CN113186190B - A blue light-mediated regulation plasmid and its construction method and application - Google Patents

Abstract

The invention discloses a blue light regulation promoter PC120 capable of being identified and combined by blue light protein SV40-VP-EL, belonging to the technical field of microorganisms. The invention also discloses a blue light mediated regulation plasmid containing the blue light regulation promoter PC120, and the plasmid also comprises a fusion gene PGAP-SV40-VP-EL for transcribing and translating the blue light protein. The invention further discloses a preparation method of the plasmid and application of the plasmid and/or the construction method in pichia pastoris regulation. The blue light mediated regulation plasmid provides a non-invasive blue light regulation mode with regulation reversibility and high space-time resolution for the induction expression of pichia pastoris, and is beneficial to widening the regulation method of pichia pastoris in production and life.

Description

A blue light-mediated regulation plasmid and its construction method and application

technical field

The invention relates to the technical field of microorganisms, in particular to a blue light-mediated regulation plasmid and a construction method and application thereof.

Background technique

The traditional regulation methods of gene expression process are mostly exogenous chemical inducers combined with soluble transcription factors to achieve artificial control of gene expression. This method is the main driving force for the rapid development of biotechnology, synthetic biology and medical research in the last century. one. Although these traditional regulatory methods can initially control the time and amount of gene expression, their limited reversibility and their inducer-based transport regulation mechanism make them have various limitations and defects, such as their potential off-target Effects, delays in transport and toxicity. Ideally, biological regulatory elements that can be quickly and accurately turned on or off at will will effectively improve the ability to analyze and regulate complex biological gene networks. The time regulation accuracy of light regulation methods can reach the millisecond range.

The EL222 light-sensitive protein derived from Erythrobacter litoralis can effectively realize the light regulation operation. EL222 consists of 222 amino acids, and the N-terminal is Light-Oxygen-Voltage (LOV), and the C-terminal is helix-turn. - Helix-turn-helix (HTH) DNA binding domain, the C-terminus of LOV is a Jα helix connecting LOV and HTH. The core of LOV is the Per-Arnt-SIM (PAS) domain, which can bind to flavin mononucleotide (FMN). Belongs to the light-induced homodimerization system. Upon blue light irradiation, the binding of FMN and LOV causes the Jα helix to wobble from PAS, thereby releasing HTH, allowing EL222 to dimerize and bind to DNA. In the dark, when the N-terminal LOV domain inhibits the DNA-bound C-terminal HTH domain, EL222 spontaneously reverses, thereby rapidly inactivating EL222. At present, the EL222 protein has been preliminarily validated in Escherichia coli and Saccharomyces cerevisiae, but its applicability has not been reported in Pichia pastoris.

As a eukaryotic organism, Pichia pastoris has many advantages of eukaryotic expression systems, such as protein processing, folding, post-translational modification, etc., and is easier to operate. Compared with eukaryotic expression systems such as mammalian cell culture and baculovirus, the Pichia pastoris expression system has the advantages of being faster, simpler, cheaper, and having higher expression levels. As a yeast, Pichia has similar molecular and genetic manipulation advantages as Saccharomyces cerevisiae, and its exogenous protein expression is ten times or even hundreds of times that of Saccharomyces cerevisiae. These many advantages make Pichia pastoris the first choice for eukaryotic protein expression systems. However, the induction and regulation of Pichia pastoris is still in the stage of exogenous chemical inducers. The most commonly used inducer, methanol, is highly toxic and flammable, which is not conducive to the application of Pichia pastoris in the fields of food, medicine, and agricultural product processing. Therefore, the development of a new light regulation method of Pichia pastoris can effectively solve the defects of traditional chemical inducers such as toxicity, instability and transport delay, and expand the methods and methods of Pichia pastoris expression regulation, which has become an urgent need in the field. solved a technical problem.

