CN113881678B

CN113881678B - C/EBPZ gene promoter and application thereof

Info

Publication number: CN113881678B
Application number: CN202111284259.XA
Authority: CN
Inventors: 张志威; 陈月婵; 高玲羽; 李泽泉
Original assignee: Shihezi University
Current assignee: Shihezi University
Priority date: 2021-11-01
Filing date: 2021-11-01
Publication date: 2024-04-12
Anticipated expiration: 2041-11-01
Also published as: CN113881678A

Abstract

The invention discloses a C/EBPZ gene promoter and application thereof, belonging to the fields of genetic engineering and molecular biology. The C/EBPZ gene promoter is a nucleotide sequence shown in SEQ ID NO:1-SEQ ID NO:6, and any one of the A1-A6 sequences shown in FIG. 6. The method for extracting DNA from chicken whole blood is used as a template, and a primer is used for carrying out PCR amplification and restriction enzyme digestion to obtain 6 chicken C/EBPZ gene promoters, and experiments prove that the 6 promoters have promoter activity in chicken preadipocytes and are regulated and controlled by a specific transcription factor KLF2, so that the method has potential application value for revealing a transcription regulation mechanism of chicken C/EBPZ, culturing low-abdomen fat broilers, revealing molecular mechanisms of various human diseases and developing medicaments for targeting C/EBPZ expression.

Description

C/EBPZ gene promoter and application thereof

Technical Field

The invention relates to the fields of genetic engineering and molecular biology, in particular to a C/EBPZ gene promoter and application thereof.

Background

CCAAT enhancer binding protein zeta (CCAAT/enhancer binding protein zeta, C/EBPZ; gene ID: 10153); also known as CBF, CBF-2, NOC1 and HSP-CBF are proteins that are ubiquitous and highly conserved in humans and animals. Although its name carries C/EBP, in fact it is not an alkaline leucine zipper (bZIP) protein, lacks significant homology to other C/EBP members, and therefore it should not be considered a C/EBP family member.

C/EBPZ was first reported as a CCAAT cassette-bound transcription factor for the human heat shock protein70 (heat shock protein, HSP 70) promoter, which is capable of promoting HSP70 promoter activity by acting on CCAAT cassette elements of the HSP70 promoter. Later studies reported that C/EBPZ might not bind directly to the CCAAT box element on HSP70 promoter through DNA sequence specificity, but rather activate promoter activity of HSP70 through NF-Y factor-generating protein binding to the CCAAT box, C/EBPZ might be a protein scaffold integrating different transcription factors to form transcription regulatory complexes.

Studies in human cells have reported that C/EBPZ is also an mRNA binding protein and nucleolin. In addition, C/EBPZ can be involved in m6A methylation of regulatory RNAs by recruiting METTL3 to the transcription initiation site (Transcriptional Start Site, TSS) of the acute myeloid leukemia (acute myeloid leukemia, AML) target gene.

Genetic studies have shown that C/EBPZ is a repeated mutant gene in AML occurrence and is associated with schizophrenia in humans. Furthermore, comparison of host coronavirus-protein interaction networks shows that C/EBPZ is involved in the viral-human protein interactions that occur in humans for SARS-CoV-1, MERS-CoV and SARS-CoV-2 viruses. In summary, C/EBPZ may be a related gene for the occurrence of various diseases and may have important biological functions, revealing that the expression control mechanism of C/EBPZ has important significance for revealing the physiological functions of human body and the molecular biological mechanism behind various diseases.

Chickens are the most common poultry to be raised by humans and have important economic value. Through the continuous efforts of breeding workers for more than 90 years, the daily gain and the feed conversion rate of the current commercial broilers reach very high levels. However, the current commercial broilers have a problem of excessive abdominal fat accumulation due to excessive selection of growth traits during long-term breeding. The accumulation of excessive abdominal fat not only affects the production efficiency of the broiler chickens and causes feed waste, but also causes sudden death of the broiler chickens and environmental pollution. The cultivation of high-quality low-fat broiler chickens is an effective means for controlling the accumulation of abdominal fat of the broiler chickens. The early-stage research result of the subject group shows that the mRNA expression level of C/EBPZ in the abdominal fat tissue of the broiler with low abdominal fat content is obviously higher than that of the broiler with high abdominal fat content; in vitro cell level studies showed that C/EBPZ is an important regulator of chicken abdominal adipose tissue formation. Regulating the expression level of the C/EBPZ gene in chicken belly adipose tissue may be a potential means of regulating broiler belly fat content.

