Chicken NRG4 gene near-end promoter and bidirectional promoter as well as construction method and application of chicken NRG4 gene near-end promoter and bidirectional promoter

Abstract

The invention discloses a chicken NRG4 gene near-end promoter and bidirectional promoter as well as a construction method and application thereof, and belongs to the technical field of gene engineering. The chicken NRG4 gene near-end promoter is any one of A1-A4 sequences of which the nucleotide sequences are as shown in SEQ ID NO: 1-SEQ ID NO: 4. According to the invention, DNA extracted from chicken whole blood is used as a template, and four chicken NRG4 gene near-end promoters are obtained by using a method of PCR amplification and restriction enzyme digestion by using primers. Wherein the promoter A1 has very strong bidirectional promoter activity. The invention also discloses that the NRG4 gene near-end promoter is regulated and controlled by a specific transcription factor C / EBP alpha, which has potential application values for revealing a transcription regulation and control mechanism of chicken NRG4, breeding low-abdominal-fat broilers, revealing molecular mechanisms after various human diseases occur and developing drugs for targeting NRG4 expression.

Description

Technical Field

[0001] The invention relates to the fields of genetic engineering and molecular biology, and in particular to a chicken NRG4 gene proximal promoter and a bidirectional promoter, and a construction method and application thereof. Background Art

[0002] Neuregulins (NRGs) are polypeptide growth factors and members of the epidermal growth factor family. The NRGs family has four members (NRG1, NRG2, NRG3, and NRG4). Neuregulin 4 (NRG4) is a newly discovered adipokine that is expressed in the lung, heart, and adipose tissue of mice, with the highest expression level in brown adipose tissue. NRG4 contains an EGF-like domain that is released after proteolytic cleavage as an autocrine / paracrine or endocrine signal. Mammalian studies have found that NRG4 mainly activates the ERBB4 signaling pathway by specifically binding to the epidermal growth factor receptor 4 (Erb-b2 receptortyrosine kinase 4, ERBB4), thereby promoting glucose uptake and angiogenesis, inhibiting inflammation, and maintaining metabolic homeostasis.

[0003] The expression level of NRG4 mRNA in adipose tissue of obese mice and humans is decreased, suggesting that it may have a protective effect against obesity. Studies on NRG4 transgenic mice have found that NRG4 can increase the mRNA expression of several adipose factors, including adiponectin, adiponectin, and vascular endothelial growth factor α (VEGFα), which are believed to have the effect of improving metabolic disorders in obesity. In addition, NRG4 can also promote the browning of white adipose tissue (WAT) and the differentiation of preadipocytes, and act on the liver through endocrine pathways to reduce liver fat synthesis. Therefore, NRG4 is a beneficial cytokine and is expected to be used in the treatment of metabolic diseases such as obesity, diabetes, insulin resistance, and non-alcoholic fatty liver disease.

[0004] Chicken is not only an important economic animal, but also an important animal model for the study of developmental biology and immunology. In the current broiler industry, due to long-term genetic selection, the growth rate and meat production of broilers have been significantly improved, but this is accompanied by excessive fat accumulation in broilers, especially excessive abdominal fat accumulation. Excessive fat accumulation will lead to decreased feed conversion efficiency, reduced meat quality, reduced economic benefits of chicken farming enterprises, and decreased consumer purchasing desire. Therefore, understanding the molecular mechanism of broiler fat production and fat growth and development, reducing excessive abdominal fat deposition, and creating new low-fat broiler breeds have become one of the goals of broiler breeding. The results of the study found that the protein secreted by the chicken NRG4 gene promotes the proliferation of chicken preadipocytes and inhibits the differentiation of chicken preadipocytes. Targeting the expression level of the NRG4 gene is a potential means to control excessive abdominal fat accumulation in broilers.