SUMMARY OF THE INVENTION

In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a blue light regulated promoter PC120, which can quickly and accurately realize the blue light-induced expression of the target gene.

The purpose of the present invention is also to provide a blue light-mediated regulation plasmid and its construction method and application, which can be used for blue light-induced expression of Pichia pastoris. high rate.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The present invention provides a blue light regulated promoter PC120, and the nucleic acid sequence of the promoter is shown in SEQ ID NO.1.

Preferably, the promoter can be recognized and bound by the blue light protein SV40-VP-EL.

Preferably, the amino acid sequence of the blue light protein SV40-VP-EL is shown in SEQ ID NO.2.

Preferably, the nucleic acid sequence of the blue light protein SV40-VP-EL is shown in SEQ ID NO.3.

The present invention also provides a blue light-mediated regulation plasmid, which comprises the above-mentioned blue light regulation promoter PC120 and a fusion gene PGAP-SV40-VP-EL that transcribes and translates blue light protein.

Preferably, the nucleic acid sequence of the fusion gene PGAP-SV40-VP-EL is shown in SEQ ID NO.4.

The present invention also provides the construction method of the above-mentioned blue light-mediated regulation plasmid, and the construction step comprises:

Taking the pAO815 plasmid as the backbone, the fusion gene PGAP-SV40-VP-EL was used to replace the sequence between the pAO815 plasmid AOX1 promoter and AOX1 promoter and AOX1 terminator to obtain the plasmid pGSVEA; then another fusion gene containing the promoter PC120 was used to replace the plasmid pGSVEA. The CGTTCGTTTGTGC sequence is sufficient.

Preferably, the fusion gene comprising the promoter PC120 also includes a reporter gene and/or a target gene.

Preferably, the fusion gene is site-specifically assembled into the pAO815 plasmid by means of GibsonAssembly.

The present invention also provides the application of the above blue light mediated regulation plasmid and/or the construction method of the above blue light mediated regulation plasmid in the induced expression of Pichia pastoris.

Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

The invention provides a novel blue light-induced regulation mode for the inducible expression method of Pichia pastoris. The invention adopts the light regulation method to induce expression of Pichia pastoris, is non-toxic and harmless, has quick response, simple operation, good reversibility, and high spatial and temporal resolution, and promotes the wide application of Pichia pastoris in the fields of food, medicine, agricultural product processing and the like. application.

The invention only needs one plasmid to realize blue light-induced expression of the target gene, and expands the methods and methods of Pichia pastoris expression regulation.

Description of drawings

Figure 1 is a schematic diagram of the mechanism of blue light regulating plasmids.

Figure 2 is a diagram of the pGSVEACA vector, in which PpHIS4 is the Pichia integration site and selection marker, AmpR is the antibiotic resistance, ori is the origin of replication, GAP promoter is the Pichia GAP promoter, and SV40 is the nuclear localization signal , VP16 is the transcriptional activation domain, EL222 is the blue light responsive protein, AOX1 terminator is the yeast AOX1 terminator, PC120 is the blue light regulated promoter, GFP is the green fluorescent protein, and ADH1 terminator is the Saccharomyces cerevisiae ADH1 terminator.

Figure 3 is the clone of Pichia pastoris transformed with pGSVEACA vector on MD plate.

Figure 4 is a blue light-regulated engineering bacteria genome PCR-verified positive clone.

Figure 5 shows the MD plates of blue light-regulated engineering bacteria induced under blue light conditions and dark conditions for 12 h, respectively. The left is the dark condition and the right is the blue light condition.

Figure 6 shows the MD shake flask culture induced by blue light-regulated engineering bacteria under blue light conditions and dark conditions, respectively, (A) is the fluorescence intensity of the experimental group Full Light and the control group Full Dark after 1 hour of blue light induction; (B) is blue light induced for 4 hours. The fluorescence intensity of Full Light in the experimental group and Full Dark in the control group; (C) is the fluorescence intensity of Full Light in the experimental group and Full Dark in the control group after 7 hours of blue light induction; (D) is the Full Light in the experimental group and the control group after 10 hours of blue light induction Fluorescence intensity of Full Dark; (E) is the trend graph of the average fluorescence intensity of samples taken every hour during the blue light induction period between the experimental group Full Light and the control group Full Dark.

