CN112266914B - Strong constitutive promoter of bumblebee candida and application thereof - Google Patents
Strong constitutive promoter of bumblebee candida and application thereof Download PDFInfo
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Abstract
The invention discloses a strong constitutive promoter of bumblebee candida and application thereof. The invention takes the bumblebee candida utilis as a research object, and predicts, clones, screens and evaluates a strong constitutive promoter. And (3) taking the enhanced green fluorescent protein as a reporter gene, driving the expression of the enhanced green fluorescent protein through the selected promoter, and analyzing the difference of green fluorescent intensity and the difference of transcription level to finally obtain a constitutive promoter PX with strong transcription activity. Compared with the known strong constitutive PGPD promoter, the promoter has the highest strength of transcriptional activity in different culture periods by using glucose or oleic acid as a unique carbon source. The experiment takes the enhanced green fluorescent protein as a report gene in the candida bumbleensis for the first time, obtains a DNA sequence with strong transcriptional activity by transcriptome level analysis, has good application prospect, enriches synthetic biological elements, and lays a theoretical foundation for the strain in the genetic engineering modification and exogenous gene expression.
Description
Technical Field
The invention relates to a strong constitutive promoter of bumblebee candida and application thereof, belonging to the technical field of analytical chemistry.
Background
Candida bumblebee (Starmerella bombicola) is a nonpathogenic haploid Candida, and sophorolipid produced by the Candida bumblebee is widely applied to the fields of agriculture, medicine, food industry and the like due to the characteristics of reducing surface tension, improving foamability, improving solubility and the like, and has a very wide application prospect in the aspect of environmental protection due to good environmental friendliness. At present, relevant researches at home and abroad basically clarify the metabolic pathway of biosynthesizing the sophorolipid by candida bombicola, and the yield of the sophorolipid is improved by a metabolic engineering method. The prior art mainly utilizes key genes for synthesizing the over-expressed sophorolipid to improve the yield of the sophorolipid, and a promoter plays an important role in the gene expression process and controls the transcription level of the genes.
Nowadays, with the development of synthetic biology, people pay more and more attention to the related research in the field of synthetic biological elements such as promoters, strong promoters have very critical function in the high-efficiency expression of heterologous genes, and the regulation and control of constitutive promoters are not influenced by external conditions, so that the promoted genes can be continuously expressed, and the promoter has good application prospect. In the candida bumblebee, the research on the promoter is less, so the application prospect of the promoter is limited to a great extent, the candida bumblebee promoter needs to be further analyzed and identified, and a strong constitutive promoter is obtained, thereby laying a foundation for the fields of exogenous gene high-efficiency expression, gene engineering, synthetic biology and the like.
Disclosure of Invention
In order to solve the problems, the invention uses a reporter gene to predict, clone, screen and evaluate the promoter of the candida bumbleensis, and obtains a strong constitutive promoter.
The first purpose of the invention is to provide a strong constitutive promoter of the candida bumbleensis, and the nucleotide sequence of the promoter is shown as SEQ ID NO. 1.
The second purpose of the invention is to provide an expression vector containing the promoter.
Furthermore, the expression vector is a vector suitable for constitutive expression of the candida bumbleensis.
Furthermore, the expression vector takes an integration type plasmid pPHP, an integration type plasmid pSHS or an integration type plasmid pAHA as a starting vector.
Further, the nucleotide sequence of the integrative plasmid pPHP is shown as SEQ ID NO. 2.
It is a third object of the present invention to provide a Candida bombicola comprising the promoter.
The fourth purpose of the invention is to provide the application of the promoter in genetic modification of candida bombicola.
Further, the application is to use the promoter to promote the expression of a constitutive gene.
Further, the application specifically comprises the following steps:
s1, carrying out fusion PCR on the promoter, the gene to be expressed and the terminator to obtain a gene fragment;
s2, connecting the gene fragment obtained in the step S1 to an integrative plasmid to obtain an integrative expression vector;
and S3, transferring the integrated expression vector obtained in the step S2 into a bumblebee candida utilis host bacterium to obtain a bumblebee candida utilis recombinant bacterium with enhanced expression of the gene to be expressed.
Furthermore, the nucleotide sequence of the terminator is shown as SEQ ID NO. 4.
