CN110950940B - Novel transcription regulation factor DcGliZ from deep-sea fungi and application thereof - Google Patents
Novel transcription regulation factor DcGliZ from deep-sea fungi and application thereof Download PDFInfo
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Abstract
The invention discloses a novel transcription regulation factor DcGliZ from deep-sea fungi and application thereof. The nucleotide sequence is shown in SEQ ID NO. 1. The invention discloses a novel deep-sea fungus Edwardsiella transcription regulation factor DcGliZ for the first time, the in vitro function of the novel deep-sea fungus Edwardsiella transcription regulation factor DcGliZ is verified, and through EMSA analysis, gel blocking strips exist in the DcGliZ and promoters pG, pM and pN of a gliotoxin biosynthesis gene. The interaction of DcGliZ and the gliotoxin biosynthesis gene promoters pN, pM and pG in vitro is proved, so that a molecular biological basis is laid for improving the gliotoxin yield and obtaining a novel gliotoxin compound by metabolic engineering means such as transcription regulation and the like in the later period.
Description
Technical Field
The invention relates to the fields of biochemistry and molecular biology, in particular to a novel deep-sea fungus Edwardsiella transcription regulation factor DcGliZ and application thereof.
Background
Deep-sea fungi have unique metabolic pathways due to their unique environments of high pressure, high salinity, hypoxia, low temperature, oligotrophism, etc., and thus become an important source for obtaining novel natural products with biological activity.
Disclosure of Invention
The first purpose of the invention is to provide a novel transcription regulatory factor DcGliZ from deep-sea fungus Edwardsiella sp.
This group obtained 8 novel gliotoxins of novel structure from the deep-sea fungus Dichotomomyces cejpii FS110, some of which had significant antitumor activity. On this basis, the subject group performed whole genome sequencing of d.cejpii FS110, predicting the gliotoxin biosynthetic gene cluster with homology lower than 20% to the known gliotoxin gene cluster.
The first purpose of the invention is to provide a transcription regulatory factor DcGliZ, the nucleotide sequence of which is shown in SEQ ID NO. 1.
The research of the inventor finds that the transcription regulatory factor DcGliZ is a transcription regulatory factor for positively regulating the biosynthesis of the mycotoxin, and the deletion of the transcription regulatory factor DcGliZ can block the biosynthesis of the mycotoxin, so that the transcription regulatory factor D cCliZ is important for the biosynthesis of the mycotoxin. The project group discovers a novel transcription regulatory factor DcGliZ from D.cejpii FS110, the homology with the known GliZ transcription regulatory factor is only 43 percent at most, and the position of the gliZ gene in the D.cejpii in a gene cluster is obviously different from the position of the GliZ gene in other fungi in the gene cluster, thus indicating a unique gliotoxin biosynthesis transcription regulatory mechanism of the D.cejpii. In order to obtain more novel gliotoxins and derivatives thereof with more remarkable activity by performing transcriptional control on the gliotoxin of the D.cejpii FS110 at a later stage, the transcriptional control function of biosynthesis of the gliotoxin and the derivatives thereof needs to be analyzed.
Therefore, the second objective of the invention is to provide the application of the transcriptional regulator DcGliZ sequence in the synthesis of gliotoxin by regulating the biosynthesis gene cluster of gliotoxin.
The third purpose of the present invention is to provide the use of the transcription regulatory factor DcGliZ in the regulation of the promoter pG of gliG, the promoter pM of gliM2 or the promoter pN of gliN.
8 novel gliotoxins with novel structures are obtained from deep sea fungus D.cejpii FS110 in the early stage of the subject group. However, the biosynthesis and transcription control mechanism of gliotoxins in d.cejpii has not yet been elucidated, and therefore, it is necessary to analyze the gliotoxin transcription control factor in d.cejpii FS 110. The invention discloses a novel deep-sea fungus Edwardsiella transcription regulation factor DcGliZ for the first time, the in vitro function of the novel deep-sea fungus Edwardsiella transcription regulation factor DcGliZ is verified, and through EMSA analysis, gel blocking strips exist in the DcGliZ and promoters pG, pM and pN of a gliotoxin biosynthesis gene. The interaction of DcGliZ and the gliotoxin biosynthesis gene promoters pN, pM and pG in vitro is proved, so that a molecular biological basis is laid for improving the gliotoxin yield and obtaining a novel gliotoxin compound by metabolic engineering means such as transcription regulation and the like in the later period.
