyceps militaris and even edible fungus genetic engineering technology at present is the lack of efficient promoter elements. The promoter is located at the 5' end of the structural gene, can be recognized and combined by RNA polymerase and other transcription factors, so as to start a DNA sequence transcribed by the gene, and is an important cis-acting element in the regulation and control of gene expression. At present, promoters commonly used in an edible fungus genetic transformation system are mainly two promoters of ras and gpd. Only the two promoters far cannot meet the requirement of homologous/heterologous gene expression of edible fungi. It is particularly important to find a stable and efficient strong promoter.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.
It is also an object of the present invention to provide a strong promoter.
Another object of the present invention is to provide a recombinant vector.
It is yet another object of the present invention to provide a host cell.
It is still another object of the present invention to provide a method for constructing a recombinant vector.
The invention further aims to provide the application of the strong promoter, the recombinant vector and the host cell.
The technical scheme provided by the invention is as follows:
a strong promoter having a base sequence represented by (a) or (b):
(a) As shown in SEQ ID NO. 1;
(b) Hybridizing with the nucleotide sequence defined in (a) under strict hybridization conditions and coding the nucleotide sequence with promoter function.
A recombinant vector comprising the strong promoter of claim 1.
Preferably, in the recombinant vector, the vector further comprises a terminator sequence, and the terminator sequence is located downstream of the strong promoter sequence.
A host cell comprising said strong promoter, or said recombinant vector.
A construction method of a recombinant vector comprises the following steps:
the primer pair shown in SEQ ID NO. 2 and SEQ ID NO. 3 is utilized to clone the nucleotide sequence shown in SEQ ID NO.1 from the Cordyceps militaris mycelium genome DNA, and the nucleotide sequence is connected to a pCAMBIA1300 plasmid vector to obtain the pCAMBIA1300 recombinant vector containing a strong promoter.
Preferably, the method for constructing a recombinant vector further comprises the following steps:
cloning a nucleotide sequence of a GPD terminator from Cordyceps militaris mycelium genome DNA by using a primer pair shown in SEQ ID NO. 4 and SEQ ID NO. 5;
and carrying out double digestion on the nucleotide sequences of the pCAMBIA1300 recombinant vector and the GPD terminator by using BamHI restriction endonuclease and HindIII restriction endonuclease respectively, then carrying out ligation reaction, and connecting the nucleotide sequence of the GPD terminator subjected to double digestion to the pCAMBIA1300 recombinant vector subjected to double digestion to form the pCAMBIA1300 recombinant vector containing the strong promoter shown in SEQ ID NO.1 and the nucleotide sequence of the GPD terminator.
The strong promoter, the recombinant vector or the host cell can express the exogenous gene in the fungal cell or locate the application of the homologous gene in the expression position of the fungal cell.
Preferably, in said use, the fungal cell is a large fungal cell.
The invention at least comprises the following beneficial effects:
the invention clones a hat strong promoter with stable and efficient expression from cordyceps militaris, constructs a plasmid vector containing the hat promoter and a transformant containing the plasmid vector, and applies the strong promoter obtained by the invention to the strong expression of control protein in cordyceps militaris in vivo so as to meet the requirement of homologous/heterologous gene expression of edible fungi and realize the large-scale production of cordyceps militaris. Meanwhile, the recombinant vector can also be applied to the positioning of key target genes.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic diagram of a vector construction strategy according to the present invention.
FIG. 2 is a gel electrophoresis diagram of the enzyme digestion verification of the hat promoter in the vector construction fragment in one embodiment of the present invention.
FIG. 3 is a gel electrophoresis diagram of enzyme cleavage validation after construction of a vector green fluorescent protein expression vector in one embodiment of the present invention.
FIG. 4 is a fluorescence image observed by a fluorescence microscope at 10X 10 times of Cordyceps militaris hyphae in one embodiment of the present invention.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The invention relates to a strong promoter for large-scale fungal protein expression, in particular to a strong promoter obtained by separating and cloning cordyceps militaris, a plasmid vector containing the strong promoter, a transformant containing the plasmid vector, and applications of the strong promoter, the plasmid vector, the transformant and the transformant in aspects of homologous or heterologous expression and gene positioning of proteins in cordyceps militaris.