The invention has the beneficial effects that:
the invention takes the bumblebee candida utilis as a research object, and predicts, clones, screens and evaluates a strong constitutive promoter. And (3) taking the enhanced green fluorescent protein as a reporter gene, driving the expression of the enhanced green fluorescent protein through the selected promoter, and analyzing the difference of green fluorescent intensity and the difference of transcription level to finally obtain a constitutive promoter PX with strong transcription activity. Compared with the known strong constitutive PGPD promoter, the promoter has the highest strength of transcriptional activity in different culture periods by using glucose or oleic acid as a unique carbon source. The experiment takes the enhanced green fluorescent protein as a report gene in the candida bumbleensis for the first time, obtains a DNA sequence with strong transcriptional activity by transcriptome level analysis, has good application prospect, enriches synthetic biological elements, and lays a theoretical foundation for the strain in the genetic engineering modification and exogenous gene expression.
Drawings
FIG. 1 is a schematic diagram of a constructed green fluorescent reporter vector of an integrative promoter;
FIG. 2 is a green fluorescence observation image of recombinant strains cultured in YPD medium;
FIG. 3 shows green fluorescence intensity data and transcription level data of recombinant strains cultured in a culture medium with glucose as a sole carbon source for 8 hours.
FIG. 4 shows green fluorescence intensity data and transcription level data of recombinant strains cultured in a culture medium with oleic acid as a sole carbon source for 8 hours.
FIG. 5 is a graph of green fluorescent protein intensity data measured for recombinant strains cultured for 80 hours using glucose and oleic acid as unique carbon sources, respectively.
Detailed Description
The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.
The following media formulations were used as examples: (1) YPD Medium (g/L): yeast powder 10, peptone 20 and glucose 20. Oleic acid medium (g/L): yeast powder 10, peptone 20 and rapeseed oil 60. (2) LB medium (g/L): yeast powder 5, peptone 10 and sodium chloride 10. (3) hygromycin-resistant medium (μ g/mL): the hygromycin mother liquor was added to YPD medium cooled to about 46 ℃ to a final concentration of 500. (4) ampicillin-resistant Medium (g/L): ampicillin was added to LB medium at a final concentration of 0.1.
Reagents and instrumentation used in the following examples: the one-step ligation kit was purchased from Nanjing Novozam, and hygromycin was purchased from Beijing Dingguo biology, inc.; DNA polymerase, a fluorescence quantitative analysis kit, a yeast Total RNA extraction kit and endonuclease are purchased from TaKaRa company; the MicroPulser electrotransfer instrument is purchased from Bio-Rad, boyle Life medicine (Shanghai) Inc.; laser confocal microscope Leica TCS SP8 (come card, germany); flow cytometer BD FACSAria iii (bidi, usa); real-time fluorescent quantitative gene amplification apparatus CFX96 (Burle, USA).
TABLE 1 primer sequences
Example 1: acquisition of PX strong constitutive promoter sequence
1. Taking wild type Candida bombicola culture solution cultured in YPD liquid culture medium for different culture time (4, 8, 12, 16 and 20 h), respectively extracting Total RNA by using a Takara RNA extraction kit, carrying out reverse transcription to obtain cDNA samples of 5 different time points, and handing the cDNA samples to Jin Weizhi Biotech limited, suzhou for transcription level determination and sequencing analysis.
2. After sample data obtained by analysis is screened and filtered by sequencing quality analysis, software is used for calculating the count value of the tested gene, and the FPKM value of each gene in each sample is calculated through Python script and FPKM (Fragments Per Kilobase Million) formula. Finally, a gene sequence with higher transcription level is obtained.
3. Comparing and analyzing the gene sequence with the whole genome of the bumblebee candida, finally selecting 1500bp upstream of the initiation codon of the gene as a promoter functional region, obtaining a promoter DNA sequence product PX by designing a specific primer, using the genome of the bumblebee candida as a template through PCR amplification by PX-F/PX-R, connecting the DNA sequence product to a pM-19T simple vector and sequencing, wherein the PX sequencing result is shown as SEQIDNO.1.
