UORF element for regulating expression of chicken insulin receptor and application of uORF element

Abstract

The invention discloses a uORF element for regulating and controlling expression of a chicken insulin receptor and application of the uORF element, and belongs to the field of molecular biology and genetic engineering, and the nucleotide sequence of the uORF element is shown as any one of SEQ ID NO.1-2; the application comprises any one of the following applications: (1) regulating and controlling expression of firefly luciferase in eukaryotic cells; the eukaryotic cells are chicken embryo fibroblasts and chicken preadipocytes; (2) the method is applied to genetic breeding of chickens, and the genetic breeding refers to breeding of broiler chickens capable of reducing insulin resistance; and (3) application in preparation of a reagent for regulating expression of the chicken insulin receptor. The expression level of INSR can be regulated and controlled, insulin sensitivity is improved, and material support is provided for research on analysis of a mechanism for regulating and controlling blood sugar by insulin.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology and genetic engineering, and particularly relates to a uORF element for regulating expression of chicken insulin receptor and application thereof. BACKGROUND

[0002] Insulin is a hormone synthesized by pancreatic beta cells. Insulin regulates the physiological processes of animals by binding to insulin receptors (INSR) of target organs. Chicken insulin receptor is encoded by a single gene spanning more than 38 kb on chromosome 28, which is composed of 21 exons. Insulin receptor is a transmembrane protein complex composed of 2 alpha subunits and 2 beta subunits. The alpha subunit is located outside the cell and provides a binding site for insulin. In mammals, changes in insulin receptor levels have been shown to have important physiological significance. If the insulin receptor cannot function properly, it will cause insulin resistance. Mice lacking insulin receptors have mild growth retardation at full term and no obvious metabolic abnormalities. However, after birth, the metabolic process deteriorates rapidly: glucose levels rise rapidly during feeding, and insulin levels rise to more than 1,000 times the normal level. People lacking insulin receptors exhibit severe intrauterine growth retardation, developmental abnormalities, and hypoglycemia. Mice lacking insulin receptors in specific cell types exhibit mild metabolic and reproductive abnormalities. Therefore, targeting the expression of the INSR receptor gene to improve the binding of insulin to INSR has important significance for improving the insulin signaling pathway, reducing insulin resistance, and improving the growth and development and reproductive performance of animals.

[0003] uORF refers to "upstream open reading frame", which is widely present in eukaryotes. Under normal circumstances, uORF can inhibit gene expression by reducing the translation efficiency of the downstream coding region or causing mRNA degradation. However, when the cell is under stress, uORF can promote the mRNA expression of certain stress-related genes. Studies have shown that uORF-based expression regulation plays an important role in the growth and development and metabolism of animals.

[0004] Chickens are not only an important economic animal, but also an important animal model for research in developmental biology and immunology. It is generally believed that chickens have natural insulin resistance, and the way in which insulin regulates blood glucose is significantly different from that of mammals, which is species-specific, which may be due to the low expression of INSR and IRS-1 proteins. There is no related report on the regulation of chicken INSR gene expression by uORF elements. SUMMARY

[0005] Based on the above deficiencies, the present application aims to provide a uORF element for regulating chicken INSR gene expression, which can regulate the expression level of INSR, improve insulin sensitivity, and provide material support for analyzing the mechanism of insulin regulating blood glucose.

[0006] The present application adopts the following technical scheme: a uORF element for regulating chicken insulin receptor expression, the nucleotide sequence of which is shown in any one of SEQ ID NO. 1-2.

[0007] Further, the present application provides a PCR primer set for amplifying the uORF element for regulating chicken insulin receptor expression according to claim 1, the sequence of primer 5'UTR-F is shown in SEQ ID NO. 3, and the sequence of primer 5'UTR-R is shown in SEQ ID NO. 4.

[0008] The present application also provides a recombinant vector comprising the uORF element for regulating chicken insulin receptor expression as described above.