The invention provides a strong promoter which is a hat promoter, and the base sequence of the strong promoter is shown as (a) or (b):
(a) As shown in SEQ ID NO. 1;
(b) Hybridizing with the nucleotide sequence defined in (a) under strict hybridization conditions and coding the nucleotide sequence with promoter function.
The invention also provides a recombinant vector, and the vector contains the strong promoter.
In one embodiment of the present invention, it is preferred that the vector further comprises a terminator sequence, said terminator sequence being located downstream of said strong promoter sequence. Preferably, the terminator is a GPD terminator. The foreign gene can be constructed between the strong promoter sequence and the GPD terminator.
In one embodiment of the present invention, the vector is preferably a pCAMBIA1300 plasmid vector.
The invention provides a host cell containing the strong promoter or the recombinant vector.
The invention also provides a construction method of the recombinant vector, which comprises the following steps:
the primer pair shown in SEQ ID NO. 2 and SEQ ID NO. 3 is utilized to clone the nucleotide sequence shown in SEQ ID NO.1 from the cordycepin genome DNA, and the nucleotide sequence is connected to the pCAMBIA1300 plasmid vector to obtain the pCAMBIA1300 recombinant vector containing the strong promoter.
In the above embodiment of the present invention, preferably, the method further includes the following steps:
cloning a nucleotide sequence of a GPD terminator from Cordyceps militaris mycelium genome DNA by using a primer pair shown in SEQ ID NO. 4 and SEQ ID NO. 5;
BamHI restriction endonuclease and HindIII restriction endonuclease are used for respectively carrying out double enzyme digestion on the pCAMBIA1300 recombinant vector and the nucleotide sequence of the GPD terminator, then ligation reaction is carried out, the nucleotide sequence of the GPD terminator after double enzyme digestion is ligated to the pCAMBIA1300 recombinant vector after double enzyme digestion, and the pCAMBIA1300 recombinant vector containing the strong promoter shown in SEQ ID NO.1 and the nucleotide sequence of the GPD terminator is formed.
The invention also provides the application of the strong promoter, the recombinant vector or the host cell in expressing exogenous genes in fungal cells or positioning homologous genes in expression positions in fungal cells.
In one embodiment of the present invention, preferably, the fungal cell is a large fungal cell.
In order to make the technical solution of the present invention better understood by those skilled in the art, the following examples are now provided for illustration:
the invention discloses a strong promoter derived from cordyceps militaris and application thereof. The invention separates and clones strong promoter from Cordyceps militaris, and the nucleotide sequence is shown in SEQ ID No. 1. The invention also provides a recombinant expression vector containing the strong promoter and terminator and a transformant containing the recombinant expression vector. The invention further discloses application of the compounds in expressing homologous or heterologous proteins in cordyceps militaris.
Example one isolation of hat promoter from Cordyceps militaris
The hat promoter is the strong promoter described in the application, and the nucleotide sequence of the hat promoter is shown as SEQ ID NO. 1.