Drawings
FIG. 1 shows the cloning of novel transcription regulatory factor DcGliZ and the construction of recombinant expression vector. lane 1,2kb Marker; lane2, gliZ gene PCR product;
FIG. 2 shows the expression and purification of DcGliZ transcriptional regulatory protein and Western blot validation; lane 1,70kDa marker; l ane2, renaturation of purified DcGliZ protein; lane 3, western blot to verify DcGliZ protein; lane 4,60kDa marker;
FIG. 3 is an EMSA analysis of purified DcGliZ protein and the promoter of the gliotoxin biosynthesis gene, wherein A is the PCR amplification product of the gliotoxin biosynthesis gene promoters pG, pI, pM, pN, pCP; b is EMSA analysis of the promoter of the biosynthesis gene of the DcGliZ protein and the gliotoxin;
FIG. 4 is a Biacore analysis of purified DcGliZ protein with the gliotoxin biosynthesis gene promoters pG, pM, pN and pCP.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
Extracting RNA of D.cejpii FS110, and carrying out reverse transcription to obtain cDNA. Amplification was carried out using gliZ F: TGTTTAACTTTAAGAAGGAGATATACAATGTCGCCATTGTCCGATTCCCAG and gliZ R: GTGGTGGTGGTGGTGGTGGAGCAGATTACAGAGCCTGCTCTGCAG as primers to obtain gliZ (FIG. 1). Inserting gliZ into pEASY-T1 vector, PCR screening positive clone of bacterial liquid, sequencing by company to obtain gliZ sequence, wherein the nucleotide sequence is shown as SEQ ID NO.1 and named as DcGliZ transcription regulation factor.
The sequence of gliZ is utilized to construct a recombinant expression vector pET22b-gliZ by utilizing homologous recombination technology (namely, the sequence of gliZ is inserted between NdeI and XhoI sites of the expression vector pET22 b), is transformed into trans5 alpha competent cells, is coated on an ampicillin resistant LB plate with 100 mu g/ml to screen out positive clones, and utilizes primers T7-F: TAATACGACTCACTATAGG and T7-ter: TAATACGACTCACTATAGG to carry out bacterial liquid PCR verification on the positive clones, and the sequencing verification construction is successful, thus obtaining trans5 alpha bacteria containing pET22 b-gliZ.
Example 2
Expression and purification of DcGliZ transcriptional regulatory factor:
frozen pET22b-gliZTrans5 alpha bacteria liquid is taken for expansion culture into 5mL ampicillin LB culture medium, shaking culture is carried out at 200rpm/min at 37 ℃ overnight, the quality of the particles is improved, 2 mul of transformed BL21 (DE 3) are taken respectively (same as 2.3.3 competent transformation scheme), the single colonies are taken for 12h of culture, 500 mul of LB culture medium added with ampicillin are added for culture until turbidity is reached, then expansion culture is carried out respectively to 200ml, 200rpm/min at 37 ℃ of shaking culture is carried out until OD value reaches 0.4-0.6, 200 mul of IPTG (isopropyl thiogalactoside) inducer is added respectively, bacterial liquid (DE 3) containing pET22b-gliZ plasmid is collected after BL21 (DE 3) liquid is induced for 4h at 37 ℃, the bacterial precipitate is dissolved in 25-30mL of 1 XPBS buffer solution, and cell disruption amplitude is carried out by an ultrasonic disrupter (37%, work 5 s/5 s) to clear (disruption on ice at 4 rpm), 15min of pET22b-gliZ plasmid containing the disrupted T22b is obtained in the form of the expression of the BL21 (DE 21 b) protein after centrifugation, the precipitation is carried out, and the expression of the BL21 b-gliZ plasmid is carried out in the form of the bacterial liquid in an ultrasonic disrupter (ice disruption form after 15-clear ice disruption.
