CN102703449A

CN102703449A - siRNA capable of restraining chicken myostatin gene expression and application thereof

Info

Publication number: CN102703449A
Application number: CN2012101607205A
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: 2012-10-03
Anticipated expiration: 2029-10-13
Also published as: CN102703449B

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

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

China's poultry output was 1506.6 ten thousand tons in 2006, was only second to the U.S., occupied the second in the world, increased by 6.5 times than 1985, and the same period, poultry output in the world's only increased by 1.4 times.According to FAO statistics, China's fryer amount of butchering is from 76.95 hundred million of being increased to 2006 of 21.29 hundred million of nineteen ninety, 55.66 hundred million of net increases.Chicken output is from 1070.1 ten thousand tons of being increased to 2006 of 266.32 ten thousand tons 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 big rising space on year-on-year basis.

White plumage fryer is the chicken kind of fast large scale commercial product seed selection, has growth soon, produce the many characteristics of meat, but meat is relatively poor.Local high-quality chicken kind is Fresh & Tender in Texture, and local flavor is good, but poor growth, and it is less to produce meat.After the World War II, the breeding of fast large-scale white plumage fryer makes much progress, and compares with nineteen fifty-seven, and the time that fast big fryer reaches market weight has shortened 69 days, only needs to go on the market in 32 days at present.High-quality chicken has abundant germ plasm resource in China, and rough Statistics has more than 100 indigenous chicken kind approximately, like 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 specialty industries that the high-quality chicken kind is produced as China's fryer for a long time have the vast market space, and are very popular.Though price is wanted expensive one times even more compared to fast large-scale white plumage fryer, its demand is still in continuous growth.This huge market is also being coveted always by external well-known fryer breeding company.But the high-quality a breed of chicken is made slow progress, and age in days is no less than 50 days when reaching 1.5Kg.At present the high-quality chicken breeding work is followed and is transmitted traditional breeding way clearly from generation to generation, paternally, maternal all adopts the breeding system of 1 year generation, chooses qualitative character by rule of thumb, sets up the breeding crowd 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 proterties 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 set up yellow feather dwarf broiler seed selection plan, each five-year plan thereafter all had the high-quality chicken of relating to breeding Program for Tackling Key Problems, in the hope of improving the meat yield and the meat matter of high quality meat chicken.

Fire in 1998 etc. inject double-stranded RNA (dsRNA) in the nematode body, and Expression of Related Genes has been produced the defective type the same with gene knockout by specific inhibition.The phenomenon that they but are suppressed expression after this genetic transcription is called RNAi (RNA interference).Its mechanism be in the biomass cells long dsrna (double-stranded RNA, dsRNA) combined by the Dicer enzyme and be cut into the siRNA molecule that length is 21-23bp (small interfering RNA, siRNA).SiRNA identification also combines the homologous sequence on the mRNA after target gene is transcribed; Guiding RNA induces reticent mixture (RNA-induced silencing complex; RISC) with the mRNA degraded, and the gene silencing after causing transcribing (posttranscriptional gene silencing, PTGS).Discovering in recent years, the RNAi phenomenon extensively is formed in the bodies of aminal and plant.Except siRNA, have loop-stem structure short hairpin RNA (short hairpin RNA, shRNA) in cell through also can becoming siRNA after the processing, and induce RNA to disturb, and action time is more lasting.Make to have the gene constructed shRNA expression vector of potential utility value like this, and import in the organism, specific expression of target gene is disturbed, thereby make the plant-animal Expression of Related Genes become possibility by long-term silence to some.Also indicating plant-animal at medical science medicine, the huge practical value of potential during biotechnology and livestock industry are produced.

Muscle generates inhibin gene, and (Myostatin MSTN) is the gene of a kind of negativity regulation and control Skeletal Muscle Growth of from mice skeletal cDNA library, filtering out 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.

Provided by the inventionly act on the siRNA that muscle generates inhibin gene, for following a) or b) or c):

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

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

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

Arbitrary said siRNA generated the application in the inhibin gene expression at the muscle that suppresses cell more than the present invention also protected.

Said muscle generates inhibin gene and can be GenBank Accession Number:NM_001001461 from 5 ' terminal the 1st to 1128 Nucleotide.

Said cell specifically can be chicken embryo sarcoplast.

Arbitrary said siRNA generated the application in the inhibin gene expression at the muscle that suppresses chicken more than the present invention also protected.

More than arbitrary said method all can be applicable to a breed of chicken.

The present invention has obtained the siRNA of effective inhibition chicken MSTN genetic expression, and it has effectively reduced the expression of MSTN through the real-time fluorescence quantitative PCR proof on molecular biology; On fetology, obtained the chick that Skelettmuskel obviously increases through the microinjection success of the test.Above presentation of results, siRNA of the present invention can be used for a breed of chicken, thereby improves the especially meat yield of high-quality chicken of fryer.

