siRNA targeting and inhibiting chicken FBXW7 expression and its application

Abstract

The invention belongs to the technical field of poultry breeding, and discloses a siRNA for targeted inhibition of chicken FBXW7 expression and an application thereof; the siRNA comprises FBXW7-834; the siRNA capable of targeted inhibition of chicken FBXW7 gene expression can effectively reduce the expression of the chicken FBXW7 gene after being transferred into primary abdominal fat cells of chickens, with an inhibition efficiency of more than 50%, and a very high knockdown efficiency; the siRNA identifies the gene function by specifically knocking down the expression of the chicken FBXW7 gene in vitro, can be used as a molecular marker in the genetic selection of abdominal fat deposition in high-quality broilers, and has great economic value and scientific research value.

Description

Technical Field

[0001] The invention belongs to the technical field of poultry breeding, and particularly relates to a siRNA for targeted inhibition of chicken FBXW7 expression and an application thereof. Background Art

[0002] The F-box and WD repeat domain 7 (FBXW7) gene is a SCF E3 ubiquitin ligase that plays an important role in cell survival, cell proliferation, tumor therapy, DNA damage repair, regulation of cell senescence, and targeted cancer therapy. FBXW7 can regulate PGC-1α stability and mitochondrial homeostasis, and FBXW7 may be one of the potential targets for the treatment of sarcopenic obesity. In poultry, there are few studies on FBXW7. In addition, there are no reports on the use of siRNA to interfere with the expression of chicken FBXW7 gene in the prior art. Summary of the invention

[0003] In view of the technical problems existing in the above-mentioned prior art, the present invention provides a siRNA capable of targetedly inhibiting the expression of chicken FBXW7 gene. After being transferred into primary abdominal fat cells of chicken, the expression of chicken FBXW7 gene can be effectively reduced, and the inhibition efficiency is more than 50%, and the knockdown efficiency is very high.

[0004] The objective of the present invention is achieved through the following technical solutions:

[0005] In a first aspect, the present invention provides a siRNA that targets and inhibits the expression of chicken FBXW7, wherein the siRNA is FBXW7-834, FBXW7-615 or FBXW7-1057;

[0006] The FBXW7-834 includes a primer having a nucleotide sequence as shown in SEQ ID NO.1 and a primer having a nucleotide sequence as shown in SEQ ID NO.2;

[0007] The FBXW7-615 includes a primer having a nucleotide sequence as shown in SEQ ID NO.3 and a primer having a nucleotide sequence as shown in SEQ ID NO.4;

[0008] The FBXW7-1057 includes a primer having a nucleotide sequence shown in SEQ ID NO.5 and a primer having a nucleotide sequence shown in SEQ ID NO.6.

[0009] The sequence is shown in the following table:

[0010]

[0011]

[0012] In a second aspect, the present invention provides the use of the siRNA targeting and inhibiting the expression of chicken FBXW7 in the genetic selection of abdominal fat deposition in high-quality broiler chickens.

[0013] The present invention has the following beneficial effects: providing a siRNA capable of targeting and inhibiting the expression of the chicken FBXW7 gene, which can effectively reduce the expression of the chicken FBXW7 gene after being transferred into primary abdominal fat cells of chickens, with an inhibition efficiency of more than 50%, and a high knockdown efficiency. The siRNA of the present invention identifies the gene function by specifically knocking down the expression of the chicken FBXW7 gene in vitro, and can be used as a molecular marker in the genetic selection of abdominal fat deposition in high-quality broilers and provide reference data for the study of metabolic diseases caused by obesity in humans, etc., and has great economic value and scientific research value. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The expression of FBXW7 in abdominal fat tissue of different strains of chickens (different letters represent significant differences (P < 0.05), the same letters represent insignificant differences (P > 0.05); ** represents significant differences (P < 0.01)); S3, H, and F analysis represent S3 strain chickens, H strain chickens, and F strain chickens;

