Chicken NRG4 gene proximal promoter and bidirectional promoter, their construction methods and applications

Abstract

The present invention discloses a chicken NRG4 gene proximal promoter and bidirectional promoter, as well as a construction method and application thereof, belonging to the technical field of genetic engineering. The chicken NRG4 gene proximal promoter is any one of the A1 - A4 sequences with the nucleotide sequences shown in SEQ ID NO: 1 - SEQ ID NO: 4. In the present invention, DNA extracted from chicken whole blood is used as a template, and four kinds of chicken NRG4 gene proximal promoters are obtained by using primers for PCR amplification and restriction enzyme digestion. Among them, the A1 promoter has strong bidirectional promoter activity. The present invention also discloses that the chicken NRG4 gene proximal promoter is regulated by a specific transcription factor C / EBPα, which has potential application value for revealing the transcriptional regulation mechanism of chicken NRG4, cultivating low-abdominal-fat broilers, and revealing the molecular mechanisms behind the occurrence of various human diseases and developing drugs targeting NRG4 expression.

Description

Technical Field

[0001] The present invention relates to the fields of genetic engineering and molecular biology, and particularly relates to a chicken NRG4 gene proximal promoter and 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 4 members (NRG1, NRG2, NRG3, and NRG4). Neuregulin 4 (NRG4) is a newly discovered adipokine, which is expressed in mouse lungs, hearts, and adipose tissues, and has the highest expression level in brown adipose tissue. NRG4 contains an EGF-like domain, which is released after proteolytic cleavage and acts as an autocrine / paracrine or endocrine signal. Mammalian studies have found that NRG4 mainly promotes glucose uptake and angiogenesis, inhibits inflammation, and maintains metabolic homeostasis by specifically binding to epidermal growth factor receptor 4 (Erb-b2 receptor tyrosine kinase 4, ERBB4) and activating the ERBB4 signaling pathway.

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

[0004] Chickens are not only an important economic animal but also an important animal model for research in 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. However, this has led to excessive fat accumulation in broilers, especially excessive abdominal fat accumulation. Excessive fat accumulation will result in a decrease in the feed conversion efficiency of chickens, a reduction in meat quality, a decrease in the economic benefits of chicken farming enterprises, and a decrease in consumer purchasing desire. Therefore, understanding the molecular mechanism of broiler fat generation 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 industry development. Research results have found that the secreted protein of 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. Studies on many animals and plants have shown that exons and introns of genes, especially proximal exons and introns, can participate in the regulation of their own gene expression through various mechanisms. At present, there is no research report on the transcriptional regulation of the proximal exons and introns of the chicken NRG4 gene. Conducting promoter analysis of the proximal exons and introns of the NRG4 gene has potential application value for revealing the transcriptional regulation mechanism of chicken NRG4, breeding low-abdominal-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 deficiencies, the present invention provides a proximal promoter of the chicken NRG4 gene, 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 solution: a proximal promoter of the chicken NRG4 gene, wherein the proximal promoter of the chicken NRG4 gene is any one of the A1 - A4 sequences shown in SEQ ID NO: 1 - SEQ ID NO: 4 in nucleotide sequence.

[0008] Furthermore, the A1 sequence of the proximal promoter of the chicken NRG4 gene 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 includes: an upstream primer shown in SEQ ID NO: 5 and a downstream primer shown in SEQ ID NO: 9 for amplifying the A1 sequence; an upstream primer shown in SEQ ID NO: 6 and a downstream primer shown in SEQ ID NO: 9 for amplifying the A2 sequence; an upstream primer shown in SEQ ID NO: 7 and a downstream primer shown in SEQ ID NO: 9 for amplifying the A3 sequence; an upstream primer shown in SEQ ID NO: 8 and a downstream primer shown in SEQ ID NO: 9 for amplifying the A4 sequence.

[0010] Another object of the present invention is to provide a kit, which is characterized by including the primer set as described above.