Detailed ways

The present invention provides a blue light regulated promoter PC120, and the nucleic acid sequence of the promoter is shown in SEQ ID NO.1. The promoter PC120 of the present invention can be recognized and combined by the blue light protein SV40-VP-EL, thereby realizing the blue light-induced expression of the target gene quickly and accurately.

The amino acid sequence of the blue light protein SV40-VP-EL of the present invention is shown in SEQ ID NO.2, and the nucleic acid sequence is shown in SEQ ID NO.3. The nucleic acid sequence of the blue light protein SV40-VP-EL in the present invention can also be the other nucleic acid sequence variants encoding the protein SV40-VP-EL.

The present invention provides a blue light mediated regulation plasmid, comprising the above blue light regulation promoter PC120 and a fusion gene PGAP-SV40-VP-EL for transcription and translation of blue light protein. PGAP in the fusion gene PGAP-SV40-VP-EL of the present invention represents Pichia pastoris GAP promoter GAP promoter, SV40 represents nuclear localization signal SV40, VP represents transcription activation domain VP16, and EL represents blue light response protein EL222.

In the present invention, the nucleic acid sequence of PGAP is the GAP1 promoter sequence of Pichia GS115 ATCC:20864 (GenBankAccessionNo:CP014716REGION:809514..809990).

The nucleic acid sequence of the fusion gene PGAP-SV40-VP-EL of the present invention is shown in SEQ ID NO.4.

The present invention also provides a method for constructing the above blue light-mediated regulating plasmid, comprising: using the pAO815 plasmid as the backbone, replacing the fusion gene PGAP-SV40-VP-EL with the sequence between the pAO815 plasmid AOX1promoter and AOX1 promoter and AOX1terminator to obtain the plasmid pGSVEA ; Replace the CGTTCGTTTGTGC sequence of the plasmid pGSVEA with another fusion gene containing the promoter PC120 to obtain the blue light regulating plasmid pGSVEACA. The pGSVEACA plasmid constructed by the present invention is shown in Figure 2.

After the plasmid pGSVEA obtained in the present invention is introduced into Pichia pastoris, it can be transcribed and translated to obtain blue light protein SV40-VP-EL. Preferably, the Pichia pastoris is Pichia pastoris GS115.

The blue light regulating plasmid pGSVEACA obtained by the present invention can be transcribed and translated to obtain blue light protein SV40-VP-EL. Under blue light irradiation, the N-terminus (photo-oxidation control domain) and C-terminus ( Helix-turn-helix DNA binding domain) interacts to dimerize EL222 and bind to the promoter PC120, turning on transcription of another fusion gene containing the promoter PC120.

In the present invention, another fusion gene comprising the promoter PC120 is PC120-GFP-TADH1, wherein PC120 represents the blue light regulated promoter PC120, GFP represents the green fluorescent protein GFP, TADH1 represents the yeast ADH1 terminator ADH1terminator, and the nucleic acid sequence of TADH1 is ADH1 terminator sequence of Saccharomyces cerevisiae Y169 (GenBank Accession No: CP033484REGION: 156786..157080). In the present invention, the green fluorescent protein GFP can be replaced with other reporter genes, and can also be replaced with other target genes.

The nucleic acid sequence of the fusion gene PC120-GFP-TADH1 of the present invention is shown in SEQ ID NO.5.

In the present invention, the two fusion genes can be assembled on the pAO815 plasmid by means of GibsonAssembly. The present invention does not limit the specific assembling manner.

