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siRNA capable of restraining chicken myostatin gene expression and application thereof

A technology for muscle formation inhibition and gene expression, applied in the field of siRNA, can solve the problems of long generation interval, slow genetic progress, low selection intensity, etc., and achieve the effect of improving meat production.

Active Publication Date: 2012-10-03
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this way, not only the generation interval is long, the selection intensity is low, but also the traits of meat yield and meat quality are hybridized with other quality traits, resulting in low breeding efficiency and slow genetic progress

Method used

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  • siRNA capable of restraining chicken myostatin gene expression and application thereof
  • siRNA capable of restraining chicken myostatin gene expression and application thereof
  • siRNA capable of restraining chicken myostatin gene expression and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1, the design and synthesis of siRNA

[0024] According to the myostatin gene (MSTN) announced by NCBI, three pairs of siRNA are designed as follows:

[0025] First pair (siRNA-9): 5'-GCUAGCAGUCUAUGUUUAUTT-3' (sequence 1);

[0026] 3'-TTCGAUCGUCAGAUACAAAUA-5' (SEQ ID NO: 2);

[0027] Second pair (siRNA-338): 5'-CCACAACCGAGACGAUUAUTT-3' (SEQ ID NO: 3);

[0028] 3'-TTGGUGUUGGCUCUGCUAAUA-5' (SEQ ID NO: 4);

[0029] The third pair (siRNA-926): 5'-GCUCCGGAGAAUGUGAAUUTT-3' (SEQ ID NO: 5);

[0030] 3'-TTCGAGGCCUCUUACACUUAA-5' (SEQ ID NO: 6);

[0031] Control siRNA (siRNA-NC): 5'-UUCUCCGAACGUGUCACGUTT-3';

[0032] 3'-TTAAGAGGCUUGCACAGUGCA-5'.

[0033] The above 4 pairs of siRNA are chemically synthesized, and the chemically synthesized siRNA is a vacuum-dried product, and the product is dried at the bottom of the tube and becomes a dry powder.

Embodiment 2

[0035] Example 2, Expression of MSTN gene in chicken embryo myoblasts after siRNA transfection

[0036] Three pairs of siRNAs prepared in Example 1 were used to carry out transfection experiments on chicken embryo myoblasts, and the specific steps were as follows:

[0037] 1. Take the 9-day-old Bailaihang egg (purchased from the experimental chicken farm of the Animal Science and Technology College of China Agricultural University) and tear the inner shell membrane, allantoic membrane and amniotic membrane of the egg with sterilized elbow tweezers, clamp out the chicken embryo, and put it in Rinse blood stains and impurities in sterile petri dishes that have been added with DPBS.

[0038] 2. Move the embryo to the dissecting microscope, use ophthalmic tweezers to remove the breast skin of the chick embryo, expose the pectoral muscle, and cut the muscle from the rib cage. Put into a Petri dish filled with DPBS solution. Separate the other half of the pectoralis musculature in...

Embodiment 3

[0048] Embodiment 3, the expression level of MSTN gene in living chicken embryo after siRNA transfection

[0049] Three pairs of siRNA prepared in Example 1 and the control siRNA (siRNA-NC) were used to perform transfection experiments on live chicken embryos, and the experimental conditions are shown in Table 1.

[0050] Table 1 Live chicken embryo transfection experiment situation

[0051] injection group

Number of eggs injected

Number of 3-day-old chick embryos collected

number of chicks

siRNA-9

47

7

2

siRNA-338

42

9

4

siRNA-926

44

8

2

siRNA-NC

23

7

0

NC

79

8

6

[0052] The 3-day-old chick embryo is the chick embryo hatched 3 days after injection.

[0053] The specific steps of live chicken embryo transfection experiment are as follows:

[0054] 1. Mix 12 μL Opti-MEM and 3 μL siRNA to make solution A; mix 12 μL Opti-MEM and 3 ...

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Abstract

The invention discloses a siRNA capable of restraining chicken myostatin (MSTN) gene expression and application of the siRNA; small interfering RNA (ribonucleic acid) acted on myostatin gene is provided by the invention and is a) or b) or c) as follows: a) double-stranded RNA composed of a sequence 1 of a sequence table and a sequence 2 of the sequence table; b) double-stranded RNA composed of a sequence 3 and a sequence 4 of the sequence table; and c) double-stranded RNA composed of a sequence 5 and a sequence 6 of the sequence table; according to the invention, the small interfering RNA effectively capable of restraining chicken MSTN gene expression is obtained and is proved to effectively reduce expression of MSTN through real-time fluorescent quantitative PCR (polymerase chain reaction) on molecular biology; chicks with obviously enlarged skeletal muscle is successfully acquired through a micro-injection test on embryology; above-mentioned results show that the small interfering RNA provided by the invention can be used for breeding the chicks, so that chicken yield of the chicks specifically high-quality chicks is increased.

Description

[0001] This application is a divisional application of the patent application with the application number 200910235403.3, the application date is October 13, 2009, and the invention title is "siRNA for inhibiting chicken myostatin gene expression and its application". technical field [0002] The invention relates to a siRNA for inhibiting chicken myostatin gene expression and application thereof. Background technique [0003] In 2006, my country's poultry meat output was 15.066 million tons, second only to the United States, ranking second in the world, an increase of 6.5 times compared with 1985, while the world's poultry meat output only increased by 1.4 times during the same period. According to FAO statistics, the number of broiler chickens slaughtered in my country increased from 2.129 billion in 1990 to 7.695 billion in 2006, a net increase of 5.566 billion. Chicken production increased from 2.6632 million tons in 1990 to 10.701 million tons in 2006, a net increase of...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/88A01K67/027
Inventor 杨宁孙研研蒋斌郑江霞陈思睿
Owner CHINA AGRI UNIV
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