[0009] The present application also provides a construction method of the recombinant vector as described above, the steps of which are as follows:

[0010] Step S1: using the PCR primer set: the sequence of 5'UTR-F is shown in SEQ ID NO. 3, and the sequence of 5'UTR-R is shown in SEQ ID NO. 4, performing PCR amplification of the uORF element for regulating chicken insulin receptor expression, and collecting the PCR product;

[0011] Step S2: using psi-CHECK2 vector as a substrate, performing single enzyme digestion with Nhe I restriction endonuclease, and connecting the PCR product to the upstream of the Renilla CDs region of the linearized psi-CHECK2 vector by homologous recombination to construct a psi-CHECK2-INSR-5'UTR-WT recombinant vector.

[0012] Another object of the present application is to provide the application of the uORF element for regulating chicken insulin receptor expression as described above, which includes any one of the following: (1) regulating the expression of firefly luciferase in eukaryotic cells; the eukaryotic cells are chicken embryo fibroblasts and chicken preadipocytes; (2) applied to the genetic breeding of chickens, the genetic breeding is to breed meat chickens with reduced insulin resistance; (3) used in the preparation of reagents for regulating the expression of chicken insulin receptor.

[0013] The uORF element of the chicken INSR gene provided by the application provides material support for chicken insulin signal research. The reporter gene activity of the uORF element start codon mutation of the chicken INSR gene provided by the application is 2.31-75.16 times that of the wild type, which is helpful for improving insulin sensitivity, revealing the post-transcriptional regulation mechanism of INSR, analyzing the mechanism of insulin regulating blood glucose, and providing a reference for human insulin resistance related research; and further provides a train of thought for analyzing the glucose regulation mechanism of chickens and the treatment of human diabetes. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Figure 1 is a schematic diagram of the uORF of INSR;

[0015] Figure 2 Figure 5 is the detection of the reporter gene activity of the series of vectors of psi-CHECK2-INSR-5'UTR-WT and uORF start codon mutation;

[0016] Figure 3 Figure 6 is the detection of the Renilla mRNA relative expression of the series of vectors of psi-CHECK2-INSR-5'UTR-WT and uORF start codon mutation. DETAILED DESCRIPTION

[0017] Example 1, construction of the uORF element reporter gene vector upstream of the INSR gene

[0018] 1. Identification of the uORF element upstream of the INSR gene

[0019] The uORF Finder was used to analyze the chicken INSR transcript 5'UTR in the NCBI database, and it was found that there were two uORF elements with different start codons but using the same stop codon, as shown in Figure 1 The long type of uORF was named uORFL (465bp), and the sequence is shown as SEQ ID NO. 1, and the short type of uORF was named uORFS (51bp), and the sequence is shown as SEQ ID NO. 2;

[0020] 2. Construction of the uORF element reporter gene vector upstream of the INSR gene

[0021] S1: Using the primer set: using the mixed cDNA of AA chicken muscle, liver, fat and brain tissue as the template, using the primer set: 5'UTR-F: the sequence is shown as SEQ ID NO. 3 and 5'UTR-R: the sequence is shown as SEQ ID NO. 4, the chicken INSR transcript 5'UTR sequence was amplified by PCR, and the primer was synthesized by Jinweizhi Biological Company. The specific reaction system is as follows:

[0022] The reaction system is as follows:

[0023]

[0024]

[0025] The PCR reaction conditions are as follows:

[0026]

[0027] The PCR product is subjected to agarose gel electrophoresis, and the AXYGEN gel recovery and purification kit is used to recover the target band for standby use.

[0028] S2: Using psi-CHECK2 vector as a substrate, single enzyme digestion is performed by using Nhe I restriction endonuclease. The PCR product is connected to the upstream of the sea cucumber CD region of the linearized psi-CHECK2 vector by a homologous recombination method to construct the psi-CHECK2-INSR-5'UTR-WT vector.

[0029] The reaction system is as follows:

[0030]

[0031] Thus, the linearized psi-CHECK2 vector that can be used for recombinant plasmid construction is obtained.