The present invention also provides the application of the blue light-mediated regulation plasmid and/or the construction method of the blue-light-mediated regulation plasmid in the induced expression of Pichia pastoris. In the present invention, the blue light regulating plasmid pGSVEACA is transformed into Pichia pastoris, so that the blue light-induced expression of the target gene can be realized, the methods and methods of Pichia pastoris expression regulation are expanded, and the application of Pichia pastoris in the fields of food, medicine, agricultural product processing and the like is promoted. wide application.

The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Example 1

In this example, the gene sequence is optimized.

The gene sequences and protein sequences of VP16 and EL222 retrieved from NCBI were optimized according to the codon preference of Pichia pastoris, and the DNA sequences were re-synthesized.

The specific optimization method is: optimize the gene according to the gene synthesis company GenScript codon optimization software OptimumGene ^TM , mainly refer to the codon preference of Pichia pastoris, and then combine the filter element and balance the GC content of the gene to finally complete the gene codon optimization. The optimization of SV40-VP-EL nucleic acid sequence, as shown in SEQ ID NO.3; PGAP-SV40-VP-EL nucleic acid sequence, as shown in SEQ ID NO.4; PC120-GFP-TADH1 nucleic acid sequence, as shown in SEQ ID NO.4 ID NO.5.

Example 2

In this example, the blue light regulating plasmid pGSVEACA was designed and constructed using the commonly used plasmid pAO815 of Pichia pastoris as the backbone.

The specific construction method is:

The PGAP-SV40-VP-EL gene sequence optimized in Example 1 was fully synthesized, and then Gibsonassembly technology was used to replace the pAO815 plasmid AOX1 promoter and the sequence between AOX1 promoter and AOX1 terminator to obtain plasmid pGSVEA;

The PC120-GFP-TADH1 gene sequence optimized in Example 1 was fully synthesized, and then the CGTTCGTTTGTGC sequence of the plasmid pGSVEA was replaced by Gibsonassembly technology to obtain the blue light regulating plasmid pGSVEACA.

In this example, the PGAP-SV40-VP-EL gene sequence does not contain a terminator sequence, so it is necessary to borrow the AOX1 terminator on the pAO815 plasmid backbone to construct a complete SV40-VP-EL open reading frame to realize SV40-VP - Constitutive expression of EL.

In this example, in order to make a single plasmid achieve the effect of blue light induction, it is necessary to insert the PC120-GFP-TADH1 gene sequence into the downstream of PGAP-SV40-VP-EL on the basis of the generated plasmid pGSVEA, so PC120-GFP is used. - The TADH1 gene sequence replaces the CGTTCGTTTGTGC sequence of the plasmid pGSVEA.

Example 3

In this example, the blue light regulating plasmid pGSVEACA was transformed, induced to express and analyzed.

(1) Plasmid extraction and quantitative detection

1) Take 10 mL of the overnight bacterial culture, centrifuge at 13,400 g for 1 min, discard the supernatant and collect the precipitate.

2) Add 500 μL of solution I, solution II and solution III in sequence, immediately and gently turn up and down 6 to 8 times, then let stand for 5 minutes, and centrifuge at 13400g for 10 minutes.

Wherein the composition of solution I is 25mM Tris-HCl (pH=8.0), 10mM EDTA, 50mM glucose;

The composition of solution II is 250 mM NaOH, 1% (W/V) SDS;

The composition of solution III was 3M potassium acetate, 5M acetic acid.

3) Add the supernatant collected in the previous step to the filter column, centrifuge at 13400g for 1 min, add 450 μL of isopropanol and mix evenly. Then add it to the adsorption column, centrifuge at 13400g for 1 min and discard the waste liquid.

4) Add 700 μL of rinsing solution to the adsorption column, centrifuge at 13400 g for 1 min and discard the waste solution.

5) After the rinsing solution was removed from the adsorption column, 150 μL of deionized water was added dropwise to the center of the adsorption membrane, and then centrifuged at 13400g for 2 minutes after standing for 2 minutes.