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

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

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

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

[0015] The present invention has the following technical effects and advantages: The 4 kinds of proximal promoters of the chicken NRG4 gene provided by the present invention all have promoter activity in chicken embryo fibroblasts, indicating that these 4 promoters can directly regulate the transcription and expression of chicken NRG4, and overexpression of C / EBPα can promote the activity of these 4 promoters. At the same time, the proximal promoter A1 of the chicken NRG4 gene provided by the present invention has bidirectional promoter activity; at the same time, the present invention also verifies that the proximal promoter of the chicken NRG4 gene 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, and revealing the molecular mechanisms behind the occurrence of various human diseases and developing drugs targeting NRG4 expression. Description of the Drawings

[0016] Figure 1 Schematic diagram for constructing a reporter gene vector of the proximal promoter of the chicken NRG4 gene and physical map of related sequence localization; wherein, LUC refers to the firefly luciferin gene.

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

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

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

[0020] Figure 5 Analysis of the effect of C / EBPα on the activities of proximal promoters of various lengths of the NRG4 gene

[0021] Figure 6 Effect of deletion or mutation of ATTACATAACC on the promotion of the activity of the proximal promoter of the NRG4 gene by overexpression of C / EBPα

[0022] Figure 7 Analysis of the expression correlation between the NRG4 and C / EBPα genes in broiler adipose tissue

[0023] Figure 8 In adipose tissue, chromatin immunoprecipitation (ChIP) was performed using a C / EBPα antibody. After recovering and purifying the chromatin DNA fragments, PCR analysis of the corresponding fragments was carried out. Compared with the irrelevant antibody IgG group, the C / EBPα antibody could significantly enrich the sequence of the proximal promoter region (+269 / +375) of the chicken NRG4 gene Detailed implementation methods

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

[0025] Example 1

[0026] Preparation of the proximal promoter of the chicken NRG4 gene

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

[0028] Using chicken genomic DNA as a template, the proximal promoter sequence of the NRG4 gene was amplified by PCR with primers NRG4-F (SEQ ID NO: 5 - SEQ ID NO: 8) and NRG4-R (SEQ ID NO: 9). See SEQ ID NO: 1 to SEQ ID NO: 4 in the sequence listing for details. The proximal promoter (A1) of the NRG4 gene was inserted in reverse into pGL3-basic, and PCR amplification was carried out with 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 products were subjected to agarose gel electrophoresis, and the target bands were recovered and purified using the AXYGEN Gel Extraction and Purification Kit for later use.

[0035] 3. Construction of the luciferase reporter gene vector for the proximal exon and intron promoters of the NRG4 gene

[0036] Using the pGL3-basic vector as a substrate, double digestion was carried out with Sma I and Xho I restriction endonucleases.

[0037] The reaction system is as follows:

[0038]

[0039] The digestion conditions were: digestion at 37°C for 1 h. A linearized pGL3-basic vector that could be used for recombinant plasmid construction was obtained.

[0040] Using the One Step Cloning Kit from Vazyme, prepare the reaction system on ice according to the instructions.

[0041] The reaction system is as follows:

[0042]

[0043]

[0044] Under the condition of 37 °C, the reaction was carried out for 30 min to obtain the recombinant product. After the reaction, the centrifuge tube was immediately placed on ice for the next transformation. The transformed product was evenly spread on an LB solid culture plate with ampicillin resistance, and the plate was inverted and cultured overnight at 37 °C. Single colonies were picked and added to an LB liquid medium containing ampicillin, and the bacteria were shaken for 4 - 5 h and then sent for sequencing. After the plasmid sequencing was verified to be correct, it was stored for later use (the vector schematic diagram is as shown in Figure 1 ).

[0045] Example 2

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

[0047] 1. Verification of the promoter activities (A1 - A4) of the proximal exons and introns of the NRG4 gene with 4 different lengths

[0048] Chicken embryo fibroblasts in good growth state were inoculated into a 48 - well cell culture plate. When the cells adhered and grew to a confluence of 70% - 90%, transfection was carried out. According to the Lipofectamine 2000 transfection reagent instruction manual, 4 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 h of transfection, according to the dual - luciferase detection system instruction manual, the firefly luciferase activity was detected, and the activity of Renilla luciferase was used for calibration. The relative activity of the reporter gene was 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 activities (P < 0.01) (as shown in Figure 2 ), indicating that the 4 wild - type NRG4 gene proximal promoter (A1 - A4) reporter gene plasmids constructed had promoter activities.