The precipitate after the disruption of BL21 (DE 3) containing pET22b-gliZ plasmid was fully dissolved with 20ml of 6M guanidine hydrochloride, centrifuged at 12000rpm/min at 4 ℃ for 15min, the supernatant was placed in a new centrifuge tube and filtered with a 0.22um filter, packed in a dialysis bag with 2.2nm pore size, and placed in a guanidine hydrochloride gradient (3M, 1M, 0.5M, 0M) renaturation solution for gradient renaturation from large to small, with the renaturation time of each gradient being 9h. After completion of dialysis, SDS-PAGE and Western Blot were performed to verify, thereby obtaining DcGliZ transcriptional regulator (DcGliZ protein).
Example 3
EMSA analysis of the interaction of DcGliZ protein with the promoter of the gliotoxin biosynthesis Gene:
biotin labeling was performed according to the instructions of the EMSA probe biotin labeling kit (bi yun sky biotechnology limited, shanghai, GS 008) of bi yun sky biotechnology limited, based on the core sequences pG, pI, pM, pN, and pCP of gliotoxin biosynthesis genes gli, gliI, gliM2, gliN, and gliP gene promoter. Wherein gliG encodes glutathione s-transferase,gliIencodes an aminotransferase, gliM2 encodes an O-methyltransferase, gliN encodes an N-methyltransferase, and gliP encodes a non-ribosomal peptide synthase. The nucleotide sequences of the promoter core sequences pG, pI, pM, pN and pCP are respectively shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.
After biotin labeling is finished, EMSA reaction is carried out, and the specific flow is as follows:
(1) The preparation of EMSA gel comprises the steps of utilizing a conventional mould for preparing protein electrophoresis gel, adding 19ml of 4% non-denatured polyacrylamide gel to prevent bubbles from being generated in the adding process, and fully and uniformly mixing the gel before adding TEMED. Adding comb teeth, standing at room temperature for 30min, and solidifying;
(2) The following binding reactions were set up:
(3) Adding the reagents in sequence according to the above sequence, standing at room temperature for 20-30min, adding 1 μ l EMSA/Gel-Shift loading buffer (colorless, 10 ×), mixing well, and loading; ( Note that: mu.l of bromophenol blue buffer and 15. Mu.l of colorless loading buffer can be mixed, so that the influence of bromophenol blue on the interaction of protein and nucleic acid can be reduced, and the color can be seen )
(4) Electrophoresis, adding 0.5 XTBE buffer, electrophoresis at constant voltage of 100V until the blue line reaches about 1.5cm from the bottom (about 50bp for probe base);
(5) Rotating the membrane, putting a nylon membrane soaked by 0.5 multiplied by TBE buffer solution and provided with positive charges on EMSA gel, taking care of no bubbles, clamping by using a wet-process electric membrane rotating device, carrying out electrophoresis by using the 0.5 multiplied by TBE buffer solution, carrying out 100V constant voltage for 43min (aiming at the length of 50-700bp of a probe), and putting the device on ice to prevent heat generation in the membrane rotating process;
(6) After the membrane is rotated, placing the membrane on a dry clean flat plate, paying attention to keep the membrane wet (not too wet, removing excessive water), crosslinking for 1.5min at the wavelength of 120ml/cm <2 > by using an ultraviolet crosslinking instrument, taking out the membrane by using tweezers, absorbing excessive water on the surface, keeping the membrane dry, and placing the membrane at room temperature for 3-5 days, or directly carrying out the next chemiluminescence experiment;
(7) Dissolving the sealing solution and the washing solution (which can be used only after completely dissolving) in 37-50 deg.C water bath, placing 15ml of sealing solution in a container with membrane, and slowly shaking for 20min by side shaking table;
(8) Adding 7.5 μ L of Streptavidin-HRP Conjugate into 15mL of the sealing solution, mixing, pouring off the sealing solution for sealing the nylon membrane, adding the prepared solution, and shaking in a side shaking table for 20min;
(9) Adding 25mL of washing liquid (5X) into 100mL of ultrapure water, and uniformly mixing for later use;
(10) Moving the nylon membrane into a new container, adding 30mL of the prepared washing solution, washing for 30min each time, and repeating for four times;
(11) Putting the nylon membrane into another container filled with 20-30mL of detection balance liquid, slowly and uniformly mixing for 5min by a side swinging table, and taking out to suck off the redundant liquid;
(12) And (3) uniformly mixing 2ml of the BeyoECL Moon A solution and 2ml of the BeyoECL Moon B solution (the mixture is ready for use), adding the mixture to the surface of a nylon guide membrane, reacting for 40 seconds, imprinting and exposing on a gel imager, and observing a strip.