>SEQ ID NO:1
CGACGCGACCGCAACATACTGCCTTCCAACGATCGGGACAGAAGAGACGGACCGA GAGATCGGGCGGATAATCGACCGGGACGCTGGGGTGACCGTGGCGTCGACAATCG CGGAGCCAACCATCGCGGAGCCGACAGTCGCCCAGTCGAAAGTCGTGGAGGCCCTA GACCTGCGCAGAGATCGCGTTCTCCTGAGGCGCGGGGAAACCGCGGACAACGGAG CTCATACAACGGCGACAGCTATGTACCACGAGGACGGGATGGACCACAGCGTGGA CGAGATGGACCACAGCGTGGAAGAGATGGACCACAGCGTGGTCGAGATGGACCAC AGCGGGGACGAGACCGCCGATCGCAGCGCTCCCCATCCGCTTCGTCTTCTAGATCT AGCTCTTCGTCGCGCAGCCGCTCGCGCTCTCCACCCCGACGCCGAGACTCGACCCG ACGCCGAGACTCACCCCGACGTGGTACTACGCTACGAAAGCGATCAAGGTCTCGAT CCCGCTCACCCCAACGCGACACGGATCGTTCTCGCCGACTTGGAGACGGAAGCAAA TCACCTGCTGCCAAAAGGGCGCGGCAGGACCCAAGAAGCAGGTCCCGGAGTAGAG GTCGATCCTCGTCCTCATCAGCCGCTTCTAGCCGTTCCCGAAGCTACTCTTCGTCAA GCCGCAGCGGCAGCCGGAGCCGGAGCCGGAGCCGGAGCCGAAGTCGAAGCCGGAC CAGGGCGCGGAAGAGTAGGAGCTCGTCAAATGAGTCGGACGAGCGACGAGGAAGC CGGAAATCGCCAGCTCGTAGACGCAACAGCTCCTCGGCATCTGCACCGCGGCGCCG CAGCTGAGCTGATAGCTCTACCGCCACATTGGGATGGTGACGCATTTGTGATCAAA CTTGAAACTGCCTTTGGATAAGAGAATGAGGGGAATGGCCAAGATTGGAACTATAA CCCTTGCTAACGCGTATCTTAGCGCAGCATCCGTTCAACATATATGAGCCGCAAATA TCATGAGCTGTCGTTGGGCAGATTGCAGGATACGAAGTAAGCACATTCCTACCGTG GCTAAGCTATAACAGTCGCATAGACCCGTTTTGTATCATTCTTCATCTTTCTTCATCT TTCTGTAACTGCCTGGACGGAGTATACAAGGTAGCTGGGCGTTGGGATACCATTGC TGCGTAGAGCCTGTGCTTGAATCGCTCTCGGACCACAACCTGTTTAGATTGCTATTA CGATCAAAATATACTTGAATTAATCTTCAACCCAATACACACATGCTTCATTCTTTG CGTCTGTTACCATCTGTTGTGTCGTGTCATTCGACCATCGTGGGGTCGCCCCACCTT GAAGTTGACGAACCCAGAGAGAGAATGCGAGCCGCCAGCTTCCGTGTTCTCCTTCA TCACAACCAGACATCAAAGACGCAACAATCCCCCCCCCCCTCCGCGAAACCCTGCA AAGTGCCAGCCGCGCGCACCATCGCAAAACGCCTCACCGCACGAAGCCCCCAAC
1) Extraction of genome DNA of cordyceps militaris mycelium
Punching hole of Cordyceps militaris grown on PDA plate culture medium with puncher, inoculating mycelium with agar block into triangular flask containing 80mL PDB culture medium, and culturing at 28 deg.C and 180rpm for 72h. Filtering cultured mycelium with sterilized three-layer lens wiping paper, washing mycelium with distilled water for 2-3 times, collecting, drying with absorbent paper, and freezing at-20 deg.C. Grinding the collected mycelium with liquid nitrogen for 2-3 times; transferring the obtained hypha powder into a 2mL centrifuge tube, and adding 700 mu L of 2% CTAB solution into each tube; turning upside down and mixing 2mL of centrifuge tube evenly, preserving heat for 60min at 65 ℃, and shaking evenly for 1 time every 10min; adding equal volume of chloroform: isoamyl alcohol (24; collecting supernatant, adding isopropanol with equal volume, mixing, standing at room temperature for 10min, centrifuging at 12000r/min for 10min, removing supernatant, adding 70% anhydrous ethanol 500 μ L to wash precipitate, drying at room temperature, and adding 50 μ L dd H 2 O completely dissolving the DNA, adding RNase A (10 mg/mL), keeping the temperature at 65 ℃ for 30min, and finally dissolving the DNA in 30 μ L ddH 2 And (4) in O.
