Chicken klf2 gene promoter and construction method and application thereof

Abstract

The application discloses a chicken KLF2 gene promoter and a construction method and application thereof, and belongs to the technical field of biology.The chicken KLF2 gene promoter comprises KLF2-A, KLF2-B, KLF2-C or KLF2-D, and the nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.4 in sequence.The activity of the chicken KLF2 gene promoter is regulated by a transcription factor CEBPZ, and overexpression of CEBPZ promotes chicken KLF2 expression, which is consistent with the KLF2 promoter reporter gene result provided by the application.The chicken KLF2 gene promoter provided by the application not only has application value for revealing the regulation mechanism of chicken KLF2 transcription and expression, verifying a high-quality broiler breeding scheme targeting the expression level of chicken KLF2, and developing a new eukaryotic protein expression system, but also can be used for screening drugs targeting KLF2 expression at a cell level.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a chicken KLF2 gene promoter, its construction method, and its application. Background Technology

[0002] Krüppel-like factors (KLFs) are a family of transcription factors with three C2H2 zinc finger structures at their carboxyl terminus. They are named for the high homology between their zinc finger domains and the Drosophila segmentation factor Krüppel. Currently, 17 KLF family members have been identified in animals, widely involved in various life processes such as embryonic development, cell differentiation, and metabolic regulation.

[0003] Krüppel-like factor 2 (KLF2, also known as lung-type KLF / LKLF) was first discovered in lung tissue and is an important member of the KLF family, involved in regulating important biological processes such as T cell maturation, endothelial cell function, and adipocyte differentiation. Recent studies have confirmed that KLF2 is also expressed in various pluripotent stem cells, and its expression level exhibits dynamic changes during embryonic development: expression begins in mouse embryos on day 7, decreases on day 11, and then significantly increases again on day 15. KLF2 knockout can lead to embryonic lethality in mice, primarily due to its association with cardiovascular developmental abnormalities such as vascular developmental defects and heart failure.

[0004] Chickens are domesticated poultry with extremely high economic value. Through long-term selective breeding, modern commercial broilers have achieved significant improvements in growth rate and feed conversion ratio. However, excessive selection for growth traits has led to excessive abdominal fat deposition, becoming a major problem in the broiler industry. Excessive abdominal fat accumulation not only reduces production efficiency and wastes feed but also increases the sudden mortality rate of broilers and causes environmental pollution. Previous research by our group has shown that the KLF2 gene is highly expressed in the abdominal adipose tissue, pancreas, and spleen of chickens; in the abdominal fat of high-fat broilers at 3, 5, and 8 weeks of age, the mRNA level of KLF2 was significantly higher than that of low-fat broilers. In vitro, during the differentiation of chicken preadipocytes, the mRNA expression of KLF2 gradually decreased with the differentiation process, while the protein level showed a trend of first increasing and then decreasing. Functional studies showed that KLF2 can inhibit the promoter activity of the PPARγ gene, while upregulating the transcription of GATA2, KLF7, and CEBPZ, inhibiting the differentiation of chicken preadipocytes and promoting their proliferation. In conclusion, KLF2 plays an important role in the regulation of fat traits in broilers and may be a key gene for controlling excessive abdominal fat deposition in broilers.

[0005] Promoters are core cis-regulatory elements that control gene transcription initiation, expression timing, tissue specificity, and expression intensity, playing a vital role in gene expression regulation and synthetic biology research. Elucidating the sequence characteristics and transcriptional regulatory function of the chicken KLF2 gene promoter is crucial for elucidating the KLF2 expression regulation mechanism and establishing precise strategies for regulating KLF2 expression, holding significant theoretical and applied implications for high-quality broiler breeding. Summary of the Invention

[0006] The purpose of this invention is to provide a chicken KLF2 gene promoter, its construction method, and its applications, to address the problems existing in the prior art. This invention provides four chicken KLF2 gene promoters of different lengths, all of which have been verified to possess promoter activity and are regulated by the specific transcription factor CEBPZ. This invention has application value for revealing the transcriptional regulatory mechanism of chicken KLF2, verifying high-quality broiler breeding programs targeting chicken KLF2 expression levels, and developing new eukaryotic protein expression systems.

