SiRNA for targeting and inhibiting expression of chicken PLCB2 gene and application thereof

Abstract

The application 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 PLCB2-409, PLCB2-2566 and PLCB2-3239; the siRNA for targeted inhibition of chicken PLCB2 gene expression can effectively reduce the expression of the chicken PLCB2 gene after being converted into chicken primary subcutaneous adipose cells, and the inhibition efficiency is more than 50%; the siRNA can be used for identifying gene functions by specifically knocking down the expression of the chicken PLCB2 gene in vitro, and can be applied to genetic selection of high-quality broiler subcutaneous fat deposition as a molecular marker, and has great economic value and scientific research value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of poultry breeding, and particularly relates to siRNA for targeted inhibition of chicken PLCB2 gene expression and application thereof. BACKGROUND

[0002] Phospholipase C beta 2 (PLCB2) is an important member of the phospholipase C (PLC) beta family. The research on PLCB2 is currently mostly focused on human diseases. Through the research on key genes and molecular mechanisms mediating atherosclerosis and abnormal shear stress of endothelial cells, it is found that PLCB2 can be used as a key gene for effectively distinguishing atherosclerotic plaques and 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, and it is considered that PLCB2 can be a potential target gene for treating melanoma. On poultry, there are few reports on PLCB2 gene, and there is no related report on knocking down chicken PLCB2 gene expression by using siRNA in the prior art. Therefore, the use of siRNA to knock down chicken PLCB2 gene expression can be used as a molecular marker for molecular genetic selection of high-quality broiler subcutaneous fat deposition, shorten the generation interval, and increase the selection progress. SUMMARY

[0003] The purpose of the present application is to solve the problems in the prior art, and to provide siRNA for targeted inhibition of chicken PLCB2 gene expression and application thereof. After being transferred into chicken primary subcutaneous fat cells, the siRNA can effectively reduce the expression of chicken PLCB2 gene, and the inhibition efficiency is more than 50%, and the knockdown efficiency is very high.

[0004] In order to achieve the above purpose, the present application is realized by the following technical scheme:

[0005] In the first aspect, the present application provides siRNA for targeted inhibition of chicken PLCB2 gene expression, wherein the siRNA comprises PLCB2-409, PLCB2-2566 and PLCB2-3239,

[0006] The sequence of the positive strand of the PLCB2-409 is GAGGUGAAGGAGUAUCUGUTT, and the sequence of the negative strand is ACAGAUACUCCUUCACCUCTT. A TT base overhang is added to the suffix of each siRNA sequence.

[0007] The sequence of the sense strand of the PLCB2-2566 is AACAAAAGGUUCUUCUUUCCATT, and the sequence of the antisense strand is GAAAGAAGAACCUUUUGUUUUTT, and a TT base overhang is added at the end of each siRNA sequence.

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

[0009] In a second aspect, the application provides the siRNA for targeting and inhibiting the expression of the chicken PLCB2 gene in the genetic selection of chicken subcutaneous fat deposition.

[0010] Preferably, the application specifically refers to designing siRNA for targeting and inhibiting the expression of the chicken PLCB2 gene, transfecting siRNA into chicken primary subcutaneous fat cells, and using siRNA to specifically knock down the expression of the chicken PLCB2 gene.

[0011] Preferably, the designed siRNA for targeting and inhibiting the expression of the chicken PLCB2 gene is diluted with 125ul DEPC water to a final concentration of 20uM / ul, and stored at -20℃ for later use.

[0012] Preferably, the siRNA is transfected into chicken primary subcutaneous fat cells according to the following specific steps:

[0013] The chicken primary subcutaneous fat cells isolated and extracted from the subcutaneous fat tissue of 21-day-old chickens are used as transfection cells. The isolated and extracted chicken primary subcutaneous fat cells are inoculated into 12-well culture plates. When the cell density grows to 60-70%, the original culture medium in the culture dish is removed and replaced with fresh serum-free culture medium. The siRNA targeting the chicken PLCB2 gene is transfected into the chicken primary subcutaneous fat cells 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 then transfect into the culture medium.

[0015] The application has the following beneficial effects: the siRNA provided in the application can target and inhibit the expression of chicken PLCB2 gene, and can effectively reduce the expression of chicken PLCB2 gene after being introduced into chicken primary subcutaneous adipocytes, and the inhibition efficiency is more than 50%, and the knockdown efficiency is high. The siRNA in the application can be used for genetic selection of high-quality broiler subcutaneous fat deposition by specifically knocking down the expression of chicken PLCB2 gene in vitro to identify gene function, and can provide reference data for research on human metabolic diseases caused by obesity, and has great economic value and scientific research value. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 PLCB2 expression in different varieties (lines) of chicken subcutaneous fat tissues; (different letters represent significant differences in different development periods of the same variety (line), P<0.05, and the same letter represents no significant difference, P>0.05; ** represents extremely significant difference in the same age of different varieties (lines), P<0.01);

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

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

[0019] Figure 4 Subcutaneous adipocyte proliferation changes after exogenous transfection of PLCB2 siRNA (** represents extremely significant difference, P<0.01);

[0020] Figure 5 Subcutaneous adipocyte lipid droplet deposition changes after exogenous transfection of PLCB2 siRNA. DETAILED DESCRIPTION

[0021] The application is further illustrated and defined by the following examples without limitation. The experimental methods in the following examples are conventional methods unless otherwise specified. The application is further illustrated below with reference to the accompanying drawings.