[0049] 2. Verification of the bidirectional promoter of the proximal exons and introns of the NRG4 gene

[0050] The well-growing chicken embryo fibroblasts were inoculated into 48-well cell culture plates. When the cells adhered and grew to a confluence of 70% - 90%, transfection was carried out. According to the Lipofectamine 2000 transfection reagent instruction manual, the reporter gene plasmids of the proximal promoter (A1) of the NRG4 gene and its reverse-inserted promoter (B1) were co-transfected into the cells with the pRL-TK plasmid at a ratio of 100:1. After 48 hours of transfection, according to the dual-luciferase detection system instruction manual, the firefly luciferase activity was detected, and the activity of Renilla luciferase was used for calibration. The relative activity of the reporter gene was 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 obvious difference in the reporter gene activity between the forward-inserted reporter gene vector pGL3-NRG4(A1) and the reverse-inserted reporter gene vector pGL3-NRG4(B1), but the promoter reporter gene activities of both were significantly higher than that of the pGL3-basic empty vector, which were 207.2 and 188.8 times that of the pGL3-basic empty vector respectively (as Figure 3 shown). This indicated that the chicken NRG4 (-122 / +452) region might be a bidirectional promoter, or this region had enhancer characteristics. The proximal promoter (A1) of the chicken NRG4 gene was cloned between the Sma I and Xho I sites upstream of the SV40 promoter of the enhancer reporter gene vector pGL3-promoter, and between the Bam HI and Sal I sites downstream of the SV40 polyA region of this vector, respectively, to construct the pGL3-promoter-NRG4-F-A1 and pGL3-promoter-NRG4-R-A1 vectors. The results showed that whether the A1 fragment was located upstream or downstream of the reporter gene, it could not enhance the activity of the SV40 promoter, but instead significantly reduced its activity (P<0.01) (as Figure 4 shown). These results indicated that the proximal promoter (A1) region of the NRG4 gene did not have the classical enhancer characteristics, suggesting that the proximal promoter (A1) region of the NRG4 gene had bidirectional promoter characteristics.

[0051] 3. Effects of transcription factor C / EBPα on the activities of proximal promoters (A1 - A4) of NRG4 genes with 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) of the important transcription factor C / EBPα that controls adipogenesis. This binding site was located in exon 2 of NRG4. 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 detection results showed that overexpression of C / EBPα could significantly increase the activity of the pGL3-NRG4 (A1-A4) reporter gene vector (P<0.01) (as Figure 5 shown), increasing by 7.2, 1.7, 5.6, and 6.8 times respectively. The results indicated that there was a binding site for C / EBPα in the proximal promoter (A1-A4) region of the NRG4 gene, which was consistent with the previous bioinformatics prediction and analysis results.

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

[0054] The mutant primers (SEQ ID NO: 12-SEQ ID NO: 15) for the C / EBPα binding site were designed using the CE Design primer design software (https: / / crm.vazyme.com / cetool / simple.html). Using the site-directed mutagenesis kit (Vazyme), the sequences of the C / EBPα binding sites (ATTACATAACC) of the pGL3-NRG4 (A4) vector were completely deleted and mutated (the C / EBPα binding site was mutated to AcTctAattaC). The vector with the C / EBPα binding site deleted was named pGL3-NRG4 (B4), and the vector with the C / EBPα binding site mutated was named pGL3-NRG4 (C4). After sequencing verification, they were used for subsequent analysis.

[0055] The well-grown chicken embryo fibroblasts were inoculated into a 48-well cell culture plate. When the cells adhered and grew to a confluence of 70% - 90%, transfection was performed. According to the Lipofectamine 2000 transfection reagent instruction manual, the reporter gene plasmids pGL3-NRG4(A4), pGL3-NRG4(B4), and pGL3-NRG4(C4) were co-transfected into the cells with the pRL-TK plasmid at a ratio of 100:1. After 48 hours of transfection, according to the dual-luciferase detection system instruction manual, the firefly luciferase activity was detected, and the Renilla luciferase activity was used for calibration. The relative activity of the reporter gene was the ratio of the firefly luciferase activity to the Renilla luciferase reporter gene activity (Fluc / Rluc). Overexpression of C / EBPα could significantly increase the reporter gene activity of the pGL3-NRG4(A4) vector by 2.16-fold (P < 0.05) (as Figure 6 shown); however, overexpression of C / EBPα had no significant effect on the reporter gene activities of pGL3-NRG4(B4) and pGL3-NRG4(C4) (P > 0.05) (as Figure 6 shown), indicating that both the deletion and mutation of the C / EBPα binding site would lead to the loss of the regulation of C / EBPα on the pGL3-NRG4(A4) vector.