2) Obtaining of hat promoter Gene fragment
Designing a primer sequence according to the information in the cordyceps militaris genome database as follows:
pHATf：5’-TTgaattcCGACGCGACCGCAACATACTG-3’(SEQ ID NO:2)
pHATr：5’-AAgagctcGTTGGGGGCTTCGTGCGGTGAG-3’(SEQ ID NO:3)
the lower case indicates the corresponding restriction enzyme cutting site, the EcoRI restriction enzyme cutting site is introduced into the upstream primer, and the SacI restriction enzyme cutting site is introduced into the downstream primer.
The PCR reaction conditions are as follows: a total of 35 cycles of 3 minutes at 98 ℃ (10 seconds at 98 ℃, 30 seconds at 58 ℃ and 1 minute at 68 ℃), and 10 minutes at 72 ℃.
The PCR reaction system is as follows: cordyceps militaris genome DNA 1 uL, taq pfu polymerase 1 uL, primers with concentration of 100mM each 0.4 uL, buffer 5 uL, dNTP with concentration of 10mM 1 uL, and water for the rest to make up to 50 uL.
Recovering hat promoter gene product 3. Mu.L from PCR gel, 1. Mu.L of pUCM-T vector, 1. Mu.L of T4 ligase buffer, mixing the rest with ddH 2 And O is complemented to 10 mu L, the recovered cloning promoter gene product and a pUCM-T vector are treated and purified by T4 ligase, ligation reaction is carried out, then the recombined pUCM-T vector is transferred to escherichia coli DH-5 alpha, positive cloning is selected, colony PCR and enzyme digestion verification are carried out, sample sending and sequencing are carried out after the positive cloning is verified to be correct, and the nucleotide sequence is SEQ ID No:1 is shown.
3) Cordyceps militaris terminator obtention
Designing a primer sequence according to the information in the cordyceps militaris genome database as follows:
tGPDf：5’-TTggatccGAGGCGGGGATGGGTAATAGAGTA-3’(SEQ ID No：4)
tGPDr：5’-TTaagcttATGGGAACACGGGAAAGAGTCG-3’(SEQ ID No： 5)
the lower case indicates the corresponding restriction enzyme cutting site, the upstream primer introduces the BamHI restriction endonuclease cutting site, and the downstream primer introduces the HindIII restriction endonuclease cutting site.
The PCR reaction conditions are as follows: 3 minutes at 98 deg.C, (10 seconds at 98 deg.C, 30 seconds at 58 deg.C, 15 seconds at 68 deg.C) for a total of 35 cycles, 10 minutes at 72 deg.C.
The PCR reaction system is as above, and the vector recovery, ligation and sequencing reaction is as above.
EXAMPLE two construction of hat promoter expression vector
The expression vector construction strategy is shown in FIG. 1.
Firstly, the pCAMBIA1300 plasmid (with green fluorescent protein gene) is double-digested by EcoRI and SacI restriction enzymes, and is connected with a promoter for double-digestion with the same EcoRI and SacI restriction enzymes, and the reaction conditions and the reaction system are shown in example 1. And transforming the ligation product to escherichia coli DH-5 alpha, coating the escherichia coli DH-5 alpha containing the recombinant plasmid vector on a flat plate containing kanamycin resistance for screening, selecting transformants for culturing, extracting plasmids and carrying out restriction enzyme digestion verification. The resulting fragments were of the correct size, as shown in FIG. 2. The vector containing the hat promoter, which was verified to be correct, was double-digested with BamHI and HindIII restriction endonucleases and ligated with GPD terminator that was also double-digested with BamHI and HindIII restriction endonucleases under the reaction conditions and reaction system shown in example 1. And transforming the ligation product into escherichia coli DH-5 alpha, coating the escherichia coli DH-5 alpha containing the recombinant plasmid vector on a plate containing kanamycin resistance for screening, selecting transformants for culture, extracting plasmids and carrying out enzyme digestion verification. The constructed vector was named pCAMBIA1300-pHAT.