[0005] In summary, NRG4 is a candidate gene for low-fat broiler breeding. Many studies on animals and plants have shown that the exons and introns of genes, especially the proximal exons and introns, can participate in regulating the expression of their own genes through various mechanisms. At present, there are no reports on the transcriptional regulation of the proximal exons and introns of the chicken NRG4 gene. The promoter analysis of the proximal exons and introns of the NRG4 gene has potential application value in revealing the transcriptional regulation mechanism of chicken NRG4, breeding low-fat broilers, revealing the molecular mechanisms behind the occurrence of some human diseases, and developing drugs targeting NRG4 expression. Summary of the invention

[0006] Based on the above shortcomings, the present invention provides a chicken NRG4 gene proximal promoter, which has promoter activity in chicken embryo fibroblasts and is regulated by a specific transcription factor CEBP / α.

[0007] The present invention adopts the following technical scheme: a chicken NRG4 gene proximal promoter, wherein the chicken NRG4 gene proximal promoter is any one of the A1-A4 sequences shown in SEQ ID NO: 1-SEQ ID NO: 4 as a nucleotide sequence.

[0008] Furthermore, the chicken NRG4 gene proximal promoter A1 sequence has bidirectional promoter activity.

[0009] Another object of the present invention is to provide a primer set for amplifying the proximal promoter of the chicken NRG4 gene as described above, the primer set comprising: an upstream primer as shown in SEQ ID NO: 5 and a downstream primer as shown in SEQ ID NO: 9 for amplifying the A1 sequence; an upstream primer as shown in SEQ ID NO: 6 and a downstream primer as shown in SEQ ID NO: 9 for amplifying the A2 sequence; an upstream primer as shown in SEQ ID NO: 7 and a downstream primer as shown in SEQ ID NO: 9 for amplifying the A3 sequence; an upstream primer as shown in SEQ ID NO: 8 and a downstream primer as shown in SEQ ID NO: 9 for amplifying the A4 sequence.

[0010] Another object of the present invention is to provide a kit, characterized in that it comprises the primer set as described above.

[0011] Another object of the present invention is to provide a method for constructing the chicken NRG4 gene proximal promoter as described above, comprising the following steps: using DNA extracted from chicken whole blood as a template, using the primer set described above to perform PCR amplification to obtain the chicken NRG4 gene proximal promoter.

[0012] Another object of the present invention is to provide a recombinant vector comprising the chicken NRG4 gene proximal promoter as described above.

[0013] Another object of the present invention is to provide a recombinant bacterium, comprising the recombinant vector described in the claims.

[0014] Another object of the present invention is to provide an application of the chicken NRG4 gene proximal promoter as described above, which is applied to any of the following items: (1) applied to initiate the expression of firefly luciferase in eukaryotic cells; the eukaryotic cells are chicken embryo fibroblasts; (2) applied to genetic breeding of chickens, wherein the genetic breeding is to breed low-abdominal fat broilers; (3) the chicken NRG4 gene proximal promoter is regulated by a specific transcription factor CEBP / α.

[0015] The present invention has the following technical effects and advantages: the four chicken NRG4 gene proximal promoters provided by the present invention all have promoter activity in chicken embryo fibroblasts, indicating that the four promoters can directly regulate the transcriptional expression of chicken NRG4, and the activity of the four promoters can be promoted by overexpressing C / EBPα. At the same time, the chicken NRG4 gene proximal promoter A1 provided by the present invention has bidirectional promoter activity; at the same time, the present invention also verifies that the chicken NRG4 gene proximal promoter is regulated by a specific transcription factor C / EBPα, which has potential application value and practical value for revealing the transcriptional regulation mechanism of chicken NRG4, cultivating low-abdominal fat broilers, revealing the molecular mechanism behind the occurrence of various human diseases, and developing drugs targeting NRG4 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the construction of the chicken NRG4 gene proximal promoter reporter gene vector and the physical map of the location of related sequences; where LUC refers to the firefly luciferin gene.

[0017] Figure 2 The schematic diagram of the construction of the chicken NRG4 gene proximal promoter reporter gene and the relative activity analysis of the luciferase reporter gene. Among them, pGL3-basic without the promoter sequence is the negative control, and "**" indicates a significant difference from pGL3-basic (P<0.01).

[0018] Figure 3 This is a bidirectional promoter activity analysis diagram of the chicken NRG4 gene proximal promoter; pGL3-basic without a promoter sequence is the negative control group, and "**" indicates a significant difference from pGL3-basic (P<0.01).