[0007] To achieve the above objectives, the present invention provides the following solution: The present invention provides a chicken KLF2 gene promoter, wherein the chicken KLF2 gene promoter includes KLF2-A, KLF2-B, KLF2-C or KLF2-D, and the nucleotide sequences are shown in SEQ ID NO.1-SEQ ID NO.4 respectively.

[0008] Optionally, the transcription factor CEBPZ regulates the activity of the chicken KLF2 gene promoter.

[0009] The present invention also provides a primer set for amplifying the chicken KLF2 gene promoter, the primer set comprising the upstream primer shown in SEQ ID NO. 6 and the downstream primer shown in SEQ ID NO. 11 for amplifying KLF2-A, the upstream primer shown in SEQ ID NO. 7 and the downstream primer shown in SEQ ID NO. 11 for amplifying KLF2-B, the upstream primer shown in SEQ ID NO. 8 and the downstream primer shown in SEQ ID NO. 11 for amplifying KLF2-C, or the upstream primer shown in SEQ ID NO. 9 and the downstream primer shown in SEQ ID NO. 11 for amplifying KLF2-D.

[0010] The present invention also provides a recombinant vector comprising the chicken KLF2 gene promoter described above.

[0011] The present invention also provides a recombinant bacterium, comprising the aforementioned recombinant vector.

[0012] The present invention also provides a kit comprising the aforementioned primer set.

[0013] The present invention also provides a method for constructing the chicken KLF2 gene promoter, including the step of performing PCR amplification using the primer set to obtain the chicken KLF2 gene promoter.

[0014] This invention also provides the application of the chicken KLF2 gene promoter in any of the following: (1) Application in initiating the expression of firefly luciferase in eukaryotic cells; (2) Application in genetic breeding targeting chicken KLF2 gene expression; (3) Application in the preparation of drugs or feed additives that target chicken KLF2 gene expression.

[0015] Optionally, the eukaryotic cells include chicken embryo fibroblasts.

[0016] Optionally, the genetic breeding includes breeding low-fat broiler chickens.

[0017] The present invention discloses the following technical effects: The autonomous promoters of the chicken KLF2 gene disclosed in this invention were obtained by PCR amplification using DNA extracted from whole chicken blood as a template, yielding five predicted chicken KLF2 promoter sequences (SEQ ID NO.1-SEQ ID NO.5), named KLF2-A, KLF2-B, KLF2-C, KLF2-D, and KLF2-E, respectively. These five promoters were then combined with the pGL4.10 vector to construct five luciferase reporter gene plasmids containing chicken KLF2 promoter sequences (KLF2-A, KLF2-B, KLF2-C, KLF2-D, and KLF2-E) of different lengths using recombination technology. Luciferase activity analysis showed that the chicken KLF2 promoters KLF2-A, KLF2-B, KLF2-C, and KLF2-D possessed autonomous promoter activity in chicken embryo fibroblast DF-1 cells. Further co-transfection analysis of candidate regulatory gene overexpression vectors showed that CEBPZ has a significant regulatory effect on the chicken KLF2 promoter. In vitro cell-level western blot and real-time PCR analyses showed that overexpression of CEBPZ promotes chicken KLF2 expression, consistent with the results of the KLF2 promoter reporter gene provided in this invention.