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

[0057] Total RNA of chicken abdominal adipose tissue was extracted using the RNAiso Plus (TaKaRa, Japan) kit. The absorbance of the total RNA sample at 260 nm and 280 nm was measured using an ultraviolet spectrophotometer, and 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). 500 ng of the extracted total RNA was taken, and the experiment was carried out according to the steps of the kit instruction manual. The reaction conditions were: 37°C for 10 min, 85°C for 5 s, and finally maintained at 4°C. After reverse transcription was completed, the cDNA product was stored at -20°C.

[0058] Operate according to the SYBR Green Pro Tag Hs Premixed qPCR Kit instruction manual (Aikerui, Hunan). Use the cDNA obtained by reverse transcription as a template, configure a 10 μL reaction system and add it to a 96-well plate. The reaction conditions are as follows: 95 °C for 30 s, 95 °C for 5 s, 60 °C for 30 s, for 40 cycles. Use the TATA-binding protein gene (TBP) as an internal reference gene, and calculate the relative expression level of the target gene mRNA using the 2-ΔΔCt method. Use Primer Premier 5.0 software to design primers for gene expression detection. The primer pair sequences are shown as SEQ ID No.16 - SEQ ID No.21. The results show that the expression of the C / EBPα gene in abdominal adipose tissue is significantly positively correlated with the expression of the NRG4 gene (r = 0.596, P < 0.01) (as Figure 7 shown), and this result supports that C / EBPα regulates the expression of the NRG4 gene in adipose tissue.

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

[0060] Collect fresh chicken abdominal adipose tissue, wash the adipose tissue with PBS, fix it with 1.25% formaldehyde for 10 min, and then add 0.125 mol / L glycine for 5 min to terminate crosslinking. According to the Simple ChIP Plus Enzymatic Chromatin IP Kit instruction manual, perform nuclear preparation and chromatin digestion, and fragment the DNA by sonication. In the IP group, use the C / EBPα antibody (Sanying, Wuhan), and IgG as a negative control. Use the magnetic bead method for chromatin immunoprecipitation, and after de-crosslinking, purify the sample DNA using a DNA purification spin column. Without antibody treatment, directly recover and purify the DNA after sonication as a positive control input. Design primers according to the position of the C / EBPα binding site (+278 / +288) in exon 2 of the NRG4 gene. The primer pair sequences are shown as SEQ ID No.22 and SEQ ID No.23. Use qPCR to detect whether the transcription factor C / EBPα binds to exon 2, and the data is expressed as a percentage of chromatin Input. The results show that compared with the IgG group treatment, the DNA fragments treated with the C / EBPα antibody are significantly enriched (as Figure 8 shown), 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 proximal promoter (A1) of the chicken NRG4 gene of the present invention has bidirectional promoter activity, and these 4 proximal promoters of the NRG4 gene all have promoter activity in chicken embryo fibroblasts, indicating that each promoter can directly regulate the transcription and expression of chicken NRG4. Overexpression of C / EBPα promotes the activity of all 4 proximal promoters of the chicken NRG4 gene. Gene expression correlation analysis shows that there is a significant positive correlation between the mRNA expression of C / EBPα and the NRG4 gene in chicken adipose tissue, and C / EBPα regulates the expression of the NRG4 gene in chicken adipose tissue by directly binding to ATTACATAACC of the promoter.

Claims

1. A chicken NRG4 gene proximal promoter, characterized in that: The chicken NRG4 gene proximal promoter is a nucleotide sequence as shown in SEQ ID NO:

1.

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

3. A primer set, characterized in that: Used to amplify the chicken NRG4 gene proximal promoter as described in claim 1, the primer set includes: an upstream primer as shown in SEQ ID NO: 5 for amplifying the promoter sequence and a downstream primer as shown in SEQ ID NO:

9.

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 method is applied in any of the following: (1) to start 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 cultivate low-abdominal fat broilers.

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