In conclusion, C/EBPZ is a regulatory gene for the occurrence of various human diseases and a candidate gene for low-fat broiler breeding. At present, no research report of C/EBPZ transcription regulation exists, and the determination of the promoter of chicken C/EBPZ gene has potential application value for revealing the transcription regulation mechanism of chicken C/EBPZ, breeding low-abdomen fat broilers, revealing the molecular mechanism behind various human diseases and developing drugs targeting C/EBPZ expression.

Disclosure of Invention

The invention aims to provide a C/EBPZ gene promoter and application thereof, which are used for solving the problems in the prior art, wherein the promoter has promoter activity in chicken preadipocytes and is regulated and controlled by a specific transcription factor KLF2, and the promoter has potential application value for revealing a transcription regulation mechanism of chicken C/EBPZ, culturing low-belly fat broilers, revealing molecular mechanisms behind various human diseases and developing medicaments for targeting C/EBPZ expression.

In order to achieve the above object, the present invention provides the following solutions:

the invention provides a C/EBPZ gene promoter, which is a nucleotide sequence shown in SEQ ID NO:1-SEQ ID NO:6, and any one of the A1-A6 sequences shown in FIG. 6.

The invention also provides a primer group for amplifying the C/EBPZ gene promoter, wherein the primer group comprises SEQ ID NO for amplifying the A1 sequence: 9 and the upstream primer set forth in SEQ ID NO:8, a downstream primer shown in FIG. 8; amplification of the A2 sequence of SEQ ID NO:10 and the sequence of SEQ ID NO:8, a downstream primer shown in FIG. 8; amplification of the A3 sequence of SEQ ID NO:11 and the sequence of SEQ ID NO:8, a downstream primer shown in FIG. 8; amplification of the A4 sequence of SEQ ID NO:12 and the upstream primer shown in SEQ ID NO:8, a downstream primer shown in FIG. 8; amplification of the A5 sequence of SEQ ID NO:13 and the upstream primer shown in SEQ ID NO:8, a downstream primer shown in FIG. 8; amplification of the A6 sequence of SEQ ID NO:14 and the upstream primer set forth in SEQ ID NO:8, a downstream primer shown in FIG. 8.

The invention also provides a recombinant vector comprising the C/EBPZ gene promoter.

The invention also provides a recombinant bacterium comprising the recombinant vector.

The C/EBPZ gene promoter disclosed by the invention not only refers to SEQ ID NO:1-6, recombinant vectors, recombinant bacteria, transgenic cell lines or expression cassettes containing the above-mentioned DNA fragments are also within the scope of the invention.

The invention also provides a kit comprising the primer group.

The invention also provides a construction method of the C/EBPZ gene promoter, which comprises the following steps:

and (3) taking DNA extracted from chicken whole blood as a template, and carrying out PCR amplification by using the primer group to obtain the C/EBPZ gene promoter.

The invention also provides an application of the C/EBPZ gene promoter, which is applied to any one of the following:

(1) Application to the initiation of firefly luciferase expression in eukaryotic cells;

(2) The method is applied to the genetic breeding of chickens;

(3) The method is applied to preparing drugs or feed additives targeting the expression of the C/EBPZ genes.

Preferably, the eukaryotic cells include chicken preadipocytes.

Preferably, the C/EBPZ gene promoter is under the control of a specific transcription factor KLF 2.

Preferably, the genetic breeding comprises raising low-fat broilers.

