Applications of mir-206 in inhibition of generation of triglyceride in porcine adipocytes and breeding of lean pigs
A mir-206, 1. The technology of mir-206 is applied in the field of application of mir-206 in inhibiting triglyceride production in porcine adipocytes and lean pig breeding, and can solve the problem of reducing the abundance of encoded proteins , to achieve the effect of reducing the accumulation of subcutaneous fat
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Embodiment 1
[0045] Embodiment 1, the preparation of porcine preadipocyte
[0046] Washing solution (pH7.2-7.4): NaCl 2g, KCl 0.05g, NaCl 2 HPO 4 12H 2 O 0.725g, KH 2 PO 4 0.05g, dilute to 250mL, add penicillin and streptomycin after autoclaving, so that the concentration of penicillin is 100IU / mL, and the concentration of streptomycin is 100μg / mL, and set aside at 4°C.
[0047] Digestive solution: 0.1g type I collagenase (Gibco), 2g bovine serum albumin, dissolved in 100mL ultrapure water, filtered through a 0.22μm filter membrane, collected filtrate, 10mL / tube for aliquot, and stored at -20°C for later use.
[0048] The longissimus dorsi muscle of 3-day-old Jiaxing black piglets was collected under aseptic conditions, washed with washing solution, and then cut into about 1mm 3 Tissue pieces; take the tissue pieces, immerse in the digestive solution, shake and digest at 37°C for 90-120min, then filter through a 200-mesh steel sieve and collect the filtrate; take the filtrate, centri...
Embodiment 2
[0050] Example 2. It was found that mir-206 may be an important factor for intramuscular fat deposition in Jiaxing black pigs
[0051] 1. Expression characteristics of miRNA in Jiaxing black pig adipocytes
[0052] The intramuscular preadipocytes and subcutaneous preadipocytes prepared in Example 1 were taken to induce adipogenic differentiation respectively, and then the cells were collected, fully washed with PBS buffer, and then RNA-seq deep sequencing was performed using BGISEQ-500 sequencing technology . Finally, 161,724,934 high-quality clean reads were completely matched with Sus scrofa miRNAs, of which 95.53±0.14% and 94.8±0.07% were in intramuscular adipocytes and subcutaneous adipocytes, respectively. The length of these clean reads is mainly 18-30nt, with a peak of 22-23nt ( figure 1 ). Compared with known small RNA databases (including miRBase and Rfam), it was found that miRNA was the largest type of small RNA in all sequenced samples, accounting for more than ...
Embodiment 3
[0062] Example 3, mir-206 is abundantly expressed in porcine intramuscular fat and is down-regulated during adipocyte differentiation
[0063] 1. mir-206 is abundantly expressed in pig intramuscular fat
[0064] The heart, liver, spleen, lung, kidney, brain, hypothalamus, subcutaneous adipocytes, and intramuscular adipocytes of 3-day-old Jiaxing black piglets were collected to extract total RNA and detect the abundance of mir-206 by qRT-PCR (U6 gene was used as an internal reference gene).
[0065] see results Figure 6 a. Figure 6 In A, 1 to 9 represent the heart, liver, spleen, lung, kidney, brain, hypothalamus, subcutaneous fat cells, and intramuscular fat cells in turn. The results showed that miR-206 could be detected in most tissues, and its abundance was much higher in intramuscular adipocytes than in subcutaneous adipocytes and other tissues.
[0066] 2. Down-regulation of mir-206 during adipocyte differentiation
[0067] The intramuscular preadipocytes and subcu...
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