Example construction of recombinant expression vector for three Green fluorescent protein GFP
1) Primers were designed based on the existing GFP sequences as follows:
GFPf：5’-ATgagctcATGGTGAGCAAGGGCGAGGAGC-3’(SEQ ID No： 6)
GFPr：5’-TTaagcttTTACTTGTACAGCTCGTCCAT-3’(SEQ ID No：7)
the lower case font in the primer sequence represents the corresponding enzyme cutting site, the SacI restriction enzyme cutting site is introduced into the upstream primer, and the BamHI restriction enzyme cutting site is introduced into the downstream primer.
2) The PCR reaction conditions were: a total of 35 cycles of 3 minutes at 98 ℃ (10 seconds at 98 ℃, 30 seconds at 58 ℃, 30 seconds at 68 ℃) and 10 minutes at 72 ℃.
Connecting the amplified gfp gene fragment to a pUCM-T vector, transforming to escherichia coli, extracting plasmids from the obtained transformant, carrying out enzyme digestion verification, and carrying out sequencing identification to obtain the correct gfp fragment as an insert. After the recombinant vector pCAMBIA1300-pHAT is subjected to double digestion by SacI and BamHI restriction enzymes, the gfp fragment is connected with the digested vector and is transformed into escherichia coli. The transformation product is smeared on a plate containing kanamycin resistance for screening, positive transformants are selected for culturing, plasmids are extracted and enzyme digestion verification is carried out. As shown in FIG. 3, the resulting fragments were of the correct size.
Example four Green fluorescent protein is expressed in Cordyceps militaris in vivo
1) The pCAMBIA1300-pHAT vector is transferred into the agrobacterium AGL1 by adopting a freeze-thaw method for transformation. Selecting a single colony of agrobacterium from a newly cultured LB plate (containing 50 mu g/mL of kanamycin), inoculating the single colony of agrobacterium into 5mL of LB containing kanamycin, and culturing overnight at the temperature of 28 ℃ and the rotating speed of 200 rpm; the next day, 200-400. Mu.L of the culture medium was transferred to 5mL of induction liquid medium AIM containing 200. Mu. Mol/L acetosyringone AS, with an initial OD of about 0.15, and cultured at 28 ℃ for 5-6 hours to reach an OD600 of 0.5-0.6.
AIM plate medium contains: 1mL of CaCl 2 ×2H 2 O(w/v)，10mL 0.01％FeSO 4 (w/v)，5mL spore elements，2.5mL 20％NH 4 NO 3 (w/v)，10mL 50％glycerol (v/v)，40mL 1M MES pH 5.5，0.8mL1.25K-Phosphate-buffer pH 4.8，10mL 20％glucose(w/v)。
2) Co-transformation: 100 μ L of the cultured Agrobacterium AGL1 strain solution and 100 μ L of the diluted spore suspension (10% concentration) 6 pieces/mL), then evenly spreading the mixed solution on an AIM plate culture medium paved with a nitrocellulose membrane (NC), and co-culturing for 72h at 23 ℃; the membrane was cut into small pieces and cultured on selective PDA medium at 25 ℃ until transformants appeared. The selective PDA culture medium contains 600 mug/mL hygromycin, 400 mug/mL cefamycin and 60 mug/mL streptomycin. After transformants grew, they were rescreened on PDA plates containing 600. Mu.g/mL hygromycin.
3) Fluorescence observation
The transformants obtained by the secondary screening were plated on PDA plate medium and cultured in the dark at 25 ℃ for 10 days. The hyphae of the transformants are picked up by toothpicks and placed on a glass slide for observation under a fluorescence microscope.
The fluorescence microscope observation shows that strong fluorescence expression exists in cordyceps militaris hyphae (figure 4), and the hat promoter separated by the method can promote the stable and efficient expression of GFP green fluorescent protein in cordyceps militaris.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable to various fields of endeavor with which the invention may be practiced, and further modifications may readily be effected therein by those skilled in the art, without departing from the general concept as defined by the claims and their equivalents, which are not limited to the details given herein and the examples shown and described herein.