[0019] Figure 4This is an analysis diagram of the enhancer activity of the proximal promoter of the chicken NRG4 gene; "**" indicates a significant difference from pGL3-promoter (P<0.01).

[0020] Figure 5 To analyze the effect of C / EBPα on the activity of proximal promoters of various lengths of NRG4 gene.

[0021] Figure 6 The deletion or mutation of ATTACATAACC causes the loss of the promoting effect of C / EBPα overexpression on the proximal promoter activity of the NRG4 gene.

[0022] Figure 7 This is a correlation analysis of the expression of NRG4 and C / EBPα genes in broiler adipose tissue.

[0023] Figure 8 Chromatin immunoprecipitation (ChIP) was performed in adipose tissue using C / EBPα antibody. After recovering and purifying the chromatin DNA fragments, PCR analysis of the corresponding fragments was performed. Compared with the irrelevant antibody IgG group, C / EBPα antibody can significantly enrich the proximal promoter region (+269 / +375) sequence of the chicken NRG4 gene. DETAILED DESCRIPTION

[0024] The chicken NRG4 gene proximal promoter disclosed in the present invention is obtained by extracting DNA from chicken whole blood as a template, and using primers to perform PCR amplification to obtain four chicken NRG4 gene proximal promoter sequences (SEQ ID NO: 1-SEQ ID NO: 4), which are named as sequences A1-A4 respectively, and constructed into a pGL3 vector by recombination technology to obtain four plasmids containing luciferase reporter gene plasmids of different lengths of chicken NRG4 gene proximal promoter sequences (A1-A4). The present invention is further described in detail below with reference to the accompanying drawings of the specification. The experimental methods used in the following examples are conventional methods unless otherwise specified, and the materials and reagents used can be obtained from commercial channels unless otherwise specified.

[0025] Example 1

[0026] Preparation of chicken NRG4 gene proximal promoter

[0027] 1. PCR amplification of the proximal promoter of the NRG4 gene

[0028] Chicken genomic DNA was used as a template, and primers NRG4-F (SEQ ID NO: 5-SEQ ID NO: 8) and NRG4-R (SEQ ID NO: 9) were used to PCR amplify the proximal promoter sequence of the NRG4 gene, as shown in the sequence table SEQ ID NO: 1 to SEQ ID NO: 4. The proximal promoter (A1) of the NRG4 gene was reversely inserted into pGL3-basic, and PCR amplification was performed using primers NRG4-F (SEQ ID NO: 10) and NRG4-R (SEQ ID NO: 11).

[0029] The reaction system is as follows:

[0030]

[0031]

[0032] The PCR reaction conditions were:

[0033]

[0034] The PCR product was subjected to agarose gel electrophoresis, and the target band was recovered using the AXYGEN gel recovery and purification kit for later use.

[0035] 3. Construction of luciferase reporter gene vectors containing proximal exon and intron promoters of NRG4 gene

[0036] The pGL3-basic vector was used as a substrate and double digested with Sma I and Xho I restriction endonucleases.

[0037] The reaction system is as follows:

[0038]

[0039] The enzyme digestion conditions were: 37°C for 1 hour to obtain a linearized pGL3-basic vector that can be used for recombinant plasmid construction.

[0040] The One Step Cloning Kit of Vazyme was used and the reaction system was prepared on ice according to the instruction manual.

[0041] The reaction system is as follows:

[0042]

[0043]

[0044] At 37°C, the reaction was continued for 30 minutes to obtain the recombinant product. After the reaction was completed, the centrifuge tube was immediately placed on ice for the next transformation. The transformation product was evenly spread on an ampicillin-resistant LB solid culture plate, the plate was inverted, and cultured at 37°C overnight. A single clone colony was selected and added to an LB liquid culture medium containing ampicillin and shaken for 4 to 5 hours before being sent for sequencing. After the plasmid sequencing was verified to be correct, it was stored for future use (the vector diagram is shown in the figure). Figure 1 shown).