[0018] In summary, this invention provides a set of promoters for the chicken KLF2 gene, which can be used to develop a reporter gene system targeting the detection of chicken KLF2 gene expression levels and can express the protein in eukaryotic cells. The chicken KLF2 promoters provided by this invention are not only valuable for elucidating the regulatory mechanism of chicken KLF2 transcriptional expression, validating high-quality broiler breeding programs targeting chicken KLF2 expression levels, and developing new eukaryotic protein expression systems, but may also be used for screening drugs targeting KLF2 expression at the cellular level. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 A schematic diagram of the construction of autonomous promoter luciferase reporter gene vectors for five chicken KLF2 genes and a physical map of the location of related sequences; where LUC refers to the firefly luciferin gene; Figure 2 This is an agarose gel electrophoresis image after PCR amplification of the predicted promoter DNA sequence of the chicken KLF2 gene. Lane M is the DNA marker. Lanes 1, 2, 4, and 5 are four different lengths of the cloned and purified predicted promoter DNA fragments of chicken KLF2 (KLF2-A, KLF2-B, KLF2-D, and KLF2-E; theoretical sizes are 1580+42bp, 1250+42bp, 251+42bp, and 131+42bp, respectively). Lane 3 is the cloned and purified KLF2-C, which did not show a clear band due to its low concentration. Lane 6 is the cloned and purified KLF2-C with increased concentration, with a theoretical size of 892+42bp. Figure 3 This is a diagram of the empty vector pGL4.10 plasmid used to construct the KLF2 gene promoter luciferase reporter gene; Figure 4 This image shows the enzyme digestion identification of the chicken KLF2 promoter luciferase reporter gene vector after successful construction; M represents the DNA marker, and lanes 1, 2, 3, 4, and 5 are the recombinant pGL4.10 plasmids digested with Xho I: pGL4.10-KLF2-A, pGL4.10-KLF2-B, pGL4.10-KLF2-C, pGL4.10-KLF2-D, and pGL4.10-KLF2-E, respectively. Figure 5 This is a diagram showing the analysis of the activity of the firefly luciferase reporter gene in the autonomous promoter of the chicken KLF2 gene; among them, the pGL4.10 plasmid without the promoter sequence is the negative control, and the pGL3-promoter is the positive control. This indicates that the group is significantly different from the negative control. P<0.05, P<0.01, P<0.001; Figure 6This is a graph showing the relative activity of the chicken KLF2 gene autonomous promoter luciferase reporter gene; cells transfected with the pGL4.10 plasmid (which does not contain the promoter sequence) serve as the negative control group, while cells transfected with the pGL3-promoter plasmid serve as the positive control group. This indicates that the group is significantly different from the negative control. P<0.05, P<0.01, P<0.001; Figure 7 To analyze DF1 cells transfected with pCMV-myc empty plasmid (EV) and overexpression plasmids pCMV-myc-GATA2 (GATA2), pCMV-myc-GATA3 (GATA3), pCMV-myc-KLF3-f1 (KLF3-f1), pCMV-myc-KLF3-f2 (KLF3-f2), pCMV-myc-KLF7 (KLF7), and pCMV-myc-CEBPZ (CEBPZ) using myc-tag antibody and β-actin antibody, M represents the protein exposure marker, and the arrows indicate the target band. Figure 8 The effect of overexpression of multiple candidate genes on the activity of the KLF2-A reporter gene in DF-1 cells is shown in the figure. Figure 9 The effect of transfection with the plasmid overexpressing CEBPZ (pCMV-myc-CEBPZ) on the activity of three different lengths of KLF2 gene promoters (KLF2-B, KLF2-C, and KLF2-D); EV represents DF1 cells transfected with pCMV-myc, and CEBPZ represents DF1 cells transfected with pCMV-myc-CEBPZ. This indicates a significant difference compared to DF1 cells transfected with empty pCMV-myc (EV). P<0.01, P<0.001; Figure 10 Real-time PCR analysis was performed to determine the level of KLF2 transcription promoted by CEBPZ overexpression in DF1 cells. EV represents DF1 cells transfected with pCMV-myc, and CEBPZ represents DF1 cells transfected with pCMV-myc-CEBPZ. P<0.05; Figure 11Western blotting analysis was performed to determine the protein levels of KLF2 in cells overexpressed with CEBPZ. M represents the protein exposure marker, EV indicates DF1 cells transfected with pCMV-myc, and CEBPZ indicates DF1 cells transfected with pCMV-myc-CEBPZ. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1: Preparation of the chicken KLF2 gene promoter 1. Extraction of genomic DNA from chicken blood 1.1 Reagent Preparation 1) Phenol:chloroform:isoamyl alcohol = 25:24:1 (prepared according to volume ratio).