Example 2: PX expression GFP recombinant plasmid construction
1. By analyzing the preference of codons of candida bombicola, a yeGFP gene (SbEGFP) (SEQ ID NO. 3) which is subjected to codon optimization and chemical synthesis is subjected to fusion PCR with a promoter PX by designing a specific primer, and finally, a green fluorescent protein expression cassette PX-yeGFP-Tsyn7 is obtained (a BamHI enzyme cutting site is inserted between the promoter and the yeGFP gene).
2. Taking an integrated plasmid pPHP which is constructed in the earlier stage of the laboratory and takes a hygromycin resistance gene (HPH) as a screening marker as a starting vector (SEQ ID NO. 2), carrying out MluI enzyme digestion, recovering the plasmid, connecting the plasmid with the expression cassette in the step 1 through C113 one-step ligase to transform escherichia coli JM109, coating the escherichia coli JM109 on an ampicillin selection plate, and finally obtaining the integrated promoter green fluorescent reporter vector pPHP-PX-yeGFP.
3. Taking a promoter green fluorescent reporter vector pPHP-PX-yeGFP as a framework, jointly digesting the vector by endonuclease MluI and BamHI, designing a specific primer PGAPD-F/PGAPD-R, taking a bumblebee candida utilis genome as a template, carrying out PCR amplification to obtain a PGPD promoter product, connecting the PGPD promoter product and the PGAPD promoter product in one step to obtain a recombinant plasmid, transferring the recombinant plasmid into JM109 competent cells, and finally obtaining the integrative promoter reporter vector pPHP-PGPD-yeGFP as shown in figure 1.
Example 3: construction of recombinant strains
1. Respectively taking the successfully constructed integrated promoter green fluorescent report vector as a template and Pxa1 gene homology arm upstream and downstream primers, carrying out PCR amplification to obtain integrated expression cassettes Pxa1-PX-yeGFPex-HPHex-Pxa1 and Pxa1-PGPD-yeGFPex-HPHex-Pxa1 DNA fragments, and purifying for later use.
2. The method for electrotransformation of bumblebee candida comprises the following steps:
1) Inoculating Candida bombicola colony in 5mL YPD liquid culture medium at 30 deg.C for 200r min -1 Culturing in shaking table for 15-20h, sucking 1mL seed solution, inoculating into 50mL YPD liquid culture medium, and culturing overnight to bacterial concentration OD 600 ＝1.0。
2) The cells were collected by centrifugation at 4 ℃ and washed with 15mL of precooled sterile water 2 times.
3) The cells were resuspended by adding 4mL of 1M sorbitol (precooling), and the cells were collected by centrifugation at 4 ℃.
4) 4mL of in-situ prepared (0.1M) LiAc solution (3500. Mu.L of water, 400. Mu.L of LiAc, 100. Mu.L of DTT) was added to suspend the cells, and the cells were allowed to stand at room temperature for 12 to 15min and collected by centrifugation.
5) Adding 4mL of sorbitol (1M), suspending and centrifugally collecting thalli; the cells were suspended by adding 250. Mu.L of sorbitol.
6) Adding 50 μ L of the bacterial suspension into 10 μ L of the fragment to be transformed, mixing in a 1.5mL EP tube, and ice-cooling for 5min; transferring the mixture to a precooled 1mm electric rotor cup, placing the mixture on ice for 3-5 min, slightly wiping the electric rotor cup, then carrying out electric shock by using an electric rotor instrument (1.5 kv, 200 omega, 5ms capacitance 25 mu L), taking out the electric rotor cup, immediately adding 1mL of precooled sorbitol to blow and suck the electric rotor cup, transferring the electric rotor cup to a 1.5mL EP tube, carrying out standing incubation for 1h in a water bath kettle at the temperature of 30 ℃.
7) Immediately taking out the bacterial suspension, sucking 200 mu L of the bacterial suspension, coating the bacterial suspension on a hygromycin-resistant YPD plate, inverting the bacterial suspension, putting the plate into an incubator at 30 ℃, and culturing for 3d until a single colony grows on the plate.
3. Identification of promoter screening strains:
1) Single colonies were picked up and cultured in 5mL YPD liquid medium at 30 ℃ for 12-24 hours until the cell density and OD600 reached 1.0 or more.
2) Centrifuging to remove supernatant, collecting thallus, adding 600 μ L sorbitol-Na 2 Suspending the thallus in EDTA solution, adding 60 μ L snailase (50 mg. ML-1), mixing, placing in 37 deg.C incubator, breaking cell wall for 4 hr, and turning once every 1 hr to completely break cell wall.