The invention discloses the following technical effects:

the C/EBPZ gene promoter disclosed by the invention is 6 promoter sequences obtained by taking DNA extracted from chicken whole blood as a template and carrying out PCR amplification by using a primer. Experiments prove that the 6C/EBPZ promoters have promoter activity in chicken preadipocytes, so that each promoter can directly regulate and control chicken C/EBPZ transcriptional expression, and over-express KLF2 promotes the activity of all 6 chicken C/EBPZ promoters. The mutant reporter plasmid pGL4.10-C/EBPZ (B6) is obtained by mutating the KLF2 binding site CGGGCGCTGCG on the luciferase reporter plasmid pGL4.10-C/EBPZ (A6) to CGGtatCTGCG by adopting a site-directed mutagenesis technology, and compared with the wild-type luciferase reporter plasmid pGL4.10-C/EBPZ (A6), the mutant reporter plasmid overexpresses KLF2 to obviously reduce the promoter promoting activity of the mutant C/EBPZ luciferase reporter plasmid pGL4.10-C/EBPZ (B6), thus indicating that CGGGCGCTGCG is a potential KLF2 acting site on the C/EBPZ promoter. In a word, the C/EBPZ gene promoter disclosed by the invention has potential application value for revealing a transcription regulation mechanism of chicken C/EBPZ, breeding low-abdomen fat broilers, revealing a molecular mechanism behind various human diseases and developing a medicine for targeting C/EBPZ expression.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of the construction of a C/EBPZ gene promoter reporter gene and a physical map of the localization of related sequences; wherein, LUC refers to firefly luciferin gene;

FIG. 2 is a schematic diagram of pGL4.10 plasmid used as the backbone of the C/EBPZ gene promoter reporter vector;

FIG. 3 is a graph showing analysis of the activity of the firefly luciferase reporter gene of the C/EBPZ gene promoter; wherein pGL4.10 without promoter sequence is a negative control, pGL3-promoter is a positive control, "x" indicates a significant difference (P < 0.01) from pGL 4.10;

FIG. 4 is a graph showing the relative activity of the luciferase reporter gene promoter of the C/EBPZ gene; wherein pGL4.10 without promoter sequence is a negative control group, pGL3-promoter is a positive control group, "x" indicates significant difference (P < 0.01) from pGL 4.10;

FIG. 5 is an identification of expression fusion proteins transfected with pCMV-Myc-GATA2, pCMV-Myc-GATA3, pCMV-Myc-KLF2 and pCMV-Myc-KLF3, respectively, in chicken preadipocytes;

FIG. 6 is an identification of the effect of GATA2, GATA3, KLF2 and KLF3 on the activity of the C/EBPZ gene A1 promoter;

FIG. 7 shows the effect of KLF2 on the activity of promoters of various lengths of the C/EBPZ gene;

FIG. 8 is a graph showing the effect of CGGGCGCTGCG motif mutations in reducing KLF2 overexpression on the promotion of C/EBPZ promoter activity.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

EXAMPLE 1 preparation of chicken C/EBPZ Gene promoter

1. Extraction of chicken genomic DNA

1) Collecting normally cultured 7 week old Ai Ba beneficial additive broiler (AA broiler), collecting blood 1ml by wing vein, adding 10 microliter anticoagulant (such as 0.5mol/L Na) ₂ EDTA) was mixed upside down in a 1.5ml EP tube.

2) Mu.l of anticoagulated chicken blood was taken in a new 1.5ml EP tube, 445. Mu.l of 1 XSET (100 mM Tris-HCl pH7.5, 100mM EDT A, after autoclaving, 1% SDS), 10. Mu.l of PK (10 mg/ml) and 25. Mu.l of SDS (10%) were added, thoroughly mixed and digested for 12 hours at 55 ℃.

3) Adding 500ul Tris saturated phenol, mixing thoroughly, and placing in a shaking table for 10min upside down.

4) Centrifuge at 12000rpm for 10min and pipette the upper layer to a new EP tube.

5) 400 μl of phenol was added: chloroform: isoamyl alcohol (24:23:1 volume ratio), are fully and evenly mixed and are placed in a shaking table to be turned upside down for 10min.

6) Centrifuge at 12000rpm for 10min and pipette the upper layer to a new EP tube.

7) 400 μl chloroform was added: isoamyl alcohol (23:1 volume ratio), are fully and evenly mixed and are placed in a shaking table to be turned upside down for 10min.

