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A promoter, gene technology, applied in the fields of genetic engineering and molecular biology
Pending Publication Date: 2022-01-04
SHIHEZI UNIVERSITY
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At present, there is no research report on the transcriptional regulation of C / EBPZ. Determining the promoter of the chicken C / EBPZ gene is helpful for revealing the transcriptional regulation mechanism of chicken C / EBPZ, cultivating low-fat broiler chickens, and revealing the molecular mechanism behind the occurrence of various human diseases. And the development of drugs targeting C / EBPZ expression has potential application value
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Embodiment 1
[0041] The preparation of embodiment 1 chicken C / EBPZ gene promoter
[0042] 1. Chicken genomic DNA extraction
[0043] 1) Take a 7-week-old Aiba Yijia broiler chicken (AA broiler) that is normally cultured, collect 1ml of blood from the wing vein, and put the collected chicken blood into a container that has been added with 10 microliters of anticoagulant (such as 0.5mol / L Na 2 EDTA) in a 1.5ml EP tube, mix by inverting up and down.
[0044] 2) Take 20 μl of anticoagulated chicken blood in a new 1.5ml EP tube, add 445 μl 1×SET (100mM Tris-HCl pH7.5, 100mM EDT A, after autoclaving, add 1% SDS), 10 μl of PK (10 mg / ml) and 25 μl of SDS (10%), mixed well, digested at 55°C for 12 hours.
[0045] 3) Add 500ul Tris saturated phenol, mix thoroughly, and place in a shaker upside down for 10min.
[0046] 4) Centrifuge at 12000rpm for 10min, and suck the upper layer into a new EP tube.
[0083] Example 2C / EBPZ gene promoter activity analysis
[0084] 1. Activity verification of six different lengths of C / EBPZ gene promoters (A1-A6)
[0085] Chicken preadipocytes in good growth state were inoculated in 12-well cell culture plates at a seeding density of 5×10 4 1 / well, after 24h, transfect 6 kinds of wild-type C / EBPZ promoter (A1-A6) reporter gene plasmids into chicken preadipocytes respectively according to the instructions according to the Fugene HD (promega) transfection reagent as the experimental group. Transfect pGL4.10 (emptyvector, EV) as negative control group, transfect pGL3-promoter as positive control group, transfect 1 μg plasmid per well, transfect three cell wells, transfect pRL-TK as internal reference, transfect each well 0.02 μg, collect the cells after 48h, according to Promega’s The Luciferase Assay System manual was used to measure the fluorescence activity, and the results showed that the constructed 6 kinds of wild-type C / EBPZ promoter ...
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Abstract
The invention discloses a C/EBPZ gene promoter and an application thereof, and belongs to the field of gene engineering and molecular biology. The C/EBPZ gene promoter is any one of A1-A6 sequences of which the nucleotide sequences are as shown in SEQ ID NO: 1-SEQ ID NO: 6. Six chicken C/EBPZ gene promoters are obtained by using DNA extracted from chicken whole blood as a template and using primers for PCR amplification and restriction enzyme digestion, and tests prove that the six promoters all have promoter activity in chicken preadipocytes and are regulated by a specific transcription factor KLF2, so that the promoter has potential application value in revealing a transcription regulation mechanism of chicken C/EBPZ, breeding low-abdominal-fat broilers, revealing molecular mechanisms of various human diseases and developing drugs for targeting C/EBPZ expression.
Description
technical field [0001] The invention relates to the fields of genetic engineering and molecular biology, in particular to a C / EBPZ gene promoter and its application. Background technique [0002] 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, is a ubiquitous and highly conserved protein in humans and animals. Despite its C / EBP name, it is in fact not a basic leucine zipper (bZIP) protein and lacks significant homology to other C / EBP members, so it should not be considered a C / EBP Member of the EBP family. [0003] C / EBPZ was first reported as a transcription factor bound to the CCAAT box of the human heat shock protein 70 (heat shock protein 70, HSP70) promoter, and it can promote the activity of the HSP70 promoter by acting on the CCAAT box element on the HSP70 promoter. Subsequent research reports showed that C / EBPZ may not directly bind to the CCAAT box element on the HSP70 p...
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