3) Centrifuging to remove supernatant, adding 500. Mu.L Tris-HCl-Na 2 EDTA solution and suspension of the cells, adding 50. Mu.L of 10% SDS solution, mixing, placing in a 65 ℃ incubator, 45min, every 10min turnover (cell wall destruction, nucleic acid lysis, protein and DNA binding destruction at higher temperature, DNA release).
4) After 45min, the bacterial liquid becomes clear, and 300. Mu.L of KAc solution (KAc 5 mol. L) is added -1 ) Mixing, and standing on ice for 1h (adjusting pH to promote protein precipitation).
5) Centrifuging to obtain 550 μ L supernatant, adding equal volume of isopropanol into new EP tube, mixing, and standing at room temperature for 15min.
6) The precipitate was collected by centrifugation and washed twice with 200. Mu.L of 70% ethanol.
7) Drying at 37 ℃ for 5min, adding 30-35 mu L of water and 3 mu L of RNA digestive enzyme (RNase A), placing in a 37 ℃ thermostat for 1h, taking the mixture as a template for PCR verification, and finally obtaining recombinant strains which are respectively named as PX-1 and PGPD-1.
Example 4:
1. culturing the recombinant strains PX-1 and PGPD-1 under the condition of taking glucose as a unique carbon source, and measuring green fluorescence intensity and transcription level respectively:
1) Culturing the recombinant strain: inoculating the two recombinant strains to 10mL YPD liquid culture medium, culturing overnight at 30 ℃, respectively transferring to 50mL liquid culture medium with glucose as a unique carbon source, culturing at 30 ℃ for 8h, enabling OD600 to reach 1.0 or above, and standing in a refrigerator at 4 ℃ for 2h.
2) 1ml of each collected cell was centrifuged, and the cells were washed 2 to 3 times with PBS buffer, and finally diluted to an appropriate concentration and tabletted.
3) The green fluorescence observation was performed using a Leica TCS SP8 laser confocal microscope with the parameters set to: wavelet: excitation =488nm, emission =510nm, and observations are shown in fig. 2.
4) According to the method, bacterial suspension with proper concentration of each recombinant strain is obtained, the flow cytometer BD FACSAria III is used for sampling and measuring the fluorescence intensity, and the detection conditions are as follows: FITC channel, excitation 488nm (blue laser irradiation), emission 530/30, data results as shown in FIG. 3 (A).
5) Taking the recombinant bacterium liquid cultured for 8 hours under the condition, extracting Total RNA and carrying out reverse transcription according to the instruction of a yeast Total RNA extraction kit, carrying out RT-qPCR (reverse transcription-polymerase chain reaction) to carry out GFP (green fluorescent protein) transcription level determination analysis by taking an Actin excited protein gene as a reference gene, and designing a specific primer Actin-F/Actin-R; q-yeGFP-F/q-yeGFP-R. A real-time fluorescent quantitative gene amplification instrument CFX96, wherein the PCR conditions are as follows: 2min at 95 ℃; 10s at 96 ℃,10 s at 60 ℃, 20s at 72 ℃ and 80 ℃; 40 cycles from 96 ℃ to 80 ℃; 5min at 61 ℃. The transcript level data is shown in FIG. 3 (B).
In the same measurement method, the recombinant strains PX-1 and PGPD-1 were cultured in a culture medium containing oleic acid as the sole carbon source, and the green fluorescence intensity and transcription intensity were measured. The measurement results are shown in FIG. 4.
2. Determination of fluorescence intensity of recombinant strains PX-1 and PGPD-1 at different time points under the condition of taking glucose or oleic acid as unique carbon source
The recombinant strains were cultured continuously in 50ml of YPD liquid medium as described above, and samples were taken at 8-hour intervals and the fluorescence intensity values of the respective recombinant strains were measured at different time points using a flow cytometer BD FACSAria III (Bidi, USA), and the results are shown in FIG. 5, in which FIG. 5 (A) shows the results of detection using glucose as a sole carbon source and FIG. 5 (B) shows the results of detection using oleic acid as a sole carbon source.