[0045] Example 2

[0046] Analysis of promoter activity of proximal exons and introns of NRG4 gene

[0047] 1. Validation of promoter activity of four different lengths of NRG4 gene proximal exons and introns (A1-A4)

[0048] Chicken embryo fibroblasts in good growth state were inoculated into 48-well cell culture plates. When the cells adhered to the wall and grew to a confluence of 70% to 90%, transfection was performed. According to the instructions of Lipofectamine 2000 transfection reagent, four wild-type NRG4 gene proximal promoter (A1-A4) reporter gene plasmids and pRL-TK plasmid were co-transfected into the cells at a ratio of 100:1. After 48 hours of transfection, the firefly luciferase activity was detected according to the instructions of the dual luciferase detection system, and the Renilla luciferase activity was used for correction. The relative activity of the reporter gene is the ratio of the firefly luciferase activity to the Renilla luciferase reporter gene activity (Fluc / Rluc). The results of the reporter gene activity analysis showed that compared with the pGL3-basic empty vector, the pGL3-NRG4 (A1-A4) reporter gene vectors all had higher activity (P<0.01) (such as Figure 2 The results showed that the four constructed wild-type NRG4 gene proximal promoter (A1-A4) reporter gene plasmids had promoter activity.

[0049] 2. Bidirectional promoter verification of proximal exons and introns of NRG4 gene

[0050] Chicken embryo fibroblasts in good growth state were inoculated into 48-well cell culture plates, and transfected when the cells adhered to the wall and grew to a confluence of 70% to 90%. According to the instructions of Lipofectamine 2000 transfection reagent, the NRG4 gene proximal promoter (A1) and its reverse inserted promoter (B1) reporter gene plasmids were co-transfected into the cells at a ratio of 100:1, respectively, and the firefly luciferase activity was detected according to the instructions of the dual luciferase detection system after 48 hours of transfection, and the Renilla luciferase activity was used for correction. The relative activity of the reporter gene is the ratio of the firefly luciferase activity to the Renilla luciferase reporter gene activity (Fluc / Rluc). The results showed that compared with the pGL3-basic vector, there was no significant difference in reporter gene activity between the forward insertion reporter gene vector pGL3-NRG4 (A1) and the reverse insertion reporter gene vector pGL3-NRG4 (B1), but the promoter reporter gene activity of both was significantly higher than that of the pGL3-basic empty vector, which was 207.2 and 188.8 times that of the pGL3-basic empty vector, respectively (e.g. Figure 3 As shown). This shows that the chicken NRG4 (-122 / +452) region may be a bidirectional promoter, or that the region has enhancer properties. The chicken NRG4 gene proximal promoter (A1) was cloned into the enhancer reporter gene vector pGL3-promoter vector between the Sma I and Xho I sites upstream of the SV40 promoter, and between the Bam HI and Sal I sites downstream of the SV40 poly A region of the vector, to construct the pGL3-promoter-NRG4-F-A1 and pGL3-promoter-NRG4-R-A1 vectors, respectively. The results showed that the A1 fragment could not enhance the activity of the SV40 promoter regardless of whether it was located upstream or downstream of the reporter gene. Instead, it significantly reduced its activity (P<0.01) (as shown). Figure 4 These results indicate that the proximal promoter (A1) region of the NRG4 gene does not have the characteristics of a classical enhancer, indicating that the proximal promoter (A1) region of the NRG4 gene has bidirectional promoter characteristics.

[0051] 3. Effect of transcription factor C / EBPα on the activity of NRG4 gene proximal promoters (A1-A4) of various lengths

[0052] First, the TFdb database was used to predict the potential transcription factor binding sites of the NRG4 (A1) fragment. The results showed that there were 135 potential transcription factor binding sites in this region, including a potential binding site (ATTACATAACC) (+278 / +288) for C / EBPα, an important transcription factor that controls adipogenesis. This binding site is located in exon 2 of NRG4. In order to verify whether C / EBPα regulates the activity of the proximal promoter of the NRG4 gene, the C / EBPα expression vector (pCMV-HA-C / EBPα) was co-transfected with the pGL3-NRG4 (A1-A4) reporter gene vector into chicken embryo fibroblasts. The reporter gene assay results showed that overexpression of C / EBPα significantly increased the activity of the pGL3-NRG4 (A1-A4) reporter gene vector (P<0.01) (e.g. Figure 5 The results showed that there was a C / EBPα binding site in the proximal promoter (A1-A4) region of the NRG4 gene, which was consistent with the previous bioinformatics prediction analysis results.

