SiRNA for targeted inhibition of chicken PLCB2 gene expression and application thereof

Abstract

The invention belongs to the technical field of poultry breeding, and discloses siRNA for targeted inhibition of chicken PLCB2 gene expression and application thereof. The siRNA comprises the following components: PLCB2-409, PLCB2-2566 and PLCB2-3239; the siRNA is capable of inhibiting the expression of the chicken PLCB2 gene in a targeted manner, the expression of the chicken PLCB2 gene can be effectively reduced after the siRNA is transferred into chicken primary subcutaneous fat cells, and the inhibition efficiency reaches 50% or above; the siRNA identifies the gene function by specifically knocking down the expression of the chicken PLCB2 gene in vitro, can be used as a molecular marker to be applied to genetic breeding of subcutaneous fat deposition of 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 PLCB2 gene expression and an application thereof. Background Art

[0002] Phospholipase Cβ2 (PLCB2) is an important member of the phospholipase C (PLC)β family. The research on PLCB2 is currently focused on human diseases. Through the study of key genes and molecular mechanisms that mediate atherosclerosis and abnormal shear stress in endothelial cells, it was found that PLCB2 can be used as a key gene to effectively distinguish atherosclerotic plaques from normal arteries. PLCB2 can regulate the expression levels of Bcl-2, Bax, caspase-3 and p53 by affecting the activation of the Ras / Raf / MAPK signaling pathway, thereby affecting cell viability and apoptosis. It is believed that PLCB2 may be a potential target gene for the treatment of melanoma. In poultry, there are few reports on the study of PLCB2 gene, and there is no related report on the use of siRNA to knock down the expression of chicken PLCB2 gene in the prior art. Therefore, the use of siRNA to knock down the expression of chicken PLCB2 gene and determine the role of PLCB2 in chicken subcutaneous fat deposition can be used as a molecular marker for molecular genetic breeding of subcutaneous fat deposition in high-quality broilers, shorten the generation interval, and increase breeding progress. Summary of the invention

[0003] The purpose of the present invention is to solve the deficiencies in the prior art and provide a siRNA for targeted inhibition of chicken PLCB2 gene expression and its application. After being transferred into chicken primary subcutaneous adipocytes, the expression of chicken PLCB2 gene can be effectively reduced, and the inhibition efficiency is more than 50%, and the knockdown efficiency is very high.

[0004] In order to achieve the above object, the present invention is achieved through the following technical solutions:

[0005] In a first aspect, the present invention provides a siRNA for inhibiting the expression of chicken PLCB2 gene, wherein the siRNA includes PLCB2-409, PLCB2-2566, PLCB2-3239,

[0006] The sequence of the PLCB2-409 sense strand is GAGGUGAAGGAGUAUCUGUTT, and the sequence of the antisense strand is ACAGAUACUCCUUCACCUCTT, and each siRNA sequence is suffixed with a TT base overhang;

[0007] The sequence of the sense strand of PLCB2-2566 is AACAAAAGGUUCUUCUUUCCATT, and the sequence of the antisense strand is GAAAGAAGAACCUUUUGUUUUTT, and each siRNA sequence is suffixed with a TT base overhang;

[0008] The sense strand sequence of the PLCB2-3239 is UCAAACUCCUCUUCUUCUGGGTT, and the antisense strand sequence is CAGAAGAAGAGGAGUUUGACGTT, and each siRNA sequence is suffixed with a TT base overhang.

[0009] In a second aspect, the present invention provides the use of the siRNA targeting and inhibiting the expression of chicken PLCB2 gene in the genetic breeding of chicken subcutaneous fat deposition.

[0010] Preferably, the application is specifically: designing siRNA for chicken PLCB2 gene expression, transfecting the siRNA into chicken primary subcutaneous adipocytes, and using the siRNA to specifically knock down chicken PLCB2 gene expression.

[0011] Preferably, the designed siRNA expressing chicken PLCB2 is diluted with 125ul of DEPC water per 1OD of siRNA to a final concentration of 20uM / ul, and is stored at -20°C for later use.

[0012] Preferably, the siRNA is transfected into primary chicken subcutaneous adipocytes, and the specific steps are as follows:

[0013] Primary chicken subcutaneous adipocytes isolated and extracted from subcutaneous fat tissue of 21-day-old chickens were used as transfected cells. The isolated and extracted primary chicken subcutaneous adipocytes were inoculated into a 12-well culture plate. When the cell density grew to 60-70%, the original culture medium in the culture dish was aspirated and replaced with fresh serum-free culture medium. The siRNA targeting the chicken PLCB2 gene was transfected into the primary chicken subcutaneous adipocytes with the help of the transfection reagent Lipofectamine 3000.