[0015] Figure 2 is the expression of FBXW7 in abdominal fat adipocytes (different letters indicate significant differences (P<0.05));

[0016] Figure 3 The expression changes of FBXW7 gene after exogenous transfection of FBXW7 siRNA (different letters represent significant differences (P<0.05), and the same letters represent no significant differences (P>0.05));

[0017] Figure 4 The changes in the proliferation of abdominal adipocytes after exogenous transfection of FBXW7 siRNA (* indicates significant difference (P < 0.05));

[0018] Figure 5 Changes in lipid droplet deposition in abdominal adipocytes after exogenous transfection of FBXW7 siRNA. DETAILED DESCRIPTION

[0019] The present invention is further described and defined by the following examples but is not limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified. The present invention is further described below in conjunction with the accompanying drawings.

[0020] Example 1 Detection of FBXW7 gene expression in abdominal adipose tissue and cells by fluorescence quantitative PCR

[0021] 1.1 Expression of FBXW7 in abdominal adipose tissue

[0022] QPCR technology was used to detect the expression of FBXW7 in abdominal adipose tissue of different strains of chickens (S3, H, and F) at different developmental stages. Figure 1 As shown, there are significant breed and developmental differences in the expression level of FBXW7 in abdominal fat tissue of different strains at different developmental stages, indicating that FBXW7 is involved in the process of abdominal fat deposition in chickens.

[0023] 1.2 Expression of FBXW7 in adipocytes

[0024] QPCR technology was used to detect the expression of FBXW7 in the proliferation and differentiation stages of abdominal adipocytes. Figure 2 As shown, the expression of FBXW7 in the proliferation stage of myoblasts was higher than that in the differentiation stage, among which the expression level of 50% in the proliferation stage was the highest and significantly higher than that of 100% in the proliferation stage and each differentiation stage (P<0.01), and there was no significant difference between 50% in the proliferation stage and 100% in the proliferation stage (P>0.05). This preliminarily indicates that FBXW7 may have the effect of inhibiting abdominal fat deposition.

[0025] Case 2 Screening of the best siRNA sequence targeting chicken FBXW7 gene

[0026] 2.1 FBXW7 siRNA design

[0027] According to the NCBI online database, the mRNA sequence information of the chicken FBXW7 gene was obtained (accession number: XM_046915934.1), and si Catch TM siRNA design software was used to design 3 pairs of siRNAs targeting the CDS region of the chicken FBXW7 gene, and each siRNA sequence was suffixed with a TT base overhang. The designed siRNA was synthesized by Suzhou Jima Biotechnology Co., Ltd. After synthesis, each 1OD of siRNA was diluted with 125ul of DEPC water, and the final concentration was 20uM / ul, and stored at -20°C for use. The sequences of the 3 pairs of siRNAs designed are shown in Table 1.

[0028] Table 1 Three pairs of siRNA sequences

[0029]

[0030] 2.2siRNA transfection of primary chicken abdominal adipocytes

[0031] Chicken abdominal adipocytes isolated and extracted from abdominal adipose tissue of 14-day-old chickens were used as transfected cells. The isolated and extracted chicken primary adipocytes were inoculated into 12-well culture plates. When the cell density grew to 70%, the original culture medium in the culture dish was aspirated and replaced with fresh culture medium. Three pairs of siRNA targeting chicken FBXW7 gene and negative control siRNA (NC) were transfected into chicken adipocytes respectively with the help of transfection reagent Lipofectamine 3000, and 3 replicate wells were set in each group.

[0032] The specific transfection system is as follows: experimental group: 150ul opti-MEM culture medium + 5ul siRNA with a final concentration of 20uM + 8ul Hiperfect transfection; control group: 100ul opti-MEM culture medium + 5ul siRNA with a final concentration of 20uM + 8ul Hiperfect transfection, incubate for 8 minutes to form a transfection complex, and transfect into the culture medium.