[0053] 4.ATTACATAACC (+278 / +288) at least partially mediates the promoting effect of C / EBPα overexpression on the activity of the proximal promoter of the NRG4 gene.

[0054] The CE Design primer design software (https: / / crm.vazyme.com / cetool / simple.html) was used to design the mutation primers of the C / EBPα binding site (SEQ ID NO: 12-SEQ ID NO: 15). The site-directed mutagenesis kit (Vazyme) was used to completely delete and mutate the C / EBPα binding site (ATTACATAACC) sequence of the pGL3-NRG4(A4 vector) (mutating the C / EBPα binding site to ActctAattaC). The vector with the deleted C / EBPα binding site was named pGL3-NRG4(B4), and the vector with the mutated C / EBPα binding site was named pGL3-NRG4(C4). After sequencing verification, it was used for subsequent analysis.

[0055] Chicken embryo fibroblasts in good growth state were inoculated into 48-well cell culture plates. When the cells adhered to the wall and grew to a confluence of 70% to 90%, transfection was performed. According to the instructions of Lipofectamine 2000 transfection reagent, pGL3-NRG4 (A4), pGL3-NRG4 (B4) and pGL3-NRG4 (C4) reporter gene plasmids were co-transfected into the cells with pRL-TK plasmid at a ratio of 100:1. After 48 hours of transfection, the firefly luciferase activity was detected according to the instructions of the dual luciferase detection system, and the Renilla luciferase activity was used for correction. The relative activity of the reporter gene is the ratio of the firefly luciferase activity to the Renilla luciferase reporter gene activity (Fluc / Rluc). Overexpression of C / EBPα can significantly increase the reporter gene activity of the pGL3-NRG4 (A4) vector by 2.16 times (P<0.05) (e.g. Figure 6 However, overexpression of C / EBPα had no significant effect on the reporter gene activity of pGL3-NRG4(B4) and pGL3-NRG4(C4) (P>0.05) (as shown in Figure 6 This indicates that both deletion and mutation of the C / EBPα binding site will lead to the loss of C / EBPα regulation on the pGL3-NRG4(A4) vector.

[0056] 5. There is a significant positive correlation between the expression of C / EBPα and neuregulin 4 genes in chicken abdominal adipose tissue

[0057] The total RNA from chicken abdominal adipose tissue was extracted using the RNAiso Plus (TaKaRa, Japan) kit, and the absorbance of the total RNA of the sample at 260nm and 280nm was measured using an ultraviolet spectrophotometer. The quality of the total RNA was evaluated according to the ratio (A260 / 280). Reverse transcription was performed using a reverse transcription kit (TaKaRa, Japan). 500ng of the extracted total RNA was taken and the experiment was performed according to the steps in the kit manual. The reaction conditions were: 37℃ for 10min, 85℃ for 5s, and finally kept at 4℃. After the reverse transcription was completed, the cDNA product was stored at -20℃.

[0058] According to the instructions of SYBR Green Pro Tag Hs premixed qPCR kit (Aikerui, Hunan), the cDNA obtained by reverse transcription was used as a template, and a 10 μL reaction system was prepared and added to a 96-well plate. The reaction conditions were as follows: 95°C for 30 s, 95°C for 5 s, 60°C for 30 s, and 40 cycles. The TATA-binding protein gene (TBP) gene was used as the internal reference gene, and the relative expression level of the target gene mRNA was calculated using the 2-ΔΔCt method. PrimerPremier 5.0 software was used to design gene expression detection primers, and the primer pair sequences are shown in SEQ ID No.16-SEQ IDNo.21. The results showed that the expression of the C / EBPα gene in abdominal adipose tissue was significantly positively correlated with the expression of the NRG4 gene (r=0.596, P<0.01) (as shown in Figure 2). Figure 7 This result supports that C / EBPα regulates the expression of NRG4 gene in adipose tissue.

[0059] 6. Chromatin immunoprecipitation (ChIP)-PCR was used to study whether C / EBPα could bind to the promoter of NRG4 proximal exons and introns.