[0022] Example 1: Fluorescent quantitative PCR detection of PLCB2 gene expression in chicken subcutaneous fat tissues and subcutaneous adipocytes

[0023] 1.1 PLCB2 gene expression in subcutaneous fat tissues

[0024] QPCR technology was used to detect the expression of PLCB2 in the subcutaneous fat tissues of recessive white-feathered chickens and sex-linked dwarf chickens at different development periods. As shown in Figure 1, the expression of PLCB2 in the subcutaneous fat tissues of recessive white-feathered chickens was significantly higher than that in the subcutaneous fat tissues of sex-linked dwarf chickens at the same development period (P<0.05).Figure 1 As shown in Fig. 1, the expression of PLCB2 in subcutaneous adipose tissue of different breeds and different development stages was significantly different, indicating that PLCB2 was involved in the process of chicken subcutaneous fat deposition.

[0025] 1.2 Expression of PLCB2 gene in fat cells

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

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

[0028] 2.1 Design of PLCB2 siRNA

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

[0030] Table 1 Sequences of 3 pairs of siRNA

[0031]

[0032]

[0033] 2.2 Transfection of siRNA into primary chicken subcutaneous fat cells

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

[0035] ​The specific transfection system is as follows: test group: 175 uL opti-MEM medium + 5 ul final concentration of 20 uM siRNA + 8 ul Hiperfect transfection; control group: 175 uL opti-MEM medium + 5 ul final concentration of 20 uM Negative + 8 ul 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 transfection for 24 h, each well was washed with PBS for 3 times, 0.6 ml TROZL was added to the culture dish, and the cells were collected. The total RNA of the transfection PLCB2 siRNA group and the control group cells was extracted by selecting the RNA isolater Total RNA Extraction Reagent kit of Nanjing Nuo Weizan Biological Technology Co., Ltd. The RNA concentration was determined by nucleic acid quantifier. The cDNA synthesis was performed according to the reverse transcription kit instruction of Dalian Bao Biological Engineering Co., Ltd.

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

[0039] The Hi Script III RT Super Mix for qPCR (+g DNA wiper) reagent of Nanjing Nuo Weizan Biological Technology Co., Ltd. was selected, and the SYBR Green I method was used for real-time fluorescent quantitative PCR detection of the efficiency of siRNA specific interference with chicken PLCB2 gene expression, and 3 repeats were set for each sample. The Q-PCR reaction program: 95℃ 15 min; 40 cycles (94℃ 15 s, 55℃ 30 s, 70℃ 30 s).

[0040] The primer sequence of PLCB2 gene fluorescent quantitative PCR is as follows:

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

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

[0043] The primer sequence of the reference GAPDH gene fluorescent quantitative PCR is 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 siRNAs in knocking down the expression of chicken PLCB2 gene is shown in Table 1. Figure 3 Compared with the control group, 3 pairs of siRNAs, PLCB2-409 and PLCB2-3329 siRNAs, can significantly inhibit the expression of PLCB2 gene in subcutaneous adipocytes (P<0.05), and the knockdown efficiency of PLCB2-409 siRNA is the best, with an inhibition efficiency of more than 50%. PLCB2-409 is selected for 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 changes in cell proliferation were detected by CCK8 kit. As shown in Table 2, after transfection of PLCB2-409 siRNA, the proliferation of chicken primary subcutaneous adipocytes was significantly increased (P<0.05). Figure 4 As shown in Table 3, after 4d of transfection, oil red O staining showed that the fat droplet deposition of adipocytes was significantly lower than that of the control group (P<0.01). Figure 5 The experiment proved that PLCB2 gene has the effect of promoting the proliferation of chicken subcutaneous adipocytes and inhibiting the fat deposition of subcutaneous adipocytes.

[0049] The above is only the preferred embodiment of the present application, but not to limit the present application, those skilled in the art, without departing from the scope of the present application, can use the above disclosed technical content to make possible changes and modifications, or modify equivalent changes and equivalent embodiments, without departing from the content of the present application, according to the technical essence of the above embodiments, any simple modification, equivalent change and modification, all fall within the scope of the present application.

Claims

1. A siRNA that targets and inhibits the expression of the chicken PLCB2 gene, characterized in that, The siRNA is PLCB2-409. The sequence of the PLCB2-409 positive chain is GAGGUGAAGGAGUAUCUGUTT, and the sequence of the negative chain is ACAGAUACUCCUUCACCUCTT.

2. The application of the siRNA that targets and inhibits the expression of the chicken PLCB2 gene as described in claim 1 in the genetic selection for inhibiting subcutaneous fat deposition in chickens.

Images