[0014] The specific transfection system is as follows: 175uL opti-MEM medium + 5ul siRNA with a final concentration of 20uM + 8ul Hiperfect transfection; incubate for 10 minutes to form a transfection complex, and transfect into the culture medium.

[0015] The present invention has the following beneficial effects: provides a siRNA capable of targeting and inhibiting the expression of the chicken PLCB2 gene, which can effectively reduce the expression of the chicken PLCB2 gene after being transferred into primary subcutaneous 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 PLCB2 gene in vitro, can be used as a molecular marker in the genetic selection of subcutaneous fat deposition in high-quality broilers, and provides 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

[0016] Figure 1 The expression of PLCB2 in subcutaneous adipose tissue of chickens of different breeds (lines); (different letters indicate significant differences in the same breed (line) at different developmental stages, P < 0.05, the same letters indicate no significant differences, P > 0.05; ** indicates extremely significant differences in different breeds (lines) at the same week of age, P < 0.01);

[0017] Figure 2 is the expression of PLCB2 in subcutaneous adipocytes (different letters indicate significant differences, P < 0.05);

[0018] Figure 3 The expression changes of PLCB2 gene after exogenous transfection of PLCB2 siRNA (different letters represent significant differences (P < 0.05), and the same letters represent no significant differences (P > 0.05));

[0019] Figure 4 The proliferation changes of subcutaneous adipocytes after exogenous transfection of PLCB2 siRNA (** indicates extremely significant difference, P < 0.01);

[0020] Figure 5 The changes in lipid droplet deposition in subcutaneous adipocytes after exogenous transfection of PLCB2 siRNA. DETAILED DESCRIPTION

[0021] 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.

[0022] Example 1 Detection of PLCB2 gene expression in chicken subcutaneous adipose tissue and subcutaneous adipocytes by fluorescence quantitative PCR

[0023] 1.1 Expression of PLCB2 gene in subcutaneous adipose tissue

[0024] The expression of PLCB2 in subcutaneous adipose tissue of recessive white-feathered chickens and sex-linked dwarf chickens at different developmental stages was detected by QPCR technology. Figure 1 As shown, there are significant breed and developmental differences in the expression level of PLCB2 in subcutaneous adipose tissue of different strains at different developmental stages, indicating that PLCB2 is involved in the process of subcutaneous fat deposition in chickens.

[0025] 1.2 Expression of PLCB2 gene in adipocytes

[0026] QPCR technology was used to detect the expression of PLCB2 gene in the proliferation and differentiation stages of subcutaneous adipocytes. Figure 2 As shown, the expression of PLCB2 gene in the proliferation stage of subcutaneous adipocytes was significantly higher than that in the differentiation stage (P < 0.05), which preliminarily indicated that PLCB2 gene may have the effect of inhibiting the deposition of subcutaneous fat in chickens.

[0027] Example 2 Screening of the best siRNA sequence targeting chicken PLCB2 gene

[0028] 2.1 PLCB2 siRNA design

[0029] According to the NCBI online database, the mRNA sequence information of the chicken PLCB2 gene was obtained (accession number: XM_040672597.2), and si Catch TM siRNA design software was used to design 3 pairs of siRNAs targeting the CDS region of the chicken PLCB2 gene, and each siRNA sequence was suffixed with a TT base overhang. The designed siRNA was synthesized by Suzhou Jima Biotechnology Co., Ltd., and 125ul of DEPC water was added to dilute each 1OD of the synthesized siRNA, with a final concentration of 20uM / ul, and stored at -20°C for use. The sequences of the 3 pairs of siRNAs designed are shown in Table 1.

[0030] Table 1 Three pairs of siRNA sequences

[0031]

[0032]

[0033] 2.2si RNA transfection of primary chicken subcutaneous adipocytes

[0034] Chicken subcutaneous adipocytes isolated and extracted from subcutaneous adipose tissue of 21-day-old chickens were used as transfected cells. The isolated and extracted primary chicken subcutaneous adipocytes were inoculated into a 12-well culture plate, and when the cell density grew to 60-70%, the original culture medium in the culture dish was aspirated and replaced with fresh serum-free culture medium, and three pairs of siRNAs targeting chicken PLCB2 gene and negative control siRNA (NC) were transfected into chicken subcutaneous adipocytes respectively with the help of transfection reagent Lipofectamine 3000, and 3 replicate wells were set in each group.

[0035] The specific transfection system is as follows: experimental group: 175uL opti-MEM culture medium + 5ul final concentration of 20uM siRNA + 8ul Hiperfect transfection; control group: 175uL opti-MEM culture medium + 5ul final concentration of 20uM Negative + 8ul Hiperfect transfection, incubate for 10 minutes to form a transfection complex, and transfect into the culture medium.

[0036] 2.3 RNA extraction and cDNA preparation

[0037] After 24 hours of transfection, each well was washed 3 times with PBS, and 0.6 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 PLCB2 siRNA transfected 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 Dalian Bao Bioengineering Co., Ltd.

