Construction and application of a gene expression system responsive to blue light regulation

Abstract

The application discloses a blue light response regulated gene expression system construction and application, and belongs to the field of synthetic biology. The application regulates the expression of genes by using a blue light activated promoter and a blue light inhibited promoter in Bacillus amyloliquefaciens. The expression level of eGFP of the promoter pBLind is increased by 1.4 times under a blue light irradiation environment compared with that under a dark environment. The expression level of eGFP of the promoter pBLrep is reduced by 10 times under a blue light irradiation environment compared with that under a dark environment, so that rapid, directional and accurate regulation of gene expression in Bacillus amyloliquefaciens is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to a blue light regulated gene expression system construction and application, and belongs to the field of synthetic biology. BACKGROUND

[0002] In microbial cells, basic processes such as gene expression and cell cycle are largely controlled by spatial and temporal oscillations. Precise spatiotemporal control of inducible and repressible gene expression systems will help fine-tune gene expression in microbial cells, thereby avoiding metabolic burden on host cells, and thus facilitating efficient synthesis of target products. Ideally, expression control systems that can be rapidly and accurately turned on or off at will will help improve our ability to perturb and regulate complex biological gene networks. Generally, exogenous chemical inducers that bind to soluble transcription factors are used to achieve artificial control of gene expression. However, due to their potential non-target effects, transport process delays, toxicity, and lack of reversibility of gene expression, their effectiveness is limited. For example, once induced, it is difficult to remove residual chemical inducers from the growth medium, which poses difficulties for studies that require precise control of gene expression at desired levels. Light stimulation is non-toxic and can be rapidly delivered to cells through precise spatiotemporal control. Gene expression levels can be easily adjusted and reversed according to the intensity of light and the duration of light exposure. An ideal optogenetic system requires light-dependent inducible and repressible devices that can work in parallel with rapid and reversible gene expression in space and time. The availability of these tools will well facilitate researchers to regulate the expression of multiple endogenous genes in a more controllable manner. SUMMARY

[0003] The present application provides a blue light regulated gene expression system and its application, based on the promoter, a blue light response based regulated gene expression system is constructed, specifically, the promoter and the developed gene expression system are used to regulate the target gene in Bacillus amyloliquefaciens.

[0004] The present application provides a blue light response gene regulation expression element, comprising: a repressor protein and a blue light response promoter; the repressor protein is located upstream of the blue light response promoter; the repressor protein is composed of a constitutive promoter P HpaII expression; the blue light response promoter contains a binding sequence of the repressor protein, the binding sequence is shown as SEQ ID NO. 4; the blue light response promoter regulates the expression of the target gene; the regulation includes activation, promotion or inhibition.

[0005] In an embodiment, the blue light response promoter contains the nucleotide sequence shown in SEQ ID NO. 1 or 2.

[0006] In an embodiment, the constitutive promoter P HpaII The nucleotide sequence of the promoter is shown as SEQ ID NO. 1.

[0007] The present application also provides a recombinant expression vector comprising the gene-regulating expression element, which can allow insertion of a target gene and expression of the target gene in a microbial cell.

[0008] In an embodiment, the expression vector includes, but is not limited to, plasmid pMA5.

[0009] The present application also provides a microbial cell comprising the gene-regulating expression element or the recombinant expression vector.

[0010] In an embodiment, the microbial cell includes, but is not limited to, a bacterial cell or a fungal cell.

[0011] In an embodiment, the bacteria include, but are not limited to, Escherichia coli, Bacillus subtilis, or Bacillus amyloliquefaciens.

[0012] In an embodiment, the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens NBΔCN, which is obtained by knocking out the cwlO gene and the ycgN gene based on Bacillus amyloliquefaciens NB; the accession number of the Bacillus amyloliquefaciens NB is CCTCC NO: M 2016346, which has been disclosed in the patent document with the publication number CN106047780B.

[0013] The present application also provides a method for regulating the expression amount of a target protein, which comprises connecting a gene encoding the target protein downstream of the blue light-responsive regulatory promoter, culturing a microorganism containing the gene in a culture medium under suitable conditions, and starting or inhibiting transcription of the target gene under the condition of blue light irradiation.

