CN102703449B

CN102703449B - siRNA capable of restraining chicken myostatin gene expression and application thereof

Info

Publication number: CN102703449B
Application number: CN 201210160720
Authority: CN
Inventors: 杨宁; 孙研研; 蒋斌; 郑江霞; 陈思睿
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2009-10-13
Filing date: 2009-10-13
Publication date: 2013-12-25
Anticipated expiration: 2029-10-13
Also published as: CN102703449A

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

A kind of chicken muscle that suppresses generates siRNA and the application thereof that inhibin gene is expressed

The application is dividing an application of application number is 200910235403.3, the applying date is on October 13rd, 2009, denomination of invention is " suppress chicken muscle and generate siRNA and the application thereof that inhibin gene is expressed " patent application.

Technical field

The present invention relates to a kind of chicken muscle that suppresses and generate siRNA and the application thereof that inhibin gene is expressed.

Background technology

Within 2006, China's poultry output is 1506.6 ten thousand tons, is only second to the U.S., occupies the second in the world, increased by 6.5 times than 1985, and the same period, poultry output in the world's only increases by 1.4 times.According to the FAO statistics, China's broiler chicken amount of butchering is from 21.29 hundred million 76.95 hundred million of being increased to 2006 of nineteen ninety, 55.66 hundred million of net increases.Chicken output is from 266.32 ten thousand tons of 1070.1 ten thousand tons of being increased to 2006 of nineteen ninety, 803.78 ten thousand tons of net increases.China's poultry output accounts for 18.7% of meat total amount at present, 1.5 kilograms of occupancy volume per persons, and world's poultry output ratio 30.3% also has the very large rising space on year-on-year basis.

White plumage broiler chicken is the chicken kind of fast large scale commercial product seed selection, has growth soon, produce the characteristics that meat is many, but meat is poor.Local high-quality chicken kind is Fresh & Tender in Texture, and local flavor is good, but poor growth is produced meat less.After World War II, the Advances in Breeding of fast large-scale white plumage broiler chicken is very fast, with nineteen fifty-seven, compares, fast large broiler chicken reach market weight time shorten 69 days, only need at present can go on the market in 32 days.High-quality chicken has abundant germ plasm resource in China, and rough Statistics approximately has more than 100 indigenous chicken kind, as three yellow chickens, numb chicken, langshan chicken, a fine breed of chicken with thick brownish feathers etc.These chicken matter are fresh and tender, good in color, smell and taste, and wherein black-bone chicken also has very high pharmaceutical use.The high-quality chicken kind, for a long time as the specialty industries of China's broiler production, has the wide market space, very popular.Although price compared to fast large-scale white plumage broiler chicken want expensive one times even more, its demand is still in continuous growth.This huge market is also being coveted always by external well-known broiler chicken breeding company.But the Advances in Breeding of high-quality chicken is slow, while reaching 1.5Kg, age in days is no less than 50 days.At present the high-quality chicken breeding work is followed and is transmitted traditional breeding way clearly from generation to generation, and paternal, the maternal breeding system that all adopts 1 year generation, choose qualitative character by rule of thumb, sets up the breeding group and carries out generation and hybridize to come seed selection.So not only the generation interval long, selection intensity is low, and meat yield and Meat Quality and the hybridization of other qualitative character disorderly joins, and causes breeding efficiency low, genetic progress is slow.Therefore need badly at present and accelerate China's high-quality chicken breeding technique system innovation.Country also quite payes attention to this, and since State Scientific and Technological Commission in 1980 sets up yellow feather dwarf broiler seed selection plan, each five-year plan thereafter has the plan of the high-quality chicken of relating to breeding project, to improving meat yield and the meat of high quality meat chicken.