8) Centrifuge at 12000rpm for 10min and pipette the upper layer to a new EP tube.

9) Adding 800 μl (2 times volume) of absolute ethanol (-20deg.C pre-cooling), shaking to obtain white flocculent precipitate;

10 8000rpm for 10min, discarding the liquid, and leaving a white precipitate at the bottom

11 1ml of 70% ethanol (-20 ℃ pre-cooling), shaking, and washing the white precipitated DNA;

12 Centrifuging at 8000rpm for 10min, discarding the liquid, and leaving a white precipitate at the bottom; the EP tube was placed upside down on the filter paper until the tube wall was free of beads.

13 200. Mu.l TE (sterilized) was added thereto and dissolved in a water bath at 55 ℃.

2. PCR amplification of C/EBPZ Gene promoter

PCR amplification was performed using the extracted chicken genomic DNA as a template, and the primers C/EBPZ-F (SEQ ID NO:9SEQ ID NO: 14) and C/EBPZ-R (SEQ ID NO: 8).

C/EBPZ-F(SEQ ID NO：9)：

cctgagctcgctagcctcgagAATTGTGTTCAGTATTACATATTTCCCTG；

C/EBPZ-F(SEQ ID NO：10)：cctgagctcgctagcctcgagCATTCTGTTTACCCATGGCCA；

C/EBPZ-F(SEQ ID NO：11)：cctgagctcgctagcctcgagGTCCCCCCTAACGCCTGC；

C/EBPZ-F(SEQ ID NO：12)：cctgagctcgctagcctcgagCGGGGGGAGGAGATGAGC；

C/EBPZ-F(SEQ ID NO：13)：cctgagctcgctagcctcgagGTTACCGCGGTGACAGGGA

C/EBPZ-F(SEQ ID NO：14)：cctgagctcgctagcctcgagGGCGGCATGATCGGCTGC。

C/EBPZ-R(SEQ ID NO：8)：ccagatcttgatatcctcgagCGCCATGGCAGGCTGCAG；

The reaction system is shown in Table 1:

TABLE 1 reaction system

The PCR reaction conditions were as shown in Table 2:

TABLE 2 reaction conditions

And (3) carrying out agarose gel electrophoresis on the PCR product, and recovering a target strip for later use by using an AXYGEN gel recovery and purification kit.

3. Construction of C/EBPZ promoter luciferase reporter vector

The pGL4.10 vector was used as a substrate for cleavage with restriction enzymes, and Xho I was used as an cleavage system, as shown in Table 3:

TABLE 3 enzyme digestion system

The enzyme digestion conditions are as follows: and the enzyme digestion is carried out for 1h at 37 ℃. A linearized pGL4.10 vector is obtained which can be used for the construction of recombinant plasmids.

The system was formulated on ice using the Vazyme One Step Cloning Kit kit according to the instructions and is as shown in Table 4:

table 4 formulation reagents

The reaction system reacts for 30min at 37 ℃ to complete plasmid recombination, a luciferase reporter gene body containing 6C/EBPZ promoters of A1-A6 shown in figure 1 is constructed, the 6 recombinant plasmids take pGL4.10 vectors purchased from PROMEGA company as a framework (shown in figure 2), 6C/EBPZ promoter sequences of A1-A6 are introduced through homologous recombination, the construction is successful, and the insertion sequence is shown in sequence table SEQ ID NO:1 to SEQ ID NO:6. the recombinant product is transformed into competent cells of escherichia coli JM109, the AMP resistance is selected and cultured, after the monoclonal is selected, the monoclonal is inoculated into LB liquid medium added with 1% AMP for culturing for 12 hours, and after the culture is performed for 12 hours, plasmid is extracted by adopting an AXYGEN plasmid extraction kit, and the plasmid is stored for standby after sequencing verification.