As can be seen from the examples, the invention takes Candida bombicola as a research object, and predicts, clones, screens and evaluates the strong constitutive promoter. And (3) taking the enhanced green fluorescent protein as a reporter gene, driving the expression of the enhanced green fluorescent protein through the selected promoter, and analyzing the difference of green fluorescent intensity and the difference of transcription level to finally obtain a constitutive promoter PX with strong transcription activity. Compared with the known strong constitutive PGPD promoter, the promoter has the highest strength of transcriptional activity in different culture periods by using glucose or oleic acid as a unique carbon source. The experiment takes the enhanced green fluorescent protein as a report gene in the candida bombicola for the first time, obtains a DNA sequence with strong transcriptional activity through transcriptome level analysis, has good application prospect, enriches synthetic biological elements, and lays a theoretical foundation for the strain in the genetic engineering modification and exogenous gene expression.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university of south of the Yangtze river
<120> bumblebee candida utilis strong constitutive promoter and application thereof
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cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt acacaaatcg cccgcagaag 2340
cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc gatagtggaa accgacgccc 2400
cagcactcgt ccgagggcaa aggaatagat tctagagtcg acatgcatgg ctttcgtgac 2460
cgggcttcaa acaatgatgt gcgatggtgt ggttcccggt tggcggagtc tttgtctact 2520
ttggttgtct gtcgcaggtc ggtagaccgc aaatgagcaa ctgatggatt gttgccagcg 2580
atactataat tcacatggat ggtctttgtc gatcagtagc tagtgagaga gagagaacat 2640
ctatccacaa tgtcgagtgt ctattagaca tactccgaga ataaagtcaa ctgtgtctgt 2700
gatctaaaga tcgattcggc agtcgagtag cgtataacaa ctccgagtac cagcaaaagc 2760
acgtcgtgac aggagcaggg ctttgccaac tgcgcaacct tgcttgaatg aggatacacg 2820
gggtgcaaca tggctgtact gatccatcgc aaccaaaatt tctgtttata gatcaagctg 2880
gtagattcca attactccac ctcttgcgct tctccatgac atgtaagtgc acgtggaaac 2940
cataggtacc tacaactacc atcagggtcg ttagtccaaa taaatggctc tccacggtct 3000
attattgcag acatacttgc ctcacccgct aagaggctat cggctgcaga cgtagggagg 3060
ttgctgtagt tttgcaaaga tatctcaatg tcagaacgat tctggttctc ggctgtttct 3120
gagacttgtc atcactttgg ttacttggtt cataacatca cagaaaaagt gaagcccgtg 3180
tgtcgaatta cagaatgcca accttttctt gaaatgattc tggccactga ttctggcctc 3240
tgctggaatc ctttgattct ggcaagtgtg caacataata cataagagag catacaaaat 3300
tagaattgta caggggttta gtagcctaga tcaatgccta atgttagttc cgtagcggct 3360
tcaatctgag gcacactacc tcctgcatct atccgaagta gcacatatgg tcataggatc 3420
gacatgtcac gtgtgtcatc tcaatcgtcg aacggcaggc gtgcaaactt ggcgtaatca 3480
tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga 3540
gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt 3600
gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 3660
atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 3720
actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 3780
gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 3840
cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 3900
ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 3960
ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 4020
ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 4080
agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 4140
cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 4200
aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 4260
gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 4320
agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 4380
ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 4440
cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 4500
tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 4560
aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata 4620
tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg 4680
atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata 4740
cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg 4800
gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct 4860
gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt 4920
tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc 4980
tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga 5040
tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt 5100
aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc 5160
atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa 5220
tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca 5280
catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca 5340
aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct 5400
tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc 5460
gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa 5520
tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt 5580
tagaaaaata aacaaatggg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc 5640
taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt 5700
cgtc 5704
<210> 3
<211> 717
<212> DNA
<213> (Artificial sequence)
<400> 3
atgagcaagg gtgaagagct tttcactggc gttgtcccta tccttgtcga gcttgacggc 60
gacgttaacg gccacaagtt cagcgtcagc ggcgagggtg aaggcgacgc tacctacggt 120
aagcttaccc tcaagttcat ctgcaccacc ggcaagcttc ctgtcccttg gcctactctc 180
gttaccactt tcggctacgg cgttcagtgc ttcgcccgtt accctgacca tatgaagcag 240
cacgacttct tcaagagcgc tatgcctgag ggctacgttc aagagcgcac catcttcttc 300
aaggacgacg gcaactacaa gacccgcgct gaagtcaagt tcgaaggcga cactctcgtc 360
aaccgcattg agctcaaggg catcgacttt aaggaggacg gcaacatcct cggccacaag 420
cttgagtaca attacaacag ccacaacgtc tacatcatgg ccgacaagca gaagaacggc 480
atcaaggtca atttcaagat ccgccacaac atcgaggacg gcagcgttca gctcgccgac 540
cactaccagc agaacactcc tatcggcgac ggccctgttc ttcttcctga caaccactac 600
cttagcactc agagcgctct tagcaaggac cctaacgaga agcgcgacca catggtcctc 660
cttgagttcg ttactgctgc cggtatcacc catggcatgg acgagcttta caagtaa 717
<210> 4
<211> 32
<212> DNA
<213> (Artificial sequence)
<400> 4
tatataactg tctagaaata aattttttca aa 32
<210> 5
<211> 46
<212> DNA
<213> (Artificial sequence)
<400> 5
tctaaactca aatctgagaa acgcgtgtcc tatggcttct gctttg 46
<210> 6
<211> 46
<212> DNA
<213> (Artificial sequence)
<400> 6
agctcttcac ccttgctcat ggatcctttt caaattaagt tttttg 46
<210> 7
<211> 46
<212> DNA
<213> (Artificial sequence)
<400> 7
tctaaactca aatctgagaa acgcgttgct accttattga agtcta 46
<210> 8
<211> 46
<212> DNA
<213> (Artificial sequence)
<400> 8
agctcttcac ccttgctcat ggatccttgt gtagagttgt ttttgt 46
<210> 9
<211> 46
<212> DNA
<213> (Artificial sequence)
<400> 9
caaaaaactt aatttgaaaa ggatccatga gcaagggtga agagct 46
<210> 10
<211> 46
<212> DNA
<213> (Artificial sequence)
<400> 10
ctcgcatgta tgcacgtcta acgcgttttg aaaaaattta tttcta 46
<210> 11
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 11
attttggaga gtttgtgact gctttatcaa 30
<210> 12
<211> 30
<212> DNA
<213> (Artificial sequence)
<400> 12
tgagatgaca cacgtgacat gtcgatccta 30
<210> 13
<211> 23
<212> DNA
<213> (Artificial sequence)
<400> 13
gtcatctgct caacgaactg tat 23
<210> 14
<211> 20
<212> DNA
<213> (Artificial sequence)
<400> 14
atgtccttct gagcggtctg 20
<210> 15
<211> 18
<212> DNA
<213> (Artificial sequence)
<400> 15
actaccttag cactcaga 18
<210> 16
<211> 18
<212> DNA
<213> (Artificial sequence)
<400> 16
agcagtaacg aactcaag 18
Claims (9)
1. The strong constitutive promoter of the candida bumbleensis is characterized in that the nucleotide sequence of the promoter is shown as SEQ ID No. 1.
2. An expression vector comprising the promoter of claim 1.
3. The expression vector of claim 2, wherein the expression vector is a vector suitable for constitutive expression of Candida bombesi.
4. The expression vector of claim 3, wherein the expression vector is derived from the integration plasmid pPHP, the integration plasmid pSHS or the integration plasmid pAHA.
5. The expression vector of claim 4, wherein the nucleotide sequence of the integrative plasmid pPHP is shown in SEQ ID NO. 2.
6. The use of the promoter of claim 1 for genetic engineering of candida bombicola.
7. The use of claim 6, wherein said use is the use of said promoter to promote expression of a constitutive gene.
8. The application according to claim 7, characterized in that it comprises in particular the following steps:
s1, carrying out fusion PCR on the promoter, the gene to be expressed and the terminator to obtain a gene fragment;
s2, connecting the gene fragment obtained in the step S1 to an integrative plasmid to obtain an integrative expression vector;
and S3, transferring the integrated expression vector obtained in the step S2 into a bumblebee candida utilis host bacterium to obtain a bumblebee candida utilis recombinant bacterium with enhanced expression of the gene to be expressed.
9. The use of claim 8, wherein the terminator has the nucleotide sequence set forth in SEQ ID No. 4.
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