Fire in 1998 etc. inject double-stranded RNA (dsRNA) in the nematode body, and the expression of genes involved, by specific inhibition, has produced the defective type the same with gene knockout.They after this genetic transcription the phenomenon of suppressed expression be called RNAi(RNA interference).Its mechanism be long dsrna (double-stranded RNA, dsRNA) in biomass cells by the Dicer enzyme in conjunction with and be cut into the siRNA molecule that length is 21-23bp (small interfering RNA, siRNA).Homologous sequence on mRNA after siRNA identifies and transcribes in conjunction with target gene, guiding RNA induces reticent mixture (RNA-induced silencing complex, RISC) mRNA is degraded, gene silencing after causing transcribing (posttranscriptional gene silencing, PTGS).Research discovery in recent years, the RNAi phenomenon extensively is formed in the bodies of aminal and plant.Except siRNA, the short hairpin RNA (short hairpin RNA, shRNA) with loop-stem structure also can become siRNA in cell after processing, and induces RNA to disturb, and action time is more lasting.Make so the gene constructed shRNA expression vector that there is potential utility value for some, and import in organism, specific expression of target gene is disturbed, thereby make the expression of animals and plants genes involved be become possibility by long-term silence.Also indicating that animals and plants are at medicine, potential huge practical value in biotechnology and livestock industry production.

Myostatin gene (Myostatin, MSTN) is the gene of a kind of negative regulate Skeletal Muscle Growth of filtering out from the mice skeletal cDNA library such as McPherron in 1997.

Summary of the invention

The purpose of this invention is to provide a kind of chicken muscle that suppresses and generate siRNA and the application thereof that inhibin gene is expressed.

The siRNA that acts on myostatin gene provided by the invention, for following a) or b) or c):

A) double-stranded RNA formed by nucleotide sequence shown in the sequence 2 of the sequence 1 of sequence table and sequence table;

B) double-stranded RNA formed by nucleotide sequence shown in the sequence 4 of the sequence 3 of sequence table and sequence table;

C) double-stranded RNA formed by nucleotide sequence shown in the sequence 6 of the sequence 5 of sequence table and sequence table.

The present invention also protects the application of above arbitrary described siRNA in the myostatin gene that suppresses cell is expressed.

Described myostatin gene can be GenBank Accession Number:NM_001001461 from the 1st to 1128 Nucleotide of 5 ' end.

Described cell specifically can be chicken embryo sarcoplast.

The present invention also protects the application of above arbitrary described siRNA in the myostatin gene that suppresses chicken is expressed.

Above arbitrary described method all can be applicable to a breed of chicken.

The present invention has obtained the siRNA of effective inhibition chicken MSTN genetic expression, on molecular biology, by real-time fluorescence quantitative PCR, proves that it effectively reduces the expression of MSTN; Test successfully and obtained the chick that skeletal muscle obviously increases by microinjection on fetology.These results suggest that, siRNA of the present invention can be used for a breed of chicken, thereby improves the especially meat yield of high-quality chicken of broiler chicken.

The accompanying drawing explanation

Fig. 1 is the rear MSTN gene by fluorescence quantitative experimental result of siRNA transfection chicken embryo sarcoplast (CEM) in embodiment 2.

The experimental group chick (a) that Fig. 2 is injection siRNA-9 in embodiment 3 is shone with control group chick (b) and corresponding the dissection.

Fig. 3 is the result that in embodiment 3, real-time fluorescence quantitative PCR detects the expression amount of Embryo Gallus domesticus phase muscle MSTN gene.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, be ordinary method.Test materials used in following embodiment, if no special instructions, be and purchase available from routine biochemistry reagent shop." % " in following examples, if no special instructions, be the quality percentage composition.