6) Collect the liquid in the tube and measure the concentration of plasmid pGSVEACA, and store at -20°C for later use.

(2) Linearization and recovery of plasmid pGSVEACA

1) Add 1/10 volume of 3M sodium acetate solution and 2.5 times volume of absolute ethanol to the sample, mix well, and place at -20°C for 1 hour. Then, centrifuge at 12,000 rpm for 10 min, and discard the supernatant.

2) Resuspend the pellet with 2.5 times the volume of pre-cooled 75% ethanol, centrifuge at 12000 rpm for 10 min, and discard the supernatant. And placed on the ultra-clean workbench to blow for 15min.

3) Add 25 μL of deionized water to the bottom of the tube to resuspend, measure the plasmid concentration, and store at -20°C for later use.

(3) Electrotransformation of Pichia pastoris GS115

1) Add 5-10 μg of the linearized plasmid pGSVEACA to the prepared Pichia GS115 competent cells, mix well, transfer to a new pre-cooled electroporation cup (0.2cm size), and bath in ice for 5 minutes.

2) Quickly take out the electric rotor cup and wipe it dry, conduct electric shock under the parameters of voltage of 1.5kV, capacitance of 25μF and resistance of 200Ω, immediately add 1mL of pre-cooled 1M sorbitol, absorb the liquid and transfer it to a new 1.5mL sterile centrifuge tube and incubate at 30°C for 1.5h.

3) After centrifugation at 12,000 rpm for 20s, most of the supernatant was discarded, and the cells were resuspended and coated on an MD screening plate, and incubated at 28°C for 2 days until a single colony visible to the naked eye appeared. The yeast transformation results are shown in Figure 3.

(4) Qualitative analysis of induced expression and fluorescence intensity of Pichia pastoris transformants

The transformed yeast transformants were randomly selected and inoculated into 5 mL of MD liquid medium for 19 h at 28°C on a shaker. Bacteria were preserved and genomic DNA was extracted, and gene positive detection primers were designed for PCR amplification to obtain positive strains. The primers used in PCR of the present invention are shown in Table 1.

Table 1 PCR primers

In Table 1, the GAP-F sequence is shown in SEQ ID NO.6, the ADH-R sequence is shown in SEQ ID NO.7, the AOX-R sequence is shown in SEQ ID NO.8, and the AOX-F sequence is shown in SEQ ID NO. .9 shown.

The results of PCR amplification are shown in Figure 4, indicating that the yeast transformants picked and cultured contain the target plasmid.

According to the PCR detection results, 3-5 positive clones were selected and streaked on the MD medium, firstly cultured in the dark environment at 28°C for 24h, and then the experimental group was induced and incubated with blue light.

After being induced by blue light for 24 hours, the experimental group and the control group were placed under the excitation light of 475nm for qualitative comparison of the fluorescence intensity. The green fluorescent protein was obtained by the intracellular transcription and translation of Pichia pastoris induced by blue light, and the PC120 promoter could successfully recognize and bind to the blue light protein SV40-VP-EL to precisely turn on the transcription and translation of the reporter gene.

(5) Flow cytometry analysis of Pichia pastoris transformants

According to the PCR detection results, select 3 to 4 positive clones, and transfer 50-100 μL of culture solution to 50 mL of MD liquid medium. Induced expression. The experimental group and the control group were sampled every 1 h after blue light induction for 10 h in total.

The samples were pretreated for flow cytometry analysis. The specific operations were: centrifuge the sample bacterial solution at 5000 rpm for 1 min at 4 °C, discard the supernatant, resuspend it with PBS, and dilute it to a state that is invisible to the naked eye. Volumes of sample dilutions were filtered through a 300-mesh filter and stored in flow analysis tubes for flow cytometry analysis.