[0014] In an embodiment, the method is to connect a gene encoding a target protein downstream of the promoter shown in SEQ ID NO. 1, culture a microorganism containing the gene in a culture medium under suitable conditions, and start transcription of the target gene under the condition of blue light irradiation.

[0015] In an embodiment, the method is to connect a gene encoding a target protein downstream of the promoter shown in SEQ ID NO. 2, culture a microorganism containing the gene in a culture medium under suitable conditions, and inhibit transcription of the target gene under the condition of blue light irradiation.

[0016] In an embodiment, the culturing is in a blue light irradiation environment.

[0017] In an embodiment, the blue light irradiation comprises continuous irradiation or intermittent irradiation.

[0018] In an embodiment, the gene of interest comprises, but is not limited to, a coding gene of green fluorescent protein.

[0019] The present application also provides applications of the gene regulatory expression element, the recombinant expression vector, the recombinant microbial cell or the method in regulating expression of a protein of interest.

[0020] Beneficial effects: The present application successfully constructs two promoters activated / inhibited by blue light in Bacillus amyloliquefaciens: a blue light activated promoter (pBLind) and a blue light inhibited promoter (pBLrep). The expression level of eGFP of pBLind is increased by 1.4 times in a blue light irradiation environment compared with that in a dark environment. The expression level of eGFP of pBLrep is decreased by 10 times in a blue light irradiation environment compared with that in a dark environment. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 Structure diagram of a recombinant expression vector responding to blue light.

[0022] Figure 2 Gel electrophoresis diagram of amplification products of a blue light induced promoter.

[0023] Figure 3 Function identification of a blue light activated promoter. A: structure diagram of a blue light activated promoter; B: analysis of expression of eGFP of a blue light activated promoter in blue light and dark conditions.

[0024] Figure 4 Function identification of a blue light inhibited promoter. A: structure diagram of a blue light inhibited promoter; B: analysis of expression of eGFP of a blue light inhibited promoter in blue light and dark conditions.

[0025] Figure 5 Expression intensity of a blue light induced promoter at different times. A: analysis of expression intensity of eGFP of a blue light activated system at different fermentation times in blue light and dark conditions. B: analysis of expression intensity of eGFP of a blue light inhibited system at different fermentation times in blue light and dark conditions.

[0026] Figure 6 Response intensity of a light induced promoter to different blue light irradiation pulses. (A: determination of response ability of a blue light activated system to express eGFP gene under different blue light pulse irradiation; B: determination of response ability of a blue light inhibited system to inhibit expression of eGFP under different blue light pulse irradiation) DETAILED DESCRIPTION

[0027] Restriction enzymes and one-step cloning enzymes were purchased from Takara and Vazyme, respectively. Multifunctional enzyme plate reader BioTek HT plate reader (Winooski, VT, USA) was used to detect the fluorescence intensity of the sample. E. coli DH5a was used for molecular cloning, E. coli GM2163 was used for demethylation, and B. amyloliquefaciens NB (strain disclosed in the literature "Efficient biosynthesis of low-molecular-weight poly-gamma-glutamic acid based on stereochemistry regulation in Bacillus amyloliquefaciens") was used for protein or gene expression.

[0028] Culture medium involved in the examples:

[0029] LB liquid medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl

[0030] LB solid medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl, 2% of agar powder

[0031] TB liquid medium: 24 g / L of yeast powder, 20 g / L of proteose peptone, 17 mmol / L of KH2PO4, 72 mmol / L of K2HPO4, 4 mL / L of glycerol

[0032] Competent preparation medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl, 0.5 M of sorbitol

[0033] Recovery medium: 10 g / L of proteose peptone, 5 g / L of yeast powder, 10 g / L of NaCl, 0.5 M of sorbitol, 0.38 M of mannitol

[0034] Electroporation buffer: 0.5 M of sorbitol, 0.5 M of mannitol, 10% of glycerol

[0035] Competent cell suspension: 0.5 M of sorbitol, 0.5 M of mannitol, 10% of glycerol, 14% of PEG-6000

[0036] Example 1 Design of a gene regulation system responsive to blue light

[0037] The gene regulation system responsive to blue light comprises a repressor protein, a blue light inducible promoter and a target gene; the repressor protein is located upstream of the blue light inducible promoter; the repressor protein is expressed by a constitutive promoter P HpaII expression, the constitutive promoter PHpaII The nucleotide sequence of the nucleotide sequence is shown as SEQ ID NO. 5; the blue light-responsive promoter contains the binding sequence of the repressor protein, and the nucleotide sequence of the binding sequence is shown as SEQ ID NO. 4; the blue light-responsive promoter regulates the expression of the target gene; the regulation includes activating and promoting the expression of the target gene, or inhibiting the expression of the target gene.