The design of embodiment 1, siRNA and synthetic

It is as follows that the myostatin gene (MSTN) of announcing according to NCBI designs three couples of siRNA:

First to (siRNA-9): 5 '-GCUAGCAGUCUAUGUUUAUTT-3 ' (sequence 1);

3 '-TTCGAUCGUCAGAUACAAAUA-5 ' (sequence 2);

Second to (siRNA-338): 5 '-CCACAACCGAGACGAUUAUTT-3 ' (sequence 3);

3 '-TTGGUGUUGGCUCUGCUAAUA-5 ' (sequence 4);

The 3rd to (siRNA-926): 5 '-GCUCCGGAGAAUGUGAAUUTT-3 ' (sequence 5);

3 '-TTCGAGGCCUCUUACACUUAA-5 ' (sequence 6);

Contrast siRNA(siRNA-NC): 5 '-UUCUCCGAACGUGUCACGUTT-3 ';

3’-TTAAGAGGCUUGCACAGUGCA-5’。

The above 4 couples of siRNA of chemosynthesis, the siRNA of chemosynthesis is decompression centrifugal drying product, product dried, in the pipe end, becomes dry powder.

Respectively 3 couples of siRNA are carried out to the experiment of embodiment 2 and embodiment 3.Before experiment, add appropriate DEPC water oscillator to mix making concentration after respectively siRNA is centrifugal is 20 μ M solution.

The expression amount of MSTN gene in chicken embryo sarcoplast after embodiment 2, siRNA transfection

With three couples of siRNA of embodiment 1 preparation, chicken embryo sarcoplast is carried out to transfection experiment respectively, concrete steps are as follows:

1, get the Bai Laihang kind egg (purchased from China Agricultural University's Animal Science And Technology test chicken house) of growing by 9 days and tear egg inner shell membrane, chorioallantoic membrane and amnion with sterilizing elbow tweezer, press from both sides out the chicken embryo, be put in the sterile petri dish that adds DPBS rinsing dehematize dirt, impurity.

2, the embryo is moved under anatomical lens, with ophthalmic tweezers, remove chicken embryo skin of chest, expose chest muscle, from thoracic cavity rib clip muscle.Put in the culture dish that DPBS solution is housed.Separate second half chest muscle meat tissue with the same manner.

3, with eye scissors, muscle tissue is shredded into to the cube meat of 1 cubic millimeter, add appropriate DPBS, spend the pipettor piping and druming of tip several times, remove supernatant after standing.

4, add the DMEM substratum containing 15%FBS in the muscle gruel, the 30s that vibrates on the vortex oscillation device, standing rear absorption supernatant cell suspension, repeat this step 3-5 time.

5, by the cell suspension piping and druming several of collecting, cell is uniformly dispersed, collecting cell filtrate after four layers of filtered through gauze.

6, cell filtrate is inoculated in large Tissue Culture Flask to 37 ℃ of CO ₂in incubator, cultivate half an hour.Make the inoblast that wherein mixes adherent.

7, take out gently Tissue Culture Flask, sucking-off upper strata cell culture fluid, after the appropriateness dilution, through Trypan Blue, counting viable count (chicken embryo sarcoplast) wherein.According to 3 * 10 ⁵the concentration of cell/mL is inoculated in the coated Tissue Culture Plate of 0.01% poly-lysine.

8, the six orifice plate Central Plains poultry embryo sarcoplast of being commissioned to train, when the cytogamy degree reaches 50%, carry out transfection.

9, wash away former substratum with DPBS before transfection, be changed to OPTI-MEM substratum (serum-free); Transfection liquid (A liquid: siRNA 100pmol+250 μ LOPTI-MEM substratum, room temperature 5 minutes; B liquid: liposome 2,000 3 μ L+250 μ L OPTI-MEM substratum, room temperature is placed 5 minutes; A, B liquid are mixed, and room temperature is placed 20 minutes) dropwise add in each culture hole; Establish control wells (NC) simultaneously, with isopyknic sterilized water, replace siRNA; Continue to cultivate, after 6 hours, change normal perfect medium.

10, collecting cell after 36 hours, extract RNA, and real time fluorescent quantitative carries out MSTN genetic expression interference effect and detects.

The results are shown in Figure 1(reaction, to set up β-actin be the internal reference gene, and the ratio of goal gene and the initial copy number of β-actin gene, as the relative expression quantity of this gene).Result shows: transfection the myoblastic MSTN gene expression amount of 3 couples of siRNA significantly lower than control group; SiRNA-9, the inhibition of siRNA-338 has reached 70%.Proved that on molecular level siRNA interference sequence of the present invention can effectively suppress the expression of MSTN gene, indirect proof its promote the effect of the growth of muscle, there is vivo applications and be worth.