The results are shown in fig. 3, and it is found that the purified DcGliZ protein has strong binding with gliG promoter, obvious retardation band exists, and has relatively weak retardation band with gliM2 and gliN coding for O-methyltransferase, which proves that there is relatively weak interaction with it, suggesting that DcGliZ regulatory protein may regulate gliotoxin biosynthesis mainly by interacting with promoter core sequence of gliG, gliM2, gliN genes.
Example 4
Biacore analysis of the interaction of the DcGliZ protein with the gliotoxin biosynthesis gene promoter:
the in-machine experiments were performed on the purified DcGliZ protein and the synthetic promoter core sequences pG, pM, pN and pCP as follows:
(1) Calculating a ligand coupling value according to the molecular weight of the DcGliZ protein and the molecular weight of the promoter nucleotide, and adopting a formula:
R L is the desired amount of ligand coupling, R max Is set to 100RU, S m For the ligand to analyte binding ratio (1:1 binding in this experiment), the analyte molecular mass is about 32.4kDa, the ligand molecular relative mass is 18kDa, and R is calculated L 55.6 at a practical coupling level of 1.5R L ＝83RU
(2) Pre-enrichment of ligand, because many carboxymethyl dextran matrixes are covalently bound on the surface of the CM5 chip and are negatively charged, so that DcGliZ protein needs to be positively charged (PI is 8.58), diluting the protein with 1 mM sodium acetate buffer solution of PH4.5, 5.0 and 5.5 to make the concentration of the ligand (DcGliZ protein) be 20 mu g/ml, performing on-machine pre-enrichment, and primarily detecting the sodium acetate buffer solution with the optimal pH and whether the expected RU value can be reached; (in this case, the ligand molecule for washing the chip is prepared by using 50mM sodium hydroxide solution)
(3) Ligand coupling, selecting sodium acetate buffer solution with the optimal PH value to dilute ligand protein, and fixing the ligand on the chip by using an amino coupling mode. The carboxymethyl dextran matrix on the surface of the CM5 chip is firstly activated by a 1:1 mixture of 1-ethyl-3-carbodiimide (EDC for short) and N-hydroxysuccinimide (NHS for short) to generate activated succinimide ester, ligand molecules containing amino groups are covalently bonded when flowing through the activated surface, the ligand is fixed on the surface of the chip, and then ethanolamine is injected into the surface of the chip to inactivate the rest of the activated ester, so that an analyte is prevented from unnecessarily reacting with the activated ester.
(4) The interaction assay begins with chip surface testing to determine if ligand molecules and analytes are bound and to determine the correct dissociation time. The promoter probes were loaded at 1024nM diluted concentration, respectively, with F4 for the experimental channel and F3 for the reference channel, and no significant sample binding was found according to Fc =4-3 for the channels with 180s binding time and 30s dissociation time (choice of regeneration conditions was Glycine-HCl 2.0). It may be due to a weak binding between the transcription factor and the promoter. The binding capacity is not very pronounced.