[0032] The One Step Cloning Kit of Vazyme is used to configure the reaction system on ice according to the instructions.

[0033] The reaction system is as follows:

[0034]

[0035]

[0036] The recombination product is obtained under the condition of 37 DEG C for 30 min. After the reaction is completed, the centrifuge tube is immediately placed on ice for the next step of transformation. The transformation product is uniformly coated on the LB solid culture plate with ampicillin resistance, the plate is inverted, and the culture is performed at 37 DEG C overnight. A single colony is picked and added to the LB liquid culture medium containing ampicillin for shaking for 14-15 h, and then sent for sequencing. After the plasmid sequencing is verified to be correct, it is stored for standby use, and is named as psi-CHECK2-INSR-5'UTR-WT.

[0037] S3: To determine the regulatory effect of uORF elements, the start codon ATG of uORFL and uORFS was mutated to TAA, respectively, or simultaneously mutated to TAA, and named as uORFLmut, uORFSmut and uORFL+Smut, respectively. To construct the reporter gene vectors of uORFLmut, uORFSmut and uORFL+Smut, according to the instructions of the point mutation kit (vazyme, C214), using psi-CHECK2-INSR-5'UTR-WT vector as a template, using primer set: uORFLmut-F (the sequence is shown as SEQ ID NO. 5) and uORFLmut-R (the sequence is shown as SEQ ID NO. 6) to construct psi-CHECK2-INSR-5'UTR-uORFLmut vector in which the ATG of uORFL was mutated to TGA; using psi-CHECK2-INSR-5'UTR-WT vector as a template, using primer set: uORFSmut-F (the sequence is shown as SEQ ID NO. 7) and ORFSmut-R (the sequence is shown as SEQ ID NO. 8) to construct psi-CHECK2-INSR-5'UTR-uORFSmut vector in which the ATG of uORFS was mutated to TGA. Continue to use psi-CHECK2-INSR-5'UTR-uORFLmut vector as a template, using primer set: uORFS-mut-F (the sequence is shown as SEQ ID NO. 7) and ORFS-mut-R (the sequence is shown as SEQ ID NO. 8) to construct psi-CHECK2-INSR-5'UTR-uORFL+Smut vector in which the ATG of uORFL and uORFS was mutated to TGA. Example 2, Detection of reporter gene activity of uORF elements upstream of INSR gene

[0038] The experiment was divided into four groups:

[0039] Group 1 of the experiment was to transfect psi-CHECK2-INSR-5'UTR-WT (hereinafter abbreviated as WT) in chicken immortalized preadipocytes (ICP2) or DF1 cells;

[0040] Group 2 of the experiment was to transfect psi-CHECK2-INSR-5'UTR-uORFLmut (hereinafter abbreviated as uORFLmut) in chicken immortalized preadipocytes (ICP2) or DF1 cells;

[0041] Group 3 of the experiment was to transfect psi-CHECK2-INSR-5'UTR-uORFSmut (hereinafter abbreviated as uORFSmut) in chicken immortalized preadipocytes (ICP2) or DF1 cells;

[0042] Experimental group 4 was to transfect psi-CHECK2-INSR-5'UTR-uORFL+Smut (hereinafter abbreviated as uORFL+Smut) into chicken immortalized preadipocytes (ICP2) or DF1 cells.

[0043] Chicken immortalized preadipocytes (ICP2) were cultured in DMEM / F12 complete medium, and DF1 cells were cultured in DMEM high glucose complete medium. The medium was changed 6 hours after transfection, and reporter gene activity (Renilla / Firefly) was detected 48 hours later. The results are shown in the table. Figure 2 .