The expression amount of MSTN gene in live body chicken embryo after embodiment 3, siRNA transfection

Respectively with the three couples of siRNA and the contrast siRNA(siRNA-NC of embodiment 1 preparation) live body chicken embryo is carried out to transfection experiment, the experiment situation is in Table 1.

Table 1 live body chicken embryo transfection experiment situation

The injection group	Injection kind of egg number	Instar chicken embryo gathered number on 3rd	Go out the chick number
				siRNA-9	47	7	2
siRNA-338	42	9	4
				siRNA-926	44	8	2
siRNA-NC	23	7	0
				NC	79	8	6

Instar chicken embryo was the rear hatching of the injection chicken embryo of 3 days on 3rd.

The concrete steps of live body chicken embryo transfection experiment are as follows:

1,12 μ LOpti-MEM and 3 μ L siRNA are mixed and made into to A liquid; 12 μ L Opti-MEM and 3 μ L liposomes 2000 are mixed and made into B liquid.

2, after hatching 5min respectively under A liquid and B liquid chamber temperature, then will under both mixed room temperatures, hatch 20min, be transfection liquid.

3, fresh white is navigated after kind of an egg (purchased from the China Agricultural University Animal Science And Technology test chicken house) equatorial plane windows, find the blastodisc central section under stereoscope, pin under area pellucida, each blastodisc is injected 1 μ L left/right rotation dye liquor, and with entering to incubate after the Parafilm sealing, incubation temperature is 37.8 ℃, humidity is 75%, egg-turning angle 45 degree, 2 hours egg-turnings once, hatching after 21 days.

Control group (NC) is set, with isopyknic sterilized water, replaces siRNA, other step is the same.

By the execution of craning one of different tests group chick, Taking Pictures recording is apparent, finds the siRNA transfection group and control group chick live body is apparent and indifference, but finds after peeling that the siRNA transfection group increases obviously than control group muscle tissue, and notable difference is arranged.Wherein Fig. 2 is shown in by the 1st group (siRNA-9 transfection group) and the photo of control group.

Get the fresh muscle tissue of instar chicken embryo on the 3rd and extract RNA, utilize real-time fluorescence quantitative PCR to detect the expression amount of MSTN gene.

The PCR primer of MSTN gene by fluorescence quantitative:

F：5’-CAGTGGATTTCGAAGCTTTTGG-3’；

R：5’-CAGGTGAGTGTGCGGGTATTT-3’；

Internal reference β-actin gene primer:

F：5’-GAGAAATTGTGCGTGACATCA-3’；

R：5’-CCTGAACCTCTCATTGCCA-3’。

The real-time fluorescence quantitative PCR detected result is shown in Fig. 3.Result shows, three siRNA transfection group MSTN gene expression amounts all significantly descend with respect to control group, prove that siRNA of the present invention can effectively disturb and suppress the expression of MSTN gene, promote the hyperplasia of Embryo Gallus domesticus phase muscle to grow, there is obvious muscle growth Increment effect on live body.

Claims

1. act on the siRNA of myostatin gene, the double-stranded RNA formed for nucleotide sequence shown in the sequence 4 of the sequence 3 by sequence table and sequence table.

2. the application of the described siRNA of claim 1 in the myostatin gene that suppresses cell is expressed; Described myostatin gene is that GenBank Accession Number:NM_001001461 is from the 1st to 1128 Nucleotide of 5 ' end; Described cell is chicken embryo sarcoplast.

3. the application of the described siRNA of claim 1 in the myostatin gene that suppresses chicken is expressed; Described myostatin gene is that GenBank Accession Number:NM_001001461 is from the 1st to 1128 Nucleotide of 5 ' end.

4. the described application be applied in a breed of chicken of claim 2 or 3.