[0027] 2) Chloroform:Isoamyl alcohol = 24:1 (prepared by volume ratio).

[0028] 3) 20 mg / mL proteinase K: Add 200 mg of proteinase K to 9.5 mL of double-distilled water, shake gently until proteinase K is completely dissolved, bring the volume up to 10 mL, and then aliquot and store at -20 °C.

[0029] 4) 10% SDS (Sodium Dodecyl Sulfate): Weigh 100g of SDS and slowly transfer it to a beaker containing 0.9L of double-distilled water. Heat to 68℃ and stir with a magnetic stirrer until completely dissolved. If necessary, adjust the pH to 7.2 with 1mol / L NaOH, and bring the volume to 1L with double-distilled water. Store at room temperature; sterilization is not required.

[0030] 5) Preparation of STE: 10 mmol / L Tris-HCl (pH 8.0), 0.1 mol / L NaCl, 1 mmol / L EDTA (pH 8.0), autoclave at 121℃ for 15 min, and store at 4℃.

[0031] 6) Preparation of 1 mol / L dithiothreitol standard (DTT): Add 5 g of dithiothreitol to 32.4 mL of deionized water, divide into small portions and store at -20℃.

[0032] 1.2 DNA Extraction 1) Collect about 1-2 mL of blood from the chicken wing vein and inject it into a centrifuge tube containing EDTA anticoagulant. Immediately invert and mix gently to prevent coagulation. Then transfer the anticoagulated blood to a 1.5 mL centrifuge tube and take 50-200 μL of whole blood.

[0033] 2) Add 0.5 mL STE, 50 μL 10% SDS and 20 μL proteinase K (20 mg / mL), then add 20 μL DTT (1 mol / L), and digest overnight in a 55°C water bath.

[0034] 3) Remove from the water bath, cool to room temperature, add 500 μL of Tris-saturated phenol, invert and mix for 10 min, then centrifuge at 15000 r / min for 10 min. Use a wide-mouth pipette (0.3 cm diameter) to transfer the aqueous phase to a 1.5 mL EP tube; do not aspirate the protein layer; extract once more with Tris-saturated phenol and collect the aqueous phase.

[0035] 4) Add 500 μL of phenol:chloroform:isoamyl alcohol (25:24:1) to the aqueous phase collected in step 3), mix by inversion for 10 min, then centrifuge at 15000 r / min for 10 min, and transfer the water-absorbing layer to a 1.5 mL EP tube; 5) Add 2 volumes of anhydrous ethanol to the aqueous phase obtained in step 4), invert and wash the white precipitate, centrifuge at 12000 r / min for 5 min, and discard the liquid.

[0036] 6) Add 1 mL of 70% ethanol to the white precipitate obtained in step 5), wash, centrifuge at 12000 r / min for 5 min, and discard the supernatant.

[0037] 7) Open the lid and let it air dry for 15 min, add 50-100 μL TE (pH 8.0), dissolve at 55℃ for 10 min, and store at -20℃ for later use.

[0038] 2. Cloning of the chicken KLF2 gene promoter region according to Figure 1 Following the design principle, primer combinations were designed as shown in Table 1. Using extracted chicken blood DNA as a template, five predicted chicken KLF2 promoter sequences (KLF2-A, KLF2-B, KLF2-C, KLF2-D, and KLF2-E, nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.5) were amplified by PCR using the primer combinations shown in Table 1. The theoretical lengths were 1622 bp (1580+42bp, the 42bp after the plus sign is the introduced homologous arm), 1292 bp (1250+42bp), 934 bp (892+42bp), 293 bp (251+42bp), and 173 bp (131+42bp), respectively. The PCR reaction system is shown in Table 2, and the PCR reaction conditions are shown in Table 3.