Description of drawings

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

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

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

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains." % " in following examples like no specified otherwise, is the quality percentage composition.

The design of embodiment 1, siRNA and synthetic

Three couples of siRNA of muscle generation inhibin gene (MSTN) design according to NCBI announces are following:

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 whiz article, product dried becomes dry-powdered in the pipe end.

Respectively 3 couples of siRNA are carried out the experiment of embodiment 2 and embodiment 3.Before the experiment, respectively an amount of DEPC water oscillator mixing of the centrifugal back adding of siRNA being processed concentration is 20 μ M solution.

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

Three couples of siRNA with embodiment 1 preparation carry out transfection experiment to chicken embryo sarcoplast respectively, and concrete steps are following:

1, gets the Bai Laihang kind egg (available from China Agricultural University's animal technical college test chicken house) of growing and tear egg inner shell membrane, chorioallantoic membrane and amnion with sterilization elbow tweezer by 9 days; 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 the anatomical lens, remove chicken embryo skin of chest, expose chest muscle, rib clip muscle from the thoracic cavity with the ophthalmology tweezer.Put in the petridish 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 1 cubic millimeter cube meat, add an amount of DPBS, the pipettor piping and druming that spends tip is removed supernatant after leaving standstill several times.

4, in the muscle gruel, add the DMEM substratum that contains 15%FBS, the 30s that vibrates on the vortex oscillation device leaves standstill the back and draws the supernatant cell suspension, repeats this step 3-5 time.

5, with the cell suspension piping and druming of collecting for several times, cell is uniformly dispersed, collecting cell filtrating after four layers of filtered through gauze.

6, cell filtrating is inoculated in the big Tissue Culture Flask 37 ℃ of CO ₂Cultivate half a hour in the incubator.Make that wherein the miscellaneous inoblast is adherent.

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

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

9, transfection is preceding with the former substratum of DPBS flush away, is 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 was placed 5 minutes; A, B liquid are mixed, and room temperature was placed 20 minutes) dropwise add in each culture hole; Establish control wells (NC) simultaneously, replace siRNA with isopyknic sterilized water; Continue to cultivate, change normal perfect medium after 6 hours.

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

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

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

Three couples of siRNA with embodiment 1 preparation carry out transfection experiment with contrast siRNA (siRNA-NC) to live body chicken embryo respectively, and the experiment situation is seen table 1.

Table 1 live body chicken embryo transfection experiment situation

Injection groups	Injection kind of egg number	Instar chicken embryo was 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 3 days chicken embryo of injection back hatching on 3rd.

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

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

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

3, fresh white is navigated after kind of an egg (available from the China Agricultural University animal technical college test chicken house) equatorial plane windows, and finds the blastodisc central section under the stereoscope, pin under the area pellucida; Each blastodisc is injected 1 μ L left and right sides transfection liquid, goes into to incubate after sealing with Parafilm, and 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, replaces siRNA with isopyknic sterilized water, other step is the same.

With the execution of craning one of different tests group chick, Taking Pictures recording is apparent, finds siRNA transfection group and the apparent and indifference of control group chick live body, but it is obvious to find after the peeling that the siRNA transfection group increases than control group muscle tissue, and notable difference is arranged.Wherein the 1st group (siRNA-9 transfection group) and the photo of control group are seen Fig. 2.

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

The PCR primer of MSTN gene by fluorescence quantitative:

F：5’-CAGTGGATTTCGAAGCTTTTGG-3’；

R：5’-CAGGTGAGTGTGCGGGTATTT-3’；

Confidential reference items β-actin gene primer:

F：5’-GAGAAATTGTGCGTGACATCA-3’；

R：5’-CCTGAACCTCTCATTGCCA-3’。

The real-time fluorescence quantitative PCR detected result is seen Fig. 3.The 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 MSTN expression of gene, promote the hyperplasia of Embryo Gallus domesticus phase muscle to grow, on live body, have tangible muscle growth increment effect.

Claims

1. act on the siRNA that muscle generates inhibin gene, be the double-stranded RNA of forming by nucleotide sequence shown in the sequence 4 of the sequence 3 of sequence table and sequence table.

2. the said siRNA of claim 1 generates the application in the inhibin gene expression at the muscle that suppresses cell.

3. application as claimed in claim 2 is characterized in that: it is that GenBankAccession Number:NM_001001461 is from 5 ' terminal the 1st to 1128 Nucleotide that said muscle generates inhibin gene.

4. like claim 2 or 3 described application, it is characterized in that: said cell is a chicken embryo sarcoplast.

5. the said siRNA of claim 1 generates the application in the inhibin gene expression at the muscle that suppresses chicken.

6. application as claimed in claim 5 is characterized in that: it is that GenBank Accession Number:NM_001001461 is from 5 ' terminal the 1st to 1128 Nucleotide that said muscle generates inhibin gene.

7. arbitrary said application that is applied in a breed of chicken in the claim 2 to 6.