The relationship between the fluorescence intensity and the induction time obtained after the samples of the experimental group and the control group were analyzed by flow cytometry are shown in Table 2 and FIG. 6 .

Table 2 Fluorescence intensity of bacteria at different times under blue light induction and dark environment

It can be seen from Table 2 and Figure 6 that the fluorescence intensity of GFP reached the highest after 8 hours of continuous irradiation with blue light, and its intensity was 16.4 times that of the control group at the same period. There is a single and uniform peak in the histogram, indicating that blue light can achieve a uniform induction effect on GS115 cells carrying the pGSVEACA plasmid. And there are not many overlapping parts of the histograms of the experimental group and the control group, indicating that the induction intensity is relatively good. Overall, the flow cytometry results indicate that the pGSVEACA plasmid can effectively achieve an efficient and uniform response of GS115 cells to blue light.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

sequence listing

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gagtttgtta ctgctgctgg tattactcac ggtatggatg aattgtataa ataaggtacc 960

gaacaaaaac tcatctcaga agaggatctg aatagcggcg gccgccatca tcatcatcat 1020

cattgagttt tagccttaga catgactgtt caagttgggc acttacgaga agtaaataag 1080

ttataaaaaa aataagtgta tacaaatttt aaagtgactc ttaggtttta aaacgaaaat 1140

tcttgttctt gagtaactct ttcctgtagg tcaggttgct ttctcaggta tagcatgagg 1200

tcgctcttat tgaccacacc tctaccggca tgccgagcaa atgcctgcaa atcgctcccc 1260

atttcaccca attgtagata tgctaactcc agcaatgagt tgatgaatct cggtgtgtat 1320

tttatgtcct cagaggacaa cacctgttgt aatcgttctt ccacacg 1367

<210> 6

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 6

gattttttgta gaaatgtctt ggtgtcctc 29

<210> 7

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 7

cgtgtggaag aacgattaca acag 24

<210> 8

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 8

ggctagcgaa gatctcccg 19

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 9

cgggagatct tcgctagcc 19

Claims (8)

1. a blue light-mediated regulation plasmid, is characterized in that, described plasmid comprises the fusion gene PGAP-SV40-VP-EL of blue light regulation promoter PC120 and transcription and translation blue light protein; The nucleic acid sequence of the fusion gene PGAP-SV40-VP-EL is shown in SEQ ID NO.4; The nucleic acid sequence of the promoter is shown in SEQ ID NO.1. 2 . The blue light-mediated regulation plasmid according to claim 1 , wherein the promoter can be recognized and bound by the blue light protein SV40-VP-EL. 3 . 3. The blue light-mediated regulation plasmid according to claim 2, wherein the amino acid sequence of the blue light protein SV40-VP-EL is shown in SEQ ID NO.2. 4. The blue light-mediated regulation plasmid according to claim 2, wherein the nucleic acid sequence of the blue light protein SV40-VP-EL is shown in SEQ ID NO.3. 5. A construction method of the blue light-mediated regulation plasmid according to any one of claims 1 to 4, wherein the construction step comprises: Taking the pAO815 plasmid as the backbone, replace the fusion gene PGAP-SV40-VP-EL with the sequence between the pAO815 plasmid AOX1promoter and AOX1promoter and AOX1terminator to obtain the plasmid pGSVEA; then replace the CGTTCGTTTGTGC sequence of the plasmid pGSVEA with another fusion gene containing the promoter PC120 That's it. 6. The construction method according to claim 5, wherein the fusion gene comprising the promoter PC120 further comprises a reporter gene and/or a target gene. 7 . The construction method according to claim 5 , wherein the fusion gene is site-specifically assembled on the pAO815 plasmid by means of GibsonAssembly. 8 . 8. The application of the blue light mediated regulation plasmid according to any one of claims 1 to 4 and/or the construction method of the blue light mediated regulation plasmid according to any one of claims 5 to 7 in inducible expression of Pichia pastoris .

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