[0038] 2.4 Real-time fluorescence quantitative PCR detection of siRNA interference efficiency

[0039] 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 PLCB2 gene expression, with 3 replicates for each sample. Q-PCR reaction program: 95℃15min; 40 cycles (94℃15s, 55℃30s, 70℃30s).

[0040] The primer sequences for PLCB2 gene fluorescence quantitative PCR are as follows:

[0041] Upstream primer (SEQ ID NO: 7): 5'-TGCCAATTCCATCAATCCTGT-3'

[0042] Downstream primer (SEQ ID NO: 8): 5'-ATAGCCTGAGTGTACTGCAA-3'

[0043] The sequences of the internal reference GAPDH gene fluorescence quantitative PCR primers are as follows:

[0044] Upstream primer (SEQ ID NO: 9): 5'-AGAAGGCTGGGGCTCATCT-3'

[0045] Downstream primer (SEQ ID NO: 10): 5'-CAATGCCAAAGTTGTCATG-3'

[0046] The efficiency of 3 pairs of siRNA in specifically knocking down chicken PLCB2 gene expression is as follows Figure 3 As shown. Compared with the control group, the three pairs of siRNA, PLCB2-409 and PLCB2-3329 siRNA, could significantly inhibit the expression of PLCB2 gene in subcutaneous adipocytes (P<0.05), among which PLCB2-409 siRNA had the best knockdown efficiency, with an inhibition efficiency of more than 50%. PLCB2-409 was selected in subsequent experiments to study the function of PLCB2 gene in chicken subcutaneous fat deposition. The sense strand of PLCB2-409 is GAGGUGAAGGAGUAUCUGUTT, and the antisense strand is ACAGAUACUCCUUCACCUCTT.

[0047] 2.5 Effect of PLCB2 gene knockdown on the expression of cell proliferation and differentiation-related genes

[0048] After 24h and 72h of PLCB2-409 siRNA transfection, the CCK8 kit was used to detect the changes in cell proliferation. Figure 4 As shown in the figure, after transfection with PLCB2-409 siRNA, the proliferation of chicken primary subcutaneous adipocytes increased significantly (P<0.05). Oil red O staining 4 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 PLCB2 gene has the effect of promoting the proliferation of chicken subcutaneous fat cells and inhibiting the lipid deposition of subcutaneous fat cells.

[0049] 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 PLCB2 gene, characterized in that: The siRNA includes PLCB2-409, PLCB2-2566, PLCB2-3239, The sequence of the PLCB2-409 sense strand is GAGGUGAAGGAGUAUCUGUTT, and the sequence of the antisense strand is ACAGAUACUCCUUCACCUCTT, and each siRNA sequence is suffixed with a TT base overhang; The sequence of the sense strand of PLCB2-2566 is AACAAAAGGUUCUUCUUUCCATT, and the sequence of the antisense strand is GAAAGAAGAACCUUUUGUUUUTT, and each siRNA sequence is suffixed with a TT base overhang; The sense strand sequence of the PLCB2-3239 is UCAAACUCCUCUUCUUCUGGGTT, and the antisense strand sequence is CAGAAGAAGAGGAGUUUGACGTT, and each siRNA sequence is suffixed with a TT base overhang.

2. Use of the siRNA targeting and inhibiting chicken PLCB2 gene expression according to claim 1 in genetic breeding of chicken subcutaneous fat deposition.

3. The use according to claim 2, characterized in that: The application is specifically as follows: designing siRNA for chicken PLCB2 gene expression, transfecting the siRNA into chicken primary subcutaneous adipocytes, and using the siRNA to specifically knock down chicken PLCB2 gene expression.

4. The use according to claim 3, characterized in that: The designed siRNA expressing chicken PLCB2 was diluted with 125ul of DEPC water per 1OD of siRNA to a final concentration of 20uM / ul and stored at -20°C until use.

5. The use according to claim 3, characterized in that: The siRNA is transfected into primary chicken subcutaneous adipocytes, and the specific steps are as follows: Primary chicken subcutaneous adipocytes isolated and extracted from subcutaneous fat tissue of 21-day-old chickens were used as transfected cells. The isolated and extracted primary chicken subcutaneous adipocytes were inoculated into a 12-well culture plate. When the cell density grew to 60-70%, the original culture medium in the culture dish was aspirated and replaced with fresh serum-free culture medium. The siRNA targeting the chicken PLCB2 gene was transfected into the primary chicken subcutaneous adipocytes with the help of the transfection reagent Lipofectamine 3000. The specific transfection system is as follows: 175uL opti-MEM medium + 5ul siRNA with a final concentration of 20uM + 8ul Hiperfect transfection; incubate for 10 minutes to form a transfection complex, and transfect into the culture medium.

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