[0038] The working principle of the blue light-responsive gene regulation system includes:

[0039] (1) When the blue light-inducible promoter has the nucleotide sequence shown as SEQ ID NO. 1, the repressor protein binding region is located upstream of the -35 region of the promoter sequence. Under blue light irradiation, the repressor protein can recruit RNAP between the -35 and -10 regions, and initiate the transcription of the target gene (as shown in Figure 3 ).

[0040] (2) When the blue light-inducible promoter has the nucleotide sequence shown as SEQ ID NO. 2, the repressor protein binding region is located between the -35 and -10 regions of the promoter sequence; under blue light irradiation, the repressor protein plays an inhibitory role by preventing the binding of RNAP, thereby inhibiting the transcription of the target gene (as shown in Figure 4 ).

[0041] Example 2 Construction of a genetic recombination vector

[0042] 1. The plasmid pUC57-EL222 carrying the light-sensitive protein gene with the nucleotide sequence shown as SEQ ID NO. 3 was synthesized by Anhui General Biotech Co., Ltd.

[0043] 2. The plasmid pMA5 was double-digested using restriction endonucleases Nde I and BamH I. The enzyme reaction system was as follows: 10x Quickcut Buffer 5 μL, restriction endonucleases Nde I and BamH I each 2 μL, total amount of plasmid pMA5 41 μL, and the total volume of the reaction system was 50 μL. The enzyme digestion was performed in a 37°C water bath for 3 h, and the enzyme digestion results were detected by 1% agarose gel electrophoresis.

[0044] 3. Gene fragment purification and recovery: the experimental steps were referred to the DNA purification and recovery kit of Vazyme, and the linearized pMA5 plasmid was recovered.

[0045] 4. The blue light-inhibitory promoter pBLind and pBLrep were placed upstream of the reporter protein eGFP and co-expressed with EL222 on the plasmid pMA5, and the specific steps were as follows:

[0046] (1) Use primer pair pM-EL222-F / R to amplify the gene fragment of repressor protein EL222 with plasmid pUC57-EL222 as template; use primer pair pM-Tamy-F and pM-TamyPBL-R and pM-TamyRep-R to amplify Tamy as the terminator of EL222 with plasmid pHY-egfp (plasmid disclosed in the literature "CRISPRi-Based Dynamic Regulation of Hydrolase for the Synthesis of Poly-gamma-Glutamic Acid with Variable Molecular Weights") as template.

[0047] (2) Design long-chain primers PBL-GFP-F and PBLrep-GFP-F to amplify fragments PBLind-gfp and PBLrep-gfp with light-inducible promoters with plasmid pHY-egfp as template. Further use primers pM-PBL-F, pM-Rep-F and pM-GFP-R to amplify PBLind-gfp and PBLrep-gfp for overlap PCR.

[0048] (3) Use Vazyme DNA purification recovery kit to purify and recover the fragments obtained in step (1) or step (2), and use primer pair pM-EL222-F and pM-GFP-R to perform overlap PCR on the purified and recovered fragments.

[0049] Table 1 Primer sequence information for constructing blue light regulation system in Bacillus amyloliquefaciens

[0050]

[0051]

[0052] Table 2 PCR amplification program

[0053]

[0054] 5, DNA ligation: Determine the concentration of the purified and recovered promoter fragment and linear vector, mix them in a mass ratio of 2:1, add 2 μL ClonExpress II One Step Cloning ligase, 4 μL Buffer, and make up the total volume to 20 μL with dd H2O, and incubate at 37°C for 30 min.

[0055] 6、Transformation: The competent cells E. coli DH5a taken out from the -80 °C refrigerator were thawed on ice for 10 min, 20 μL of the ligation product was added, mixed, and then ice-bathed for 30 min. Then, it was placed in a 42 °C constant temperature water bath for 90 s, quickly placed in an ice water bath for 2 min, and then 1000 μL of LB medium (without antibiotics) was added, mixed thoroughly, and then incubated at 37 °C, 200 rpm for 1 h. Finally, 200 μL of the recovered bacterial solution was spread on LB solid medium containing 100 μg / mL of ampicillin sodium antibiotic, and incubated at 37 °C for 12 h. When single colonies grew on the plate, single colonies were picked for colony PCR verification to screen positive transformants.