[0039] Table 1 Primer combinations used for amplifying the chicken KLF2 promoter. Table 2. PCR reaction system used for amplifying the chicken KLF2 promoter Table 3. PCR reaction conditions for amplifying the chicken KLF2 promoter. The PCR products were subjected to agarose gel electrophoresis, and the target band was recovered using the AXYGEN gel purification kit. The results of agarose gel electrophoresis of the recovered and purified target band are shown below. Figure 2 As shown, lanes 1, 2, 4, and 5 represent four different lengths of the predicted chicken KLF2 promoter DNA fragments after cloning and purification (KLF2-A, KLF2-B, KLF2-D, and KLF2-E; theoretical sizes of 1580+42bp, 1250+42bp, 251+42bp, and 131+42bp, respectively). Lane 3 shows the cloned and purified KLF2-C, which did not show a clear band due to its low concentration. Lane 6 shows the cloned and purified KLF2-C with an increased concentration and a theoretical size of 892+42bp. It is evident that the sizes of all recovered and purified predicted promoter fragments meet the design expectations.

[0040] 3. Construction of chicken KLF2 promoter luciferase reporter gene vector Using the pGL4.10 carrier purchased from PROMEGA as the skeleton (e.g.) Figure 3 (As shown) Construct a chicken KLF2 promoter luciferase reporter gene vector.

[0041] First, the pGL4.10 plasmid was digested with the restriction endonuclease XhoI. The digestion system is detailed in Table 4. The digestion conditions were: 37℃ for 2 h. The digested pGL4.10 plasmid was then subjected to agarose gel electrophoresis. The XhoI-digested pGL4.10 plasmid was recovered using the AXYGEN gel purification kit to obtain the linearized pGL4.10 fragment.

[0042] Table 4. Enzyme digestion system used for linearization of pGL4.10 plasmid Using the linearized pGL4.10 fragment and the recovered and purified chicken KLF2 promoter fragment, the homologous recombination reaction system shown in Table 5 was prepared on ice according to the instructions of the Vazyme OneStep Cloning Kit.

[0043] Table 5. Plasmid Recombination System for Chicken KLF2 Promoter Luciferase Reporter Gene Construction The recombinant system was reacted at 37℃ for 30 min to complete plasmid recombination. After obtaining the recombinant plasmid, it was transformed into Escherichia coli JM109 competent cells. The transformed competent cells were then seeded onto LB solid medium containing Amp resistance for resistance selection culture.

[0044] After culturing bacteria at 37°C for 12 hours, single colonies were picked and cultured in LB liquid medium containing 1% Amp. After 10 hours of culture, bacteria were collected. Plasmids were extracted using the AXYGEN plasmid miniprep kit, and the recovered plasmids were digested with Xho1 at 37°C for 1 hour. The digestion system was analyzed by agarose gel electrophoresis. The electrophoresis results are shown below. Figure 4 As shown, the digested plasmid released a 4200bp DNA fragment (expected size of the empty vector) and a DNA fragment of the corresponding size. After confirming the enzyme digestion was correct, Sanger sequencing of the plasmid showed that all five successfully constructed chicken KLF2 luciferase reporter gene vectors successfully inserted the corresponding DNA fragments, indicating that the chicken KLF2 promoter luciferase reporter gene vectors were successfully constructed.