[0044] In DF1 and ICP2, the reporter gene activity of the uORFL+Smut vector was the highest, followed by the uORFLmut vector and the uORFSmut vector, and the reporter gene activity of the WT vector was the lowest. In DF1, the reporter gene activity of the uORFL+Smut vector was ∼75.16 times that of the WT vector; the reporter gene activity of the uORFLmut vector was ∼26.25 times that of the WT vector; and the reporter gene activity of the uORFSmut vector was ∼5.78 times that of the WT vector ( Figure 2 A). In ICP2, the reporter gene activity of the uORFL+Smut vector was ∼32.85-fold higher than that of the WT vector; the reporter gene activity of the uORFLmut vector was ∼5.76-fold higher than that of the WT vector; and the reporter gene activity of the uORFSmut vector was ∼2.31-fold higher than that of the WT vector ( Figure 2 B).

[0045] In order to determine that the change in the activity of the vector reporter gene that causes the uORF start codon mutation is uORF-mediated post-transcriptional regulation, the INSR-5'UTR inserted upstream of the sea renilla is excluded as a promoter-mediated transcriptional regulation that affects the change in reporter gene activity. The WT, uORFLmut, uORFSmut, and uORFL+Smut vectors were transfected into ICP2 and DF1 respectively using the same method. Cell RNA was collected after 48 hours, and the mRNA expression of sea renilla and firefly was detected by qPCR. The specific primer sequences are shown in SEQ ID NO.9-12. Firefly was used as the internal reference gene, and the relative expression of sea renilla mRNA was calculated by -△△ct. qPCR found that in DF1, the relative mRNA expression of sea renilla overexpressing the uORFSmut vector was significantly lower than that of the other three vectors (P < 0.01, Figure 3 A); In ICP2, the relative mRNA expression level of Renilla overexpressing uORFSmut vector was significantly lower than that of the other three vectors (P<0.05, Figure 3 B) There was no significant difference in the relative expression levels of Renilla mRNA among overexpression of WT, uORFLmut, and uORFL+Smut vectors.

[0046] Conclusion: The results of the reporter gene showed that the mutation of the start codon of the uORF increased the relative expression of the sea urchin protein, but had no significant effect or even inhibited the relative expression of the sea urchin mRNA. Therefore, the results can indicate that the uORF of INSR can negatively regulate the expression of INSR through post-transcriptional regulation, that is, the uORF element upstream of the INSR gene regulates the expression of INSR.

Claims

1. A uORF element for regulating the expression of chicken insulin receptor, characterized in that: Its nucleotide sequence is shown in any one of SEQ ID NO.1-2.

2. A PCR primer set for amplifying a uORF element regulating chicken insulin receptor expression as claimed in claim 1, characterized in that: The sequence of primer 5'UTR-F is shown in SEQ ID NO. 3, and the sequence of primer 5'UTR-R is shown in SEQ ID NO.

4.

3. A recombinant vector, characterized in that The invention comprises a uORF element for regulating the expression of chicken insulin receptor as claimed in claim 1.

4. The method for constructing a recombinant vector according to claim 3, wherein: Here are the steps: Step S1: using a PCR primer set: the sequence of 5'UTR-F is shown in SEQ ID NO.3 and the sequence of 5'UTR-R is shown in SEQ ID NO.4, PCR amplifying the uORF element regulating the expression of chicken insulin receptor, and collecting the PCR product; Step S2: Using the psi-CHECK2 vector as a substrate, single enzyme digestion was performed using the NheⅠ restriction endonuclease, and the PCR product was connected to the upstream of the Renilla CDs region of the psi-CHECK2 linearized vector by homologous recombination to construct the psi-CHECK2-INSR-5'UTR-WT recombinant vector.

5. The use of a uORF element for regulating the expression of chicken insulin receptor according to claim 1, comprising any one of the following: (1) regulating the expression of firefly luciferase in eukaryotic cells; the eukaryotic cells are chicken embryo fibroblasts and chicken preadipocytes; (2) being used in chicken genetic breeding, wherein the genetic breeding is to cultivate broilers with reduced insulin resistance; (3) being used in the preparation of a reagent for regulating the expression of chicken insulin receptor.

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