[0056] 7. Demethylation: The successfully constructed plasmid was extracted from the positive transformant obtained in step 6 according to the Vazyme plasmid extraction kit instructions. The competent cells E. coli GM2163 taken out from the -80 °C refrigerator were thawed on ice for 10 min, 20 μL of the constructed plasmid was added, mixed, and then ice-bathed for 30 min. Then, it was placed in a 42 °C constant temperature water bath for 90 s, quickly placed in an ice water bath for 2 min, and then 1000 μL of LB medium (without antibiotics) was added, mixed thoroughly, and then incubated at 37 °C, 200 rpm for 1 h. Finally, 200 μL of the recovered bacterial solution was spread on LB solid medium containing 100 μg / mL of ampicillin sodium antibiotic, and incubated at 37 °C for 12 h. When single colonies grew on the plate, single colonies were picked for colony PCR verification to screen positive transformants, and the demethylated plasmid was obtained.

[0057] Example 3 Construction of Recombinant Bacillus amyloliquefaciens

[0058] 1. Construction of Bacillus amyloliquefaciens NBΔCN: Using Bacillus amyloliquefaciens NB as the starting strain, based on the cwlO gene (nucleotide sequence as shown in Gene ID: 12205061) and ycgN gene (nucleotide sequence as shown in Gene ID: 12963445) sequences of Bacillus amyloliquefaciens, a 20bp target sequence was selected using gRNAFinder. The cwlO and ycgN genes were knocked out using the dual-plasmid CRISPR-Cas9n method (related plasmids are disclosed in the literature Development of a robust Bacillus amyloliquefaciens cell factory for efficient poly-(glutamic acid) production from Jerusalem artichoke). The Bacillus amyloliquefaciens NB is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 2016346 and has been published in the patent document with publication number CN106047780B.

[0059] 2. Preparation of competent cells for *Bacillus amyloliquefaciens* NBΔCN: *Bacillus amyloliquefaciens* NBΔCN, stored in glycerol tubes at -80℃, was streaked onto LB agar plates and incubated at 37℃ for 12 h. Single colonies were picked and incubated in 10 mL of competent cell preparation medium at 37℃ for 12 h. 1 mL of the seed culture was inoculated into a 500 mL shake flask containing 100 mL of competent cell preparation medium and incubated at 37℃ until OD... 600 The concentration of competent cells was 0.5. After incubating the culture medium on ice for 20 min, centrifuged at 8000×g for 10 min at 4°C to collect competent cells. The collected cells were resuspended in electroporation buffer and centrifuged at 8000×g for 10 min at 4°C. The supernatant was removed, and this step was repeated 4 times. An appropriate amount of suspension was added to the centrifuged cells to adjust the concentration of competent cells to 1×10⁻⁵. 10 Aliquot (100 μL per tube) and store at -80°C.

[0060] 3. After cleaning the 2mm electroporation cuvette with 75% alcohol, air-dry it under UV light and pre-cool it on ice. Mix 100ng of the plasmid DNA constructed in Example 2 with 100μL of competent cells and transfer it to the pre-cooled electroporation cuvette. Set the voltage of the electroporator to 2.5-2.9kV and the electroporation time to 4ms. After electroporation, quickly add 500μL of resuscitation medium to the cuvette and transfer it to a sterile centrifuge tube. Incubate at 37℃ with shaking for 3h and then spread it on the corresponding plates. Once a single colony has grown on the plate, pick a single colony for colony PCR verification to screen for positive transformants. The verified positive transformants are named Bacillus amyloliquefaciens NBΔCN(pMA5-EL222-pBLind-eGFP) and Bacillus amyloliquefaciens NBΔCN(pMA5-EL222-pBLrep-eGFP), respectively.