SEQUENCE LISTING
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> strong promoter derived from cordyceps militaris and application thereof
<130> 2020
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 1509
<212> DNA
<213> Cordyceps militaris cordyces militaris (L.ex Fr.) Link.
<400> 1
cgacgcgacc gcaacatact gccttccaac gatcgggaca gaagagacgg accgagagat 60
cgggcggata atcgaccggg acgctggggt gaccgtggcg tcgacaatcg cggagccaac 120
catcgcggag ccgacagtcg cccagtcgaa agtcgtggag gccctagacc tgcgcagaga 180
tcgcgttctc ctgaggcgcg gggaaaccgc ggacaacgga gctcatacaa cggcgacagc 240
tatgtaccac gaggacggga tggaccacag cgtggacgag atggaccaca gcgtggaaga 300
gatggaccac agcgtggtcg agatggacca cagcggggac gagaccgccg atcgcagcgc 360
tccccatccg cttcgtcttc tagatctagc tcttcgtcgc gcagccgctc gcgctctcca 420
ccccgacgcc gagactcgac ccgacgccga gactcacccc gacgtggtac tacgctacga 480
aagcgatcaa ggtctcgatc ccgctcaccc caacgcgaca cggatcgttc tcgccgactt 540
ggagacggaa gcaaatcacc tgctgccaaa agggcgcggc aggacccaag aagcaggtcc 600
cggagtagag gtcgatcctc gtcctcatca gccgcttcta gccgttcccg aagctactct 660
tcgtcaagcc gcagcggcag ccggagccgg agccggagcc ggagccgaag tcgaagccgg 720
accagggcgc ggaagagtag gagctcgtca aatgagtcgg acgagcgacg aggaagccgg 780
aaatcgccag ctcgtagacg caacagctcc tcggcatctg caccgcggcg ccgcagctga 840
gctgatagct ctaccgccac attgggatgg tgacgcattt gtgatcaaac ttgaaactgc 900
ctttggataa gagaatgagg ggaatggcca agattggaac tataaccctt gctaacgcgt 960
atcttagcgc agcatccgtt caacatatat gagccgcaaa tatcatgagc tgtcgttggg 1020
cagattgcag gatacgaagt aagcacattc ctaccgtggc taagctataa cagtcgcata 1080
gacccgtttt gtatcattct tcatctttct tcatctttct gtaactgcct ggacggagta 1140
tacaaggtag ctgggcgttg ggataccatt gctgcgtaga gcctgtgctt gaatcgctct 1200
cggaccacaa cctgtttaga ttgctattac gatcaaaata tacttgaatt aatcttcaac 1260
ccaatacaca catgcttcat tctttgcgtc tgttaccatc tgttgtgtcg tgtcattcga 1320
ccatcgtggg gtcgccccac cttgaagttg acgaacccag agagagaatg cgagccgcca 1380
gcttccgtgt tctccttcat cacaaccaga catcaaagac gcaacaatcc ccccccccct 1440
ccgcgaaacc ctgcaaagtg ccagccgcgc gcaccatcgc aaaacgcctc accgcacgaa 1500
gcccccaac 1509
<210> 2
<211> 29
<212> DNA
<213> Artificial sequence
<400> 2
ttgaattccg acgcgaccgc aacatactg 29
<210> 3
<211> 30
<212> DNA
<213> Artificial sequence
<400> 3
aagagctcgt tgggggcttc gtgcggtgag 30
<210> 4
<211> 32
<212> DNA
<213> Artificial sequence
<400> 4
ttggatccga ggcggggatg ggtaatagag ta 32
<210> 5
<211> 30
<212> DNA
<213> Artificial sequence
<400> 5
ttaagcttat gggaacacgg gaaagagtcg 30
<210> 6
<211> 30
<212> DNA
<213> Artificial sequence
<400> 6
atgagctcat ggtgagcaag ggcgaggagc 30
<210> 7
<211> 29
<212> DNA
<213> Artificial sequence
<400> 7
ttaagctttt acttgtacag ctcgtccat 29
Claims (8)
1. A strong promoter having a base sequence represented by (a) or (b):
(a) As shown in SEQ ID NO. 1;
(b) Hybridizing the nucleotide sequence defined in (a) under strict hybridization conditions and coding the nucleotide sequence with the function of a promoter.
