CN101984058B - Pouch pig SLA-2 genes and application thereof - Google Patents
Pouch pig SLA-2 genes and application thereof Download PDFInfo
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Abstract
The invention relates to pouch pig SLA-2 genes and application thereof. The application comprises amplification, sequence determination and analysis of four SLA-2 allelic genes cDNA of pouch pigs of a Chinese characteristic variety. The base sequences of the four SLA-2 allelic genes are expressed as SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3 and SEQ ID No: 4. The gene sequences and genetic molecular characteristics and molecular evolution maps obtained based on the gene sequence can be used for construction of a pouch pig gene bank, variety identification of the pouch pigs and guidance on genetic breeding of the pouch pigs.
Description
Technical field
The present invention relates to amplification, sequencing and the analysis of China's feature breed pocket pig SLA-2 allelotrope (SLA-2-HB) cDNA, and utilize it to carry out evaluation of pocket pig variety and optimization genetic breeding.
Background technology
The pocket pig is the small-sized monoid of Min pig, is distributed in sealing mountain area, the west of Liaoning, gains the name because of it exactly likes " pocket ".This article boar is evolved for a long time from numerous self-fertile naturally, existing more than 300 year history; This article boar wildness is strong, has good resistance against diseases; This kind pork matter is delicious in addition, and sense of taste sweet-smelling, custom have the title of " northern fragrant pig ".Classified as first-grade state protection pig kind by the Ministry of Agriculture in 1987, be put into " national livestock and poultry genetic resources protection catalogue ", be confirmed as " national livestock and poultry genetic resources protection kind ", and be the unique pig kind that obtains state guarantee in the Northeast in 2006 in 2002.Because the pocket pig is not also developed and utilizes market, it is protected to plant at present and relies on country to drop into fully, and cost is expensive, and guarantor's kind of pressure is very big.In order to keep the stable of pocket pig quantity, except from numerous self-fertile, every year will be from some some pocket pigs that are scattered of farmers in mountain area purchase.But to its kind differentiate with artificial breeding all lack of scientific effectively instruct, this breeds for pocket pig commercialization breed and brings a huge difficult problem.
The present inventor to the investigation of pocket pig resource, considers this kind particular performances and status through for a long time, infers that it also has unique status and background on evolving.In addition; (major histocompatibility complex MHC), is referred to as swine leukocyte antigen (swine leukocyte antigen again to the mhc of pig; SLA), wherein SLA-2 (being called SLA-B again) is an important function gene.Piontkivska etc. (2003) report, vertebrate MHC molecule is represented the evolution characteristic of species, infers that in view of the above pig MHC quasi-molecule critical function gene SLA-2 might reflect the molecular evolution background of pocket pig.Based on this, the present inventor intends and starting with from the SLA-2 gene of pocket pig, researches and develops variety discriminating method accurately and reliably pointedly, and is the data that strain optimization of pocket pig and cultivation new variety provide directiveness.
Summary of the invention
One of the object of the invention is to provide pocket pig SLA-2 gene order, and the contriver through RT-PCR amplification pocket pig SLA-2 gene cDNA, is cloned into the pMD-18T carrier, then order-checking through design specific primers again.The result shows, clones 4 allelotrope of pocket pig SLA-2 gene altogether, i.e. SLA-2-HB01～04, and its base sequence is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
The present inventor utilizes the RT-PCR amplification to obtain above-mentioned pocket pig SLA-2 allelotrope, and the used primer sequence that increases is:
Upstream primer S1:5 '-AGATGCGGGTCAGGGGCCCTCAAG-3 ' (SEQ ID NO:5);
Downstream primer S2:5 '-CAGTCCCCACAAGGC AGCTGTCTC-3 ' (SEQ ID NO:6);
Amplification PCR program is:
94℃?5min；
94 ℃ of 45s, 64 ℃ of 30s, 72 ℃ of 1min, 3 circulations;
94 ℃ of 45s, 62 ℃ of 30s, 72 ℃ of 1min, 3 circulations;
94 ℃ of 45s, 60 ℃ of 30s, 72 ℃ of 1min, 31 circulations;
72℃?10min。
Another object of the present invention provides and is used for the method that genetic breeding was differentiated and instructed to the pocket pig variety.4 allelotrope sequences of the pocket pig SLA-2 gene that obtains based on above-mentioned clone; The contriver assembles DDBJ/EMBL/GenBank and goes up other 44 SLA-2 allelotrope except that the pocket pig; Utilize Gentyx 9.0 (Software Development co.; Ltd) aminoacid sequence of derivation gene, DNAMAN Version5.2.2 (Lynnon Biosoft) carry out the sequence contrast to each allelotrope of SLA-2; (Mega software Co., neighbor-joining method Ltd) is drawn the molecular evolution collection of illustrative plates of each gene through Mega2 at last.