EXAMPLE 2C/EBPZ Gene promoter Activity assay

1. Activity verification of six different-length C/EBPZ Gene promoters (A1-A6)

Inoculating chicken preadipocytes with good growth state into 12-hole cell culture plate with inoculation density of 5×10 ⁴ After 24h each well, 6 wild-type C/EBPZ promoter (A1-A6) reporter plasmids were transfected into chicken preadipocytes as experimental groups according to the instructions according to Fugene HD (Promega) transfection reagent, pGL4.10 (empty vector, EV) as negative control group, pGL3-promoter as positive control group, 1. Mu.g plasmid per well, three cell wells, pRL-TK as internal control, 0.02. Mu.g per well, cells were collected after 48h transfection according to Promega companyLuciferase Assay System, the results of which showed that the constructed 6 wild-type C/EBPZ promoter (A1-A6) reporter plasmids had promoter activity (FIGS. 3 and 4).

2. Verification of transcription factors GATA2, GATA3, KLF2 and KLF3 over-vector expression proteins

Inoculating chicken preadipocytes with good growth state into 6-hole cell culture plate with inoculation density of 1×10 ⁵ Eukaryotic expression vectors pCMV-Myc-GATA2, pCMV-Myc-GATA3, pCMV-Myc-KLF2 and pCMV-Myc-KLF3 were transfected into chicken preadipocytes, respectively, 24h after each well, 2.0. Mu.g of plasmid was transfected per well, cells were collected after 48h of transfection, and the transfection was verified by Western Blot techniqueWhether somatic chicken preadipocytes express Myc-tagged fusion proteins indicated that transfection of pCMV-Myc-GATA2, pCMV-Myc-GATA3, pCMV-Myc-KLF2 and pCMV-Myc-KLF3 successfully expressed chicken GATA2, GATA3, KLF2 and KLF3 fusion proteins in chicken preadipocytes (FIG. 5).

1. Influence of transcription factors GATA2, GATA3, KLF2 and KLF3 on the activity of the C/EBPZ gene promoter (A1).

Inoculating chicken preadipocytes with good growth state into 12-hole cell culture plate with inoculation density of 5×10 ⁴ After 24h per well, each set of plasmids was transfected into chicken preadipocytes according to Fugene HD (promega) instructions, grouped as shown in table 5 below:

TABLE 5 grouping

Each group was repeated three times, and after 48 hours, the cells were harvested according to Promega CorpLuciferase Assay System, the fluorescence activity was measured. The results show that: overexpression of GATA2, GATA3 and KLF2 promoted C/EBPZ gene promoter (A1) activity (fig. 6), and overexpression of KLF3 inhibited C/EBPZ gene promoter (A1) activity (fig. 5).

5. Prediction of KLF2 binding site on the promoter sequence of the C/EBPZ gene.

Considering that the regulation and control effects of KLF2 on the constructed longest C/EBPZ promoter A1 are most obvious in four transcription factors, the JASPAR database is utilized to predict the binding site of the transcription factor KLF2 on the longest C/EBPZ gene promoter A1 sequence obtained by the invention. The results are shown in Table 6: indicating that the C/EBPZ gene promoter A1 sequence has a plurality of KLF2 transcription factor binding sites. One of them is located within the shortest C/EBPZ gene promoter A6 sequence interval (-50 bp/-40 bp) and thus is present on 6 different length C/EBPZ gene promoters simultaneously.

TABLE 6 predicted results of transcription factor KLF2 binding site on promoter A1 sequence of C/EBPZ gene

6. Effect of transcription factor KLF2 on the activity of promoters (A1-A6) of various lengths of C/EBPZ gene.

Inoculating chicken preadipocytes with good growth state into 12-hole cell culture plate with inoculation density of 5×10 ⁴ After 24h per well, each set of plasmids was transfected into chicken preadipocytes according to Fugene HD (promega) instructions, the groupings being shown in table 7:

TABLE 7 grouping and reagent dosage thereof

Each group was repeated three times, and after 48 hours, the cells were harvested according to Promega CorpLuciferase Assay System, the fluorescence activity was measured. The results show that: overexpression of KLF2 promoted the activity of all 6-clock different length C/EBPZ gene promoters (FIG. 7).