[0061] Example 4: Application of blue light-responsive gene regulation in Bacillus amyloliquefaciens

[0062] The positive bacteria *B. amy NBΔCN* (pMA5-EL222-pBLind-eGFP) and *B. amy NBΔCN* (pMA5-EL222-pBLrep-eGFP) constructed in Example 3 were streaked onto antibiotic-resistant plates and cultured overnight. The control strain *Bacillus amyloliquefaciens* NBΔCN was activated by streaking onto antibiotic-free LB agar plates. Single colonies were picked and inoculated into small shake flasks containing 5 mL of LB medium and cultured at 37°C for 12 h to obtain seed culture. The seed culture was inoculated at a rate of 1% into 50 mL shake flasks containing 10 mL of LB medium and cultured under blue light and in darkness, respectively. The blue light irradiance was 161.6 K (mW / cm²). 2 Irradiation was performed at 30W for 24 hours. After 24 hours of culture, samples were collected by centrifugation, the supernatant was discarded, and the bacterial cells were washed twice with 0.9% physiological saline and appropriately diluted. The OD of the cells was measured using a Synergy H1 microplate reader (BioTek, Berton Instrument Co., Ltd., USA). 600 Fluorescence intensity. The relative fluorescence intensity of eGFP was measured at an emission wavelength of 530 nm and an excitation wavelength of 490 nm, with a gain of 100. Fluorescence intensity versus OD 600 The ratio is defined as the expression intensity of the promoter.

[0063] The results show that the relative fluorescence intensity of the strain B. am NBACN(pMA5-EL222-pBLind-eGFP) containing the blue light-activated promoter (pBLind) in the blue light irradiation environment is 1.4 times higher than that in the dark environment, indicating that when there is blue light irradiation, EL222 recruits RNAP under the stimulation of blue light, activates the expression of the eGFP gene. In the absence of blue light, EL222 loses activity, thus reducing the transcription of the target gene. However, the relative fluorescence intensity of the strain B. am NBACN(pMA5-EL222-pBLind-eGFP) in the dark environment is 2 times higher than that of the control strain, and there is a certain leaky expression.

[0064] The recombinant strain B. am NBACN(pMA5-EL222-pBLrep-eGFP) containing the blue light-repressed promoter (pBLrep) has a fluorescence intensity that is 6.1 times lower when cultured in a blue light irradiation environment than when cultured in a dark environment, indicating that under blue light irradiation, EL222 can bind to the promoter region and occupy the space for RNAP binding to prevent transcription initiation.

[0065] This example demonstrates that EL222 can act as a transcription initiation repressor in B. amyloliquefaciens and play a role in inhibiting the expression of target genes in response to blue light. When the EL222 binding region is located upstream of the -35 region of the promoter sequence (pBLind), EL222 acts as a blue light-dependent activator. When the EL222 binding region is located between the -35 and -10 regions of the promoter sequence (pBLrep), EL222 acts as a blue light-dependent inhibitor.

[0066] Example 5 Application of the blue light-responsive gene regulation system under different blue light conditions

[0067] Precise regulation of gene expression plays a crucial role in efficiently improving the synthesis efficiency of target products. In order to verify the parameters of the blue light-regulated gene expression system constructed in B. amyloliquefaciens under different blue light irradiation conditions, two groups of experiments were conducted.

[0068] First group: the positive bacteria B. amyl NBACN(pMA5-EL222-pBLind-eGFP) and B. amyl NBACN(pMA5-EL222-pBLrep-eGFP) constructed in Example 3 were streaked on the resistant plates, incubated overnight, and the control strain B. amyl NBACN was streaked on the LB solid plate without resistance to activate. Single colonies were picked and inoculated into 5 mL LB medium in a small flask, and incubated at 37°C for 12 h; the seed liquid was inoculated into 10 mL LB medium in a 50 mL flask at an inoculation amount of 1%, and incubated under blue light irradiation and in the dark. Samples were taken at 6 h, 12 h, 24 h, 36 h and 48 h of fermentation, and the relative fluorescence intensity was determined. After sampling, the supernatant was removed by centrifugation to collect the bacterial cells, which were washed twice with 0.9% physiological saline and appropriately diluted. The OD 600 and fluorescence intensity of the cells were measured using a Synergy H1 microplate reader (BioTek, Berton Instrument Co., Ltd., USA). The fluorescence intensity of eGFP was measured at an emission wavelength of 530 nm and an excitation wavelength of 490 nm, and the gain value was 100. The ratio of fluorescence intensity to OD 600 was defined as the expression strength of the promoter.