(5) Multiple cycle kinetic studies were performed by setting a concentration gradient of promoter core sequences (both 50 bp): 0nM, 4nM (two replicates each), 16nM, 64nM, 256nM, 1024nM, 6 gradients per promoter and 4nM replicates, glycine-HCl 2.0 regeneration buffer, machine experiment, final 1: fitting was performed in the manner of 1.
(6) The experimental data were analyzed, including the amount of ligand coupling, kinetics, and level of binding and dissociation validation.
Biacore on-machine experiment results show that the promoter affinity of DcGliZ protein and GliN (pN) is strongest, and K is D Is 1.045 multiplied by 10 -7 The binding force of the promoter is close to that of GliG (pG), and is K D Is 1.596 multiplied by 10 -7 Followed by the promoter of the GliM2 gene (pM), K D Is 1.623 multiplied by 10 -7 (FIG. 4). There was substantially no interaction with the promoter of the gliP (pCP) gene. It is predicted that DcGliZ protein may regulate gliotoxin biosynthesis mainly by interacting with the promoter core sequence of the gliG, gliM2, gliN genes.
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> a novel transcription regulatory factor DcGliZ from deep-sea fungi and application thereof
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1362
<212> DNA
<213> Erysia sp FS110 (Dichotomomyces cejpii FS 110)
<400> 1
atgtcgccat tgtccgattc ccagtccacg aagccggtta agcctcgcaa gttacgggcc 60
gcttgtgatg cctgtcaccg ttccaagact cggtgttccg gtgggaaccc ctgcacgcgg 120
tgtcgagaat acctgagctc gtgtagttac agttattccg tccgttgcgg caaacctaag 180
ggatcccgct gtcgcaagac cttggagcgg gaacagcggg tggctgctgc tgctgccgcc 240
aaccgcgacc acctccatat ccataatcat tcccgcacgc aatcgcaatc gcacgcggcg 300
gaaccggtga acccgccgcc accggacccc gtcgcacgga cggaggagct acttccaggc 360
cagccggcct caaacgcccc ttcgccccgc gacgactcct ccctctgctt ctccgacgac 420
gttttgcgct ctccggcgga ggtggagcgg acccaagccg acaccacaat aagcgacacc 480
attaccgcag tcgcggaccc gctctcattc ggtgacctcg acgccagcga caacacgggg 540
ttccccctaa attggatgct cccggatctc tcagttgacg cattagaggc actctcactg 600
ccctcgtcta atccaaacga cctcgccaat gccctcttcg tacccggcac ctgggggaca 660
cacggactcg agacgacaac aaccacagaa acccaaaacc ccctactgcc acgagatccc 720
tgcaagtgtc tcctggcgct gacgacgctg acgacctcac gggatatcac cgcgcccacg 780
gtacggcagt gcgacgccac cctcgtcttc gtgcgcaact tctcccgcgc cttttgcgcc 840
ttctaccagt gcgcctactg tcccaaggac gccggtagca tcagcctcgc cgtgacgacg 900
ctgcagctgg cgacaagcgc cctggagacc acgatgcgac accatgtccg ctgcagcgtc 960
cgtgtcgacg gcggggccat taagcctaca gtcttcgacc cagacgacgt ctgctcctcg 1020
tttgcagggg ccgggctact actacccgct gggggcggca acgcaacacc cctcccgttc 1080
caactaggca tctaccacac ctccggggag gacgacgagg aacaaagcca gatcatgagc 1140
gtgctgatcc ggtccgcggt gcggcggtta ctgggcgtct gctggccagt ttgggatctg 1200
ttgcggtcga tcccgggttc ttgtgaccgg tctggggggt ttgagtcgtc caaactgtct 1260
tctctgagtc tggataatct tttagaattt tcgtcggcgg aagtggctca attgcggagg 1320
acgctggtac aactgcagag caggctctgt aatctgctct ag 1362
<210> 2
<211> 261
<212> DNA
<213> Edwardsiella FS110 (Dichroomyes cejpii FS 110)