2. A recombinant vector comprising the strong promoter of claim 1.
3. The recombinant vector of claim 2, further comprising a terminator sequence, said terminator sequence being located downstream of said strong promoter sequence.
4. A host cell comprising the strong promoter of claim 1, or the recombinant vector of claim 3 or 4.
5. A method for constructing a recombinant vector, comprising the steps of:
the primer pair shown in SEQ ID NO. 2 and SEQ ID NO. 3 is utilized to clone the nucleotide sequence shown in SEQ ID NO.1 from the genome DNA of the cordyceps militaris mycelium, and the nucleotide sequence is connected to the pCAMBIA1300 plasmid vector to obtain the pCAMBIA1300 recombinant vector containing the strong promoter.
6. The method for constructing a recombinant vector according to claim 5, further comprising the steps of:
cloning a nucleotide sequence of a GPD terminator from genome DNA of cordyceps militaris mycelia by using a primer pair shown in SEQ ID NO. 4 and SEQ ID NO. 5;
and carrying out double digestion on the nucleotide sequences of the pCAMBIA1300 recombinant vector and the GPD terminator by using BamHI restriction endonuclease and HindIII restriction endonuclease respectively, then carrying out ligation reaction, and connecting the nucleotide sequence of the GPD terminator subjected to double digestion to the pCAMBIA1300 recombinant vector subjected to double digestion to form the pCAMBIA1300 recombinant vector containing the nucleotide sequences of the strong promoter and the GPD terminator shown in SEQ ID NO. 1.
7. Use of a strong promoter according to claim 1, a recombinant vector according to claim 2 or a host cell according to claim 4 for expressing an exogenous gene or for locating the expression position of an homologous gene in a fungal cell.
8. Use according to claim 7, wherein the fungal cell is a large fungal cell.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103114089A (en) * | 2012-12-05 | 2013-05-22 | 天津工业生物技术研究所 | Strong promoter from trichoderma reesei as well as expression vector and application thereof |
| US20200399325A1 (en) * | 2018-02-12 | 2020-12-24 | Lonza Ltd | Host cell for producing a protein of interest |
| CN113151017A (en) * | 2021-03-30 | 2021-07-23 | 浙江工业大学 | Recombinant cordyceps militaris for over-expressing cordycepin |
-
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103114089A (en) * | 2012-12-05 | 2013-05-22 | 天津工业生物技术研究所 | Strong promoter from trichoderma reesei as well as expression vector and application thereof |
| US20200399325A1 (en) * | 2018-02-12 | 2020-12-24 | Lonza Ltd | Host cell for producing a protein of interest |
| CN113151017A (en) * | 2021-03-30 | 2021-07-23 | 浙江工业大学 | Recombinant cordyceps militaris for over-expressing cordycepin |
Non-Patent Citations (2)
| Title |
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| ENSEMBL FUNGI: "Gene: CCM_04549", pages 1 - 2, Retrieved from the Internet <URL:https://nov2020-fungi.ensembl.org/Cordyceps_militaris_cm01_gca_000225605/Gene/Sequence?db=core;g=CCM_04549;r=JH126401:2948843-2950138;t=EGX93177> * |
| ENSEMBL FUNGI: "Gene: CCM_06191", pages 1 - 2, Retrieved from the Internet <URL:https://nov2020fungi.ensembl.org/Cordyceps_militaris_cm01_gca_000225605/Gene/Summary?db=core;g=CCM_06191;r=JH126402:3699099-3699808;t=EGX92031> * |
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