[0045] SEQ ID NO.1 (KLF2-A): SEQ ID NO.2(KLF2-B): SEQ ID NO.3(KLF2-C): CGTTGGTTTGGGCGCAGCAAATGGGAACTGCCGACCCCTGAGCTCCCACTGCACACACAGCTCTTCCCGCAGGGAGACGGCCGGGAGGCACCGGGCAGCTCTGCGCTGCCGTGGGGCATCGCGGGGAGGGCAGAGCAGCGCCCGCACCCTGCCCCGTCAGCTCACACTCTGCTTCTGGGGTCGGGGCTGAGGCCGGCCGGGAGGGGATCAAACCCCCCACTCCCACCTCGCTGGGACCAGCGGCTTCCCGCCGCGCTGAGGGTCCCTCGCGGTGTGTGCGGGCCGTGGGCCCACCGCCGTCACCCCGCGTCACGGACGGTCCCCCCACACTCCTCGACCCCAACTCCCACGGGGATCCTCGCGGCGCCCCCAGGACGGGGGTCCCTCACATCGCCCCTTCGCAGTGCGCACAGACGCGGACCCATCAGTGCCCCCAGCCCCGGGACCCCCGGTCCGAGGTGTGGGGGGAGGGAGGGAAAGGGGGGGGGTCCCGGCTCCGCTCCCCGCGCTGGGGGGGGGGCGGGGGGGGGGCGGTCAGGGGCCGCCTTTGGGCCCCGCTCCGCTCCGCCCCGCTCGGGCTGCGGCCGCCGCCGCCGCGTCCTTTTATACCGGGGCTAAATTTAGGCTGCGCCCGAGCCGCTGCCTCCCCGCCCGCCCCGTCCGGGACGCGTTTCCTCCGCTCCCTGCAATTGGCCTGAGGGCGCCCGCGCCCGCGTTATAAGGTGGGAGGGCGGCTCTGCGCGCCGCGTCCCGCCCGGCCGCACCATGGCGCTGAGCGATACCATCCTGCCCTCCTTCGCCACCTTCGCCAGTCCCTGCCGCGAGAAAGCGCTCCACGAAGTGAGTGCGGCCCCGGCCCGGCTGAGCCCCGCTGAGCCCCCGCGGCTCTGCC; SEQ ID NO.4(KLF2-D): GCCTCCCCGCCCGCCCCGTCCGGGACGCGTTTCCTCCGCTCCCTGCAATTGGCCTGAGGGCGCCCGCGCCCGCGTTATAAGGTGGGAGGGCGGCTCTGCGCGCCGCGTCCCGCCCGGCCGCACCATGGCGCTGAGCGATACCATCCTGCCCTCCTTCGCCACCTTCGCCAGTCCCTGCCGCGAGAAAGCGCTCCACGAAGTGAGTGCGGCCCCGGCCCGGCTGAGCCCCGCTGAGCCCCCGCGGCTCTGCC; SEQ ID NO. 5 (KLF2-E): CACCATGGCGCTGAGCGATACCATCCTGCCCTCCTTCGCCACCTTCGCCAGTCCCTGCCGCGAGAAAGCGCTCCACGAAGTGAGTGCGGCCCCGGCCCGGCTGAGCCCCGCTGAGCCCCCGCGGCTCTGCC; SEQ ID NO. 6 (KLF2-A forward primer): cctgagctcgctagcctcgagGTACTTCGTTACCATGCATTCAGTG; SEQ ID NO. 7 (KLF2-B forward primer): ctgagctcgctagcctcgagGATCGCAGAGGAAATATGGAGG; SEQ ID NO. 8 (KLF2-C forward primer): cctgagctcgctagcctcgagCGTTGGTTTGGGCGCAGC; SEQ ID NO. 9 (KLF2-D forward primer): cctgagctcgctagcctcgagGCCTCCCCGCCCGCCCCG; SEQ ID NO. 10 (KLF2-E forward primer): cctgagctcgctagcctcgagCACCATGGCGCTGAGCGA; SEQ ID NO. 11 (reverse primer): ccagatcttgatatcctcgagGGCAGAGCCGCGGGGGCT.

[0046] Example 2: Analysis of Chicken KLF2 Gene Promoter Activity Chicken embryo fibroblasts (DF-1) in good growth condition were seeded into ONOBATE 24-well cell culture plates at a seeding density of 2.5 × 10⁻⁶. 4 Cells were transfected per well. After 24 h, the empty pGL4.10 plasmid, pGL3-promoter, and pGL4.10 luciferase reporter gene plasmids of five recombinant chicken KLF2 gene promoters (KLF2-A, KLF2-B, KLF2-C, KLF2-D, and KLF2-E) were transfected into cells according to the Vazyme ExFect Transfection Reagent manufacturer's instructions. Cells transfected with the empty pGL4.10 plasmid served as the negative control, and cells transfected with the pGL3-promoter plasmid served as the positive control. Each well was transfected with 500 ng of firefly luciferase reporter gene plasmid and simultaneously with 45 ng of pRL-TK plasmid as an internal control. Each group was repeated at least three times. Cells were harvested 48 h after transfection and transfected using Promega's Dual-Globe assay. ® The Luciferase Assay System reagent was used to determine luciferase activity.