<400> 2
gtttctatgt ctatatcgga ggagtgatca acggcgtccg aaaattcggc ccgcggctgt 60
cttagggctg gcctgcaggt gtagggttca ttatgaaaga ctggtagtat tatgctgcta 120
tttagtcaaa agcagaagaa cagtttgtct ttagaccttg aatagatatc tgtcaagaca 180
agagccaaga aagtcccatc accatggcag acatgaccga acgaccttct gatctcgttg 240
tggacaggct ggttctcttc g 261
<210> 3
<211> 278
<212> DNA
<213> Erysia sp FS110 (Dichotomomyces cejpii FS 110)
<400> 3
attatgcgtt tgtatatttg aggcaaggca taccagaaac agtcgttata tattcagttt 60
ccttgagatg ctacattcgg tggccgaaag gggatatgcc gacgaaagga tgtcgattct 120
tcttcatcgg cctccgaata tgttcgagac tggcatatcc cagtttactt atacagagga 180
accatatttc aaccctctag ctgaatctgc cttgtctacc ttcccaaatc caggtggatt 240
gatcttgcca gtttatgacc acaatagagg agactcag 278
<210> 4
<211> 274
<212> DNA
<213> Erysia sp FS110 (Dichotomomyces cejpii FS 110)
<400> 4
tagtctatca ctgaacagtt gaatgaattt gtttcaagac agctaaagct ccattctagc 60
cgtacgtact ggtagttctc cgcatagaaa catatatctc tctgcttggt caaagtcagg 120
tatcgccgac aagtcttaat atctgcagag tattcggtga ccgaaatagg gtcggaatat 180
aagccatata tatcactgtt ctggaaatca agaaaatata acagttgaat actcactttt 240
ctaacattcc aacaatcagt tgagctcgat caca 274
<210> 5
<211> 302
<212> DNA
<213> Erysia sp FS110 (Dichotomomyces cejpii FS 110)
<400> 5
ttacttcttg gtctactccg tattgtgaat cgaaatgaga tagaagtata gttctactgt 60
actggtttct atctgcttaa aataaataca attgtgcctt ctatacagat cagtatgtag 120
aatgaactga tagacctcca ggaggttctt cctccgagcc tgcaacgccc aagaggagaa 180
ctgtgcatac gccaggacag attttcggcc tccgaaaagg actgaggata gtatctgcca 240
tgtttatata taactgaggg aatagaccag gtagataaca gcaaaatcaa ctaaacatca 300
tc 302
<210> 6
<211> 179
<212> DNA
<213> Edwardsiella FS110 (Dichroomyes cejpii FS 110)
<400> 6
tgatttgggc cgtctggctg taagacaaca ggcaaacaca tctgatgtgg tgtccgggga 60
ggacggcata tgaagcatcc gaattcctca tatcaaaaga tcctgtattc aacaatcaga 120
ccaccatgtc tacttcctcc cgtcctatct gatcgtcgtc tctgttcctc atctcaatc 179
Claims (1)
1. The transcriptional control factor DcGliZ is used for controllinggliGThe promoter of (1)pG、gliM2The promoter of (1)pM、gliNThe promoter of (1)pNThe use of (1), wherein the transcription regulatory factor passes throughgliGThe promoter of (1)、gliM2Of (2) orgliNThe promoter of (1) is interacted to regulate the biosynthesis of gliotoxin, wherein the nucleotide sequence of the transcription regulation factor DcGliZ is shown in SEQ ID NO.1, and the promoterpG、pM、pNThe nucleotide sequences of (A) are respectively shown as SEQ ID NO.2, SEQ ID NO.4 and SEQ ID NO. 5.
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| CN107858353A (en) * | 2017-11-10 | 2018-03-30 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of angstrom moral bacterium FS110 glutathione sulfurtransferase gene GliG promoters and its application |
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| CN112608931A (en) * | 2020-12-24 | 2021-04-06 | 广东省微生物研究所（广东省微生物分析检测中心） | Deep-sea fungus FS140 anti-gliotoxin self-protection gene GliM and application thereof |
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