7. Mutation of the binding site (-50 bp/-40 bp) of the transcription factor of KLF2 in the A6 sequence of the C/EBPZ gene promoter

The KLF2 binding site CGGGCGCTGCG in the C/EBPZ promoter (A6) sequence was mutated to CGGtatCTGCG by China Hua big gene company using pGL4.10-C/EBPZ (A6) as a template and site-directed mutagenesis. The mutated sequence was subjected to KLF2 binding site analysis using the JASPAR database. The results show that: the KLF2 binding site on the mutated C/EBPZ promoter (A6) disappeared, the mutated sequence was named C/EBPZ promoter (B6) (nucleotide sequence shown as SEQ ID NO: 7), and the vector was named pGL4.10-C/EBPZ (B6).

8. CGGGCGCTGCG (-50 bp/-40 bp) mediates at least partial promotion of the activity of the C/EBPZ promoter by over-expression of KLF2

Inoculating chicken preadipocytes with good growth state into 12In the cell culture plate, the inoculation density is 5 multiplied by 10 ⁴ After 24h per well, each set of plasmids was transfected into chicken preadipocytes according to Fugene HD (promega) instructions, the groupings being shown in table 8:

table 8 groups and amounts of reagents used therein

Each group was repeated three times, and after 48 hours, the cells were harvested according to Promega CorpLuciferase Assay System, the fluorescence activity was measured. The results show that: although the over-expression of KLF2 has a significant promoting effect on the activity of both the unmutated C/EBPZ promoter reporter gene A6 and CGGGCGCTGCG (-50 bp/-40 bp) mutated C/EBPZ promoter B6, the promoting effect of KLF2 on mutated C/EBPZ promoter reporter gene B6 is significantly lower than that on wild type C/EBPZ promoter A6 (P<0.01, fig. 8).

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Sequence listing

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gggcctcgcg gcgaggcagg gaggagcggg ccggcggtta ccgcggtgac agggaagccg 1260

atggcggccg gcgaggccca gggccgtggc caggagtgcg cggcacgccg gcggcggcgg 1320

catgatcggc tgcatcaccg ccggtcgctc ctccgcgccg gaagcgggcg ctgcgcctgc 1380

gtcaccgagc cggcttcctg cagcctgcca tggcg 1415

<210> 3

<211> 799

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gtccccccta acgcctgcgc tccagatccc tcacaccttc attgcccttc tctggacacg 60

ctctggacac gctccaggac ctcagtgtac tttttgtagc acggggccca aaactcaaca 120

cagtactcga ggtgcagccc caaatctttc ggcgaatgcc ttccaacggg agctcaagaa 180

cgaaaagaaa cctaagcctc gctgagcggt acgcgagggc tgctcgtcac ctctccaaat 240

atctggctga tctccggcga ggtgatgaag tcctctccga cgccgccccg ccgcgtgtag 300

tagccctgcg gggggaggag atgagcacca aagggccgca gcgctcagtt cggcccccgc 360

ccggccccgc cgcacctgcc cggggttggt gagcgcttcc cgcatgtact ccgccaccgt 420

caccggcccg gtggcgcgca gcttcagcag cagatgccgc agcactccgc cggcctcgcc 480

gctccccgcg gcctccacct cgccccctgc gcccgaactg agccgcgccg ccaacggagc 540

taggagagag gagaggaggg gggctcaggg ccgcccggcc cgccgggcct cgcggcgagg 600

cagggaggag cgggccggcg gttaccgcgg tgacagggaa gccgatggcg gccggcgagg 660

cccagggccg tggccaggag tgcgcggcac gccggcggcg gcggcatgat cggctgcatc 720

accgccggtc gctcctccgc gccggaagcg ggcgctgcgc ctgcgtcacc gagccggctt 780

cctgcagcct gccatggcg 799

<210> 4

<211> 491

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

cggggggagg agatgagcac caaagggccg cagcgctcag ttcggccccc gcccggcccc 60

gccgcacctg cccggggttg gtgagcgctt cccgcatgta ctccgccacc gtcaccggcc 120

cggtggcgcg cagcttcagc agcagatgcc gcagcactcc gccggcctcg ccgctccccg 180

cggcctccac ctcgccccct gcgcccgaac tgagccgcgc cgccaacgga gctaggagag 240

aggagaggag gggggctcag ggccgcccgg cccgccgggc ctcgcggcga ggcagggagg 300

agcgggccgg cggttaccgc ggtgacaggg aagccgatgg cggccggcga ggcccagggc 360

cgtggccagg agtgcgcggc acgccggcgg cggcggcatg atcggctgca tcaccgccgg 420

tcgctcctcc gcgccggaag cgggcgctgc gcctgcgtca ccgagccggc ttcctgcagc 480

ctgccatggc g 491

<210> 5

<211> 179

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gttaccgcgg tgacagggaa gccgatggcg gccggcgagg cccagggccg tggccaggag 60