[0047] The results are as follows Figure 5 As shown, compared with the negative control, DF1 cells transfected with four of the five chicken KLF2 gene promoter luciferase reporter gene plasmids (pGL4.10-KLF2-A, pGL4.10-KLF2-B, pGL4.10-KLF2-C, and pGL4.10-KLF2-D) exhibited significantly increased luciferase activity (P<0.05, two-tailed unpaired). t (Test), but DF1 cells transfected with pGL4.10-KLF2-E did not show a significant increase in luciferase activity compared to the negative control (P>0.05, two-tailed unpaired). t test).

[0048] The relative luciferase activity was analyzed, and the results are as follows: Figure 6 As shown, cells transfected with pGL4.10-KLF2-A, pGL4.10-KLF2-B, pGL4.10-KLF2-C, and pGL4.10-KLF2-D all exhibited significantly increased relative luciferase activity compared to the negative control (P<0.05, two-tailed unpaired). tThe test showed that the chicken KLF2 promoter sequences KLF2-A, KLF2-B, KLF2-C, and KLF2-D possess autonomous promoter activity. DF1 cells transfected with pGL4.10-KLF2-E did not show a significant increase in relative luciferase activity compared to the negative control (P>0.05, two-tailed unpaired). t The test results indicate that the KLF2-E promoter does not exhibit significant autonomous promoter activity.

[0049] Example 3: Effect of transcription factor CEBPZ on chicken KLF2 gene promoter activity Chicken embryo fibroblasts (DF-1) in good growth condition were inoculated at a density of 2.5 × 10⁶ cells / year. 4 / Wells were passaged in ONOBATE 24-well cell culture plates. After 24 hours of passage, chicken embryo fibroblast DF-1 cells were co-transfected with chicken GATA2, chicken GATA3, chicken KLF7, chicken KLF3 protein 347 aa subtype KLF3-f1 (NCBI reference protein sequence: XP_015141159.1), chicken KLF3 protein 335 aa subtype KLF3-f2 (NCBI reference protein sequence: XP_046773224.1), chicken CEBPZ overexpression plasmid, and pCMV-myc empty vector (EV) and chicken KLF2 promoter A reporter gene plasmid (pGL4.10-KLF2-A) using Vazyme ExFect Transfection Reagent according to the transfection system shown in the table. Cells were collected 48 hours after transfection, and Western blot results are shown in the table. Figure 7 As shown, 48 h after transfection with each overexpression plasmid, the expression level of the corresponding protein of the myc tag in DF1 cells was significantly higher than that in cells transfected with the pCMV-myc empty vector (EV).

[0050] Use Dual-Glo ® The Luciferase Assay System kit was used to detect luciferase activity, and the results are as follows: Figure 8 As shown, compared with the empty vector group (EV), the CEBPZ overexpression plasmid significantly promoted the activity of the chicken KLF2 promoter in chicken embryo fibroblast DF-1 cells (P<0.05). Overexpression of both KLF3 proteins inhibited the activity of the KLF2 promoter in cells (P<0.05). Overexpression of GATA2 or GATA3 did not significantly change the activity of the KLF2 promoter in cells (P>0.05). KLF7 overexpression promoted the activity of the KLF2 promoter, but the promoting effect was not as high as that of CEBPZ overexpression.

[0051] Table 6. Transfection systems for screening the effects of multiple candidate transcription factors on chicken KLF2 promoter activity. CEBPZ was further investigated as a candidate factor for regulating KLF2 promoter activity. The regulatory effect of CEBPZ overexpression on the activity of three other autonomous KLF2 promoters (KLF2-B, KLF2-C, and KLF2-D) was studied using luciferase reporter gene technology. First, well-grown chicken embryo fibroblasts (DF-1) were seeded at a density of 2.5 × 10⁻⁶ cells / year. 4 Cells were passaged in ONOBATE 24-well cell culture plates. After 24 hours of passage, when cell confluence reached 70%-80%, transfection was performed. Before transfection, the medium was replaced with fresh complete culture medium. The transfection system was prepared using Vazyme ExFect Transfection Reagent according to Table 7. After preparation, the cells were gently mixed by pipetting and allowed to stand at room temperature for 20 minutes. The transfection complex was then added evenly to each well and gently mixed. After culturing for another 48 hours, cells were collected and transfected using Dual-Globe Imaging. ® The Luciferase Assay System kit was used to detect luciferase activity, with three replicates per group. Results showed that, compared to the empty vector group, overexpression of CEBPZ significantly activated the activity of the chicken KLF2 gene promoters KLF2-B and KLF2-C. Figure 9 However, the effect of CEBPZ overexpression on the activity of the KLF2 promoter KLF2-D was not significantly different from that of the control group (P>0.05, two-tailed unpaired). t test).