[0033] 2.3 RNA extraction and cDNA preparation

[0034] After 24 hours of transfection, each well was washed 3 times with PBS, and 0.8 ml of TROZL was added to the culture dish to collect the cells. The RNA isolater Total RNA Extraction Reagent of Nanjing Novozyme Biotechnology Co., Ltd. was used to extract the total RNA of the cells in the transfected FBXW7 si RNA group and the control group. The RNA concentration was determined by a nucleic acid quantifier. cDNA synthesis was performed according to the instructions of the reverse transcription kit of Shanghai Shenggong Biotechnology Co., Ltd.

[0035] 2.4 Real-time fluorescence quantitative PCR detection of siRNA interference efficiency

[0036] Hi ScriptⅢRT Super Mix for qPCR (+g DNA wiper) reagent from Nanjing Novozyme Biotechnology Co., Ltd. was selected, and the SYBR Green Ι method was used for real-time fluorescence quantitative PCR to detect the efficiency of siRNA specific interference with chicken FBXW7 gene expression. Three replicates were set for each sample. Q-PCR reaction program: 95℃15min; 40 cycles (94℃15s, 55℃30s, 70℃30s).

[0037] Upstream primer (SEQ ID NO.7): 5'-TCTTGACACCTCCATCCGAGT-3'

[0038] Downstream primer (SEQ ID NO.8): 5'-ACTGTAGAATCAGCGTTCCCA-3'

[0039] 2.5 The primer pair nucleic acid sequences for detecting chicken internal reference GAPDH gene expression are as follows:

[0040] Upstream primer (SEQ ID NO.9): 5'-AGAAGGCTGGGGCTCATCT-3'

[0041] Downstream primer (SEQ ID NO.10): 5'-CAATGCCAAAGTTGTCATG-3'

[0042] The efficiency of 3 pairs of siRNA in specifically knocking down chicken FBXW7 gene expression is as follows Figure 3 As shown. Figure 3 It can be seen that compared with the control group, the three pairs of siRNA can significantly inhibit the expression of FBXW7 gene in cells (P<0.05), among which FBXW7-834 siRNA has the best interference efficiency, with an inhibition efficiency of more than 50%. FBXW7-834 was selected in subsequent experiments to study the function of FBXW7 gene in chicken fat deposition. The sense strand of FBXW7-834 is GGGAGGAACGGAGAGAUAATT, and the antisense strand is UUAUCUCUCCGUUCCUCCCTT.

[0043] 2.6 Effects of FBXW7 gene knockdown on the expression of cell proliferation and differentiation-related genes

[0044] After 24h and 72h of FBXW7-834 siRNA transfection, the CCK8 kit was used to detect the changes in cell proliferation. Figure 4 As shown in the figure, after transfection with FBXW7-834 siRNA, the proliferation of chicken primary adipocytes was significantly increased (P<0.05). Oil red O staining 3 days after transfection showed that the deposition of lipid droplets in adipocytes was significantly lower than that in the control group (P<0.01). Figure 5 The experiment proved that the FBXW7 gene can promote the proliferation of abdominal fat cells in chickens and inhibit the fat deposition of abdominal fat cells.

[0045] The above description is only a preferred embodiment of the present invention, but is not intended to limit the present invention. Those skilled in the art may make possible changes and modifications to the present invention using the technical contents disclosed above without departing from the scope of the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. A siRNA targeting and inhibiting the expression of chicken FBXW7, characterized in that: The siRNA is FBXW7-834, the sequence of its sense strand is GGGAGGAACGGAGAGAUAATT, the sequence of its antisense strand is UUAUCUCUCCGUUCCUCCCTT, and each siRNA sequence is suffixed with a TT base overhang.

2. Use of the siRNA targeting and inhibiting the expression of chicken FBXW7 as claimed in claim 1 in the genetic breeding of abdominal fat deposition in high-quality broiler chickens.

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