tgcgcggcac gccggcggcg gcggcatgat cggctgcatc accgccggtc gctcctccgc 120

gccggaagcg ggcgctgcgc ctgcgtcacc gagccggctt cctgcagcct gccatggcg 179

<210> 6

<211> 100

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

ggcggcatga tcggctgcat caccgccggt cgctcctccg cgccggaagc gggcgctgcg 60

cctgcgtcac cgagccggct tcctgcagcc tgccatggcg 100

<210> 7

<211> 100

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ggcggcatga tcggctgcat caccgccggt cgctcctccg cgccggaagc ggtatctgcg 60

cctgcgtcac cgagccggct tcctgcagcc tgccatggcg 100

<210> 8

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

ccagatcttg atatcctcga gcgccatggc aggctgcag 39

<210> 9

<211> 50

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

cctgagctcg ctagcctcga gaattgtgtt cagtattaca tatttccctg 50

<210> 10

<211> 42

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

cctgagctcg ctagcctcga gcattctgtt tacccatggc ca 42

<210> 11

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

cctgagctcg ctagcctcga ggtcccccct aacgcctgc 39

<210> 12

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

cctgagctcg ctagcctcga gcggggggag gagatgagc 39

<210> 13

<211> 40

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

cctgagctcg ctagcctcga ggttaccgcg gtgacaggga 40

<210> 14

<211> 39

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

cctgagctcg ctagcctcga gggcggcatg atcggctgc 39

Claims

1. The C/EBPZ gene promoter is characterized in that the nucleotide sequence of the C/EBPZ gene promoter is shown as SEQ ID NO:1-SEQ ID NO:6, and any one of the A1-A6 sequences shown in FIG. 6.

2. A primer set for amplifying the C/EBPZ gene promoter of claim 1, said primer set comprising the sequence of SEQ ID NO:9 and the upstream primer set forth in SEQ ID NO:8, a downstream primer shown in FIG. 8; amplification of the A2 sequence of SEQ ID NO:10 and the sequence of SEQ ID NO:8, a downstream primer shown in FIG. 8; amplification of the A3 sequence of SEQ ID NO:11 and the sequence of SEQ ID NO:8, a downstream primer shown in FIG. 8; amplification of the A4 sequence of SEQ ID NO:12 and the upstream primer shown in SEQ ID NO:8, a downstream primer shown in FIG. 8; amplification of the A5 sequence of SEQ ID NO:13 and the upstream primer shown in SEQ ID NO:8, a downstream primer shown in FIG. 8; amplification of the A6 sequence of SEQ ID NO:14 and the upstream primer set forth in SEQ ID NO:8, a downstream primer shown in FIG. 8.

3. A recombinant vector comprising the C/EBPZ gene promoter of claim 1.

4. A recombinant bacterium comprising the recombinant vector of claim 3.

5. A kit comprising the primer set of claim 2.

6. A method of constructing a C/EBPZ gene promoter according to claim 1, comprising the steps of: the DNA extracted from chicken whole blood is used as a template, and PCR amplification is performed by using the primer set of claim 2 to obtain the C/EBPZ gene promoter.

7. Use of the C/EBPZ gene promoter according to claim 1, in any of the following applications:

(1) Application to the initiation of firefly luciferase expression in eukaryotic cells; the eukaryotic cells are chicken preadipocytes;

(2) The method is applied to the genetic breeding of chickens; the genetic breeding is to cultivate low-abdomen fat broilers;

the C/EBPZ gene promoter is regulated and controlled by a specific transcription factor KLF 2.