[0052] Table 7. Transfection systems for regulating chicken KLF2 promoter activity using CEBPZ. The effect of transfecting chicken CEBPZ-overexpressing plasmid (pCMV-myc-CEBPZ) into chicken embryo fibroblast DF-1 cells for 48 h on KLF2 expression levels was further investigated using real-time PCR and Western blot. The results of real-time PCR analysis are as follows: Figure 10 As shown, the KLF2 expression level in chicken embryo fibroblast DF-1 cells overexpressing CEBPZ was significantly higher than that in the empty vector (pCMV-myc, EV) group (P<0.05). Western blot results are shown below. Figure 11As shown, 48 hours after transfection with pCMV-myc-CEBPZ plasmid, the expression levels of CEBPZ and KLF2 in DF1 cells were significantly higher than those in cells transfected with the empty vector. This indicates that overexpression of CEBPZ in chicken embryo fibroblast DF-1 cells can promote the expression level of intracellular KLF2, which is consistent with the results of the KLF2 promoter reporter gene activity analysis provided in this invention.

[0053] In summary, the chicken KLF2 promoter fragment provided by this invention has eukaryotic promoter activity, and the activity of the reporter gene constructed based on this promoter is consistent with the results of expression analysis of intracellular KLF2 basal transcriptional activity.

[0054] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A chicken KLF2 gene promoter, characterized in that, The chicken KLF2 gene promoter includes KLF2-A, KLF2-B, KLF2-C or KLF2-D, with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.

4.

2. The chicken KLF2 gene promoter as described in claim 1, characterized in that, The transcription factor CEBPZ regulates the activity of the chicken KLF2 gene promoter.

3. A primer set for amplifying the chicken KLF2 gene promoter as described in claim 1 or 2, characterized in that, The primer set includes the upstream primer shown in SEQ ID NO. 6 and the downstream primer shown in SEQ ID NO. 11 for amplifying KLF2-A, the upstream primer shown in SEQ ID NO. 7 and the downstream primer shown in SEQ ID NO. 11 for amplifying KLF2-B, the upstream primer shown in SEQ ID NO. 8 and the downstream primer shown in SEQ ID NO. 11 for amplifying KLF2-C, or the upstream primer shown in SEQ ID NO. 9 and the downstream primer shown in SEQ ID NO. 11 for amplifying KLF2-D.

4. A recombinant vector, characterized in that, Includes the chicken KLF2 gene promoter as described in claim 1 or 2.

5. A recombinant bacterium, characterized in that, Includes the recombinant vector as described in claim 4.

6. A reagent kit, characterized in that, Includes the primer set as described in claim 3.

7. The method for constructing the chicken KLF2 gene promoter as described in claim 1 or 2, characterized in that, The method includes the step of using the primer set described in claim 3 to perform PCR amplification to obtain the chicken KLF2 gene promoter.

8. The use of the chicken KLF2 gene promoter as described in claim 1 or 2 in any of the following: (1) Application in initiating the expression of firefly luciferase in eukaryotic cells; (2) Application in genetic breeding targeting chicken KLF2 gene expression; (3) Application in the preparation of drugs or feed additives that target chicken KLF2 gene expression.

9. The application as described in claim 8, characterized in that, The eukaryotic cells include chicken embryo fibroblasts.

10. The application as described in claim 8, characterized in that, The genetic breeding includes the development of low-fat broiler chickens.

Images