[0069] Second group: the positive bacteria B. amyl NBACN(pMA5-EL222-pBLind-eGFP) and B. amyl NBACN(pMA5-EL222-pBLrep-eGFP) constructed in Example 3 were streaked on the resistant plates, and the control strain B. amyl NBACN was streaked on the LB solid plate without resistance to activate and incubated for 12 h. Single colonies were picked and inoculated into 5 mL LB medium in a small flask, and incubated at 37°C for 12 h; the seed liquid was inoculated into 10 mL LB medium in a 50 mL flask at an inoculation amount of 1%, and the illumination pulse on-off period was changed in a constant 60-minute cycle, and the percentage of time control is indicated in the brackets: 60 minutes off (0%), 5 minutes on; 55 minutes off (8.33%), 15 minutes on; 45 minutes off (25%), 30 minutes on; 30 minutes off (50%), 45 minutes on; 15 minutes off (75%) and 60 minutes on (100%), to investigate the influence of different blue light illumination conditions on the expression strength of the two systems.

[0070] The results show that in the first group of experiments, the fluorescence intensity of the recombinant bacteria B. amyl NBACN (pMA5-EL222-pBLind-eGFP) containing the blue light activated system increases with the increase of time in the dark. At 24 h, the blue light activated system has the best regulation ability, and under the blue light irradiation, the blue light regulated gene expression system makes the fluorescence intensity increase by 1.4 times. The fluorescence intensity of the blue light inhibited system B. amyl NBACN (pMA5-EL222-pBLrep-eGFP) cultured in the dark environment reaches the highest at 36 h, which is 57254.90, and has an inhibition ability of about 10 times compared with that in the blue light environment.

[0071] In the second group of experiments, the fluorescence expression intensity of the recombinant bacteria B. amyl NBACN (pMA5-EL222-pBLind-eGFP) containing the blue light activated system gradually increases with the increase of the illumination pulse duration. When the blue light pulse on-off period increases from 50% (30 minutes on; 30 minutes off) to 75% (45 minutes on; 15 minutes off), the expression of the blue light activated system rapidly increases, and in this range, the regulation ability of the blue light on the blue light activated system is the most sensitive. When the pulse period further increases from 75% to 100%, the activation gradually saturates. When the blue light pulse on-off period increases from 50% (30 minutes on; 30 minutes off) to 75% (45 minutes on; 15 minutes off), the fluorescence expression intensity of the recombinant bacteria B. amyl NBACN (pMA5-EL222-pBLrep-eGFP) containing the blue light inhibited system rapidly decreases, and in this range, the regulation ability of the blue light on the blue light inhibited system is the most sensitive. As can be seen from the curve fitting model, the induced switching point can be set to 50% of the pulse illumination.

[0072] The above results show that the blue light responsive gene regulation system constructed in the present application can realize the control of the expression of the target gene in Bacillus amyloliquefaciens by changing the blue light irradiation conditions. The blue light inhibited system in Bacillus amyloliquefaciens has a large regulation range (4893.71-57254.90) and strong inhibition ability (about 10 times). At the same time, when the blue light pulse on-off period is from 50% to 75%, the rapid regulation of the expression of the target gene can be realized.

[0073] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.

Claims

1. A gene expression regulatory element that responds to blue light, characterized in that, include: Repressor proteins and promoters that respond to blue light; The repressor protein is located upstream of the promoter that responds to blue light; The nucleotide sequence of the blue light-responsive promoter is shown in SEQ ID NO.2; The repressor protein is composed of a constitutive promoter. P HpaII Expression; the blue light-responsive promoter contains a binding sequence of the repressor protein, as shown in SEQ ID NO.4; the blue light-responsive promoter regulates the expression of the target gene; the regulation includes activation, promotion, or inhibition; the constitutive promoter P HpaII The nucleotide sequence is shown in SEQ ID NO.

5.

2. A recombinant expression vector containing the gene regulatory expression element of claim 1, characterized in that, The recombinant expression vector allows for the insertion of the target gene and enables its expression in microbial cells.

3. The recombinant expression vector according to claim 2, characterized in that, The expression vector includes plasmid pMA5.

4. The application of the gene regulatory expression element of claim 1, or the recombinant expression vector of claim 2, in regulating the expression of the target protein.

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