[0060] Fresh chicken abdominal adipose tissue was collected, washed with PBS, fixed with formaldehyde at a final concentration of 1.25% for 10 min, and then 0.125 mol / L glycine was added for 5 min to terminate crosslinking. According to the Simple ChIP Plus EnzymaticChromatin IP Kit instructions, nucleus preparation and chromatin digestion were performed, and DNA fragmentation was treated with ultrasound. The IP group used C / EBPα antibody (Mitaka, Wuhan), IgG as a negative control, chromatin immunoprecipitation by magnetic beads, and sample DNA was purified using a DNA purification centrifugal column after crosslinking. No antibody treatment was performed, and the purified DNA was directly recovered after ultrasound as a positive control input. Primers were designed according to the C / EBPα binding site (+278 / +288) position of exon 2 of the NRG4 gene. The primer pair sequences are as shown in SEQ ID No.22 and SEQ ID No.23. qPCR was used to detect whether the transcription factor C / EBPα binds to exon 2, and the data are expressed as a percentage of chromatin input. The results showed that compared with the IgG group, the DNA fragments treated with C / EBPα antibody were significantly enriched (e.g. Figure 8 ), indicating that C / EBPα directly binds to the sequence in the proximal promoter (+269 / +375) region of the chicken NRG4 gene.

[0061] The above experiments prove that the chicken NRG4 gene proximal promoter (A1) of the present invention has bidirectional promoter activity, and these four NRG4 gene proximal promoters all have promoter activity in chicken embryo fibroblasts, indicating that each promoter can directly regulate the transcriptional expression of chicken NRG4, and overexpression of C / EBPα promotes the activity of all four chicken NRG4 gene proximal promoters. Gene expression correlation analysis shows that there is a significant positive correlation between the mRNA expression of C / EBPα and NRG4 genes in chicken adipose tissue, and C / EBPα regulates the expression of chicken adipose tissue NRG4 gene by directly binding to the promoter's ATTACATAACC.

Claims

1. A chicken NRG4 gene proximal promoter, characterized in that: The chicken NRG4 gene proximal promoter is any one of the A1-A4 sequences shown in SEQ ID NO: 1-SEQ ID NO: 4 as the nucleotide sequence.

2. A chicken NRG4 gene proximal promoter as claimed in claim 1, characterized in that: The chicken NRG4 gene proximal promoter A1 sequence has bidirectional promoter activity.

3. A primer set, characterized in that: Used to amplify the chicken NRG4 gene proximal promoter as claimed in claim 1, the primer set comprises: an upstream primer as shown in SEQ ID NO: 5 and a downstream primer as shown in SEQ ID NO: 9 for amplifying the A1 sequence; an upstream primer as shown in SEQ ID NO: 6 and a downstream primer as shown in SEQ ID NO: 9 for amplifying the A2 sequence; The upstream primer shown in SEQ ID NO: 7 and the downstream primer shown in SEQ ID NO: 9 are used to amplify the A3 sequence; the upstream primer shown in SEQ ID NO: 8 and the downstream primer shown in SEQ ID NO: 9 are used to amplify the A4 sequence.

4. A kit, characterized in that: Comprising the primer set as claimed in claim 3.

5. A method for constructing the chicken NRG4 gene proximal promoter as claimed in claim 1, characterized in that: The following steps are involved: The DNA extracted from chicken whole blood was used as a template and PCR amplification was performed using the primer set as claimed in claim 3 to obtain the proximal promoter of the chicken NRG4 gene.

6. A recombinant vector, characterized in that: It comprises the chicken NRG4 gene proximal promoter as described in claim 1.

7. A recombinant bacterium, characterized in that: Comprising the recombinant vector according to claim 6.

8. An application of the chicken NRG4 gene proximal promoter as claimed in claim 1, characterized in that: The invention is applied in any of the following items: (1) to initiate the expression of firefly luciferase in eukaryotic cells; the eukaryotic cells are chicken embryo fibroblasts; (2) to genetic breeding of chickens, wherein the genetic breeding is to breed low-abdominal fat broilers; (3) the proximal promoter of the chicken NRG4 gene is regulated by a specific transcription factor CEBP / α.

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