Through analyzing the gene molecule characteristic of pocket pig SLA-2-HB; It is the characteristic amino acid sites; The contriver finds that pocket pig SLA-2-HB gene is characteristic amino acid, specifying information such as following table 47,48,67,68,74,97,119,138,240 of its coding region:
| The position | Amino acid | The amino acid English name | Amino acid abbreviations |
| 47 | Phenylalanine(Phe) | Phenylalanine | F or Phe |
| 48 | Isoleucine | Isoleucine | I or Ile |
| 67 | L-Ala | Alanine | A or Ala |
| 68 | Methionin | Lysine | K or Lys |
| 74 | Stimulina | Glutamine | Q or Gln |
| 97 | L-asparagine | Asparagine | N or Asn |
| 119 | Isoleucine | Isoleucine | I or Ile |
| 138 | L-arginine | Arginine | R or Arg |
| 240 | Serine | Serine | S or Ser |
According to these sites, can carry out the discriminating of pocket pig variety.
According to the allelic molecular evolution tree of pocket pig SLA-2, can analyze and draw the strain near or far away, thereby can select to carry out the advantage cross-breeding with its sibship.
According to mentioned above, pocket pig SLA-2 gene order of the present invention and resulting based on this gene molecule characteristic and molecular evolution collection of illustrative plates can be used for the structure of pocket pig gene pool, the genetic breeding of pocket pig is differentiated and instructed to the kind of pocket pig.
Description of drawings
Accompanying drawing 5 width of cloth of the present invention,
Fig. 1 is pocket pig SLA-2-HB 01 gene and amino acid sequence analysis result;
Fig. 2 is pocket pig SLA-2-HB 02 gene and amino acid sequence analysis result;
Fig. 3 is pocket pig SLA-2-HB 03 gene and amino acid sequence analysis result;
Fig. 4 is pocket pig SLA-2-HB 04 gene and amino acid sequence analysis result;
Fig. 5 is that pocket pig SLA-2-HB molecular evolution concerns collection of illustrative plates;
Wherein, underlined amino acid is the specific amino acid site of pocket pig SLA-2-HB gene in Fig. 1～4.
Embodiment
Below with the mode of specific embodiment technical scheme of the present invention is further described, this partial content does not limit content of the present invention in any form.The employed pocket pig of this part content sample picks up from Liaoning Province's pocket pig seed farm; Employed instrument comprise high speed freezing centrifuge (J-250, BECKMAN), the PCR appearance (iCycler, BIORAD), nucleic acid electrophoresis apparatus (DYY-7C; Beijing 61 instrument companies), gel imaging system (UVP GDS-8000; U.S. UVP company), uv analyzer (WD-9403C, Beijing 61 instrument companies), airbath vibrator (HZQ-C, Dongming, Harbin City Medical Instruments factory) etc.
Embodiment 1: clone's pocket pig SLA-2 allelotrope
1, design of primers
Utilize the RT-PCR above-mentioned pocket pig SLA-2 allelotrope that increases, the used primer sequence that increases is:
Upstream primer S1:5 '-AGATGCGGGTCAGGGGCCCTCAAG-3 ' (SEQ ID NO:5);
Downstream primer S2:5 '-CAGTCCCCACAAGGC AGCTGTCTC-3 ' (SEQ ID NO:6);
2, extract the total RNA of pocket pig
Gather the pocket pig spleen, adopt TRIZOL test kit (BS409, Canada Bio Basic Inc.) to extract the total RNA of nucleic acid, method is carried out according to the test kit explanation.
3、RT-PCR
As the reverse transcription primer, (M-MLV, RNase H-, precious biotechnology (Dalian) ltd) explains that with the nucleic acid reverse transcription be cDNA according to the reverse transcription test kit with Oligo dT; Then with pcr amplification primer S1/S2 amplification SLA-2.
Amplification RT-PCR program is:
94℃?5min；
94 ℃ of 45s, 64 ℃ of 30s, 72 ℃ of 1min, 3 circulations;
94 ℃ of 45s, 62 ℃ of 30s, 72 ℃ of 1min, 3 circulations;
94 ℃ of 45s, 60 ℃ of 30s, 72 ℃ of 1min, 31 circulations;
72℃?10min。
The amplification purified test kit of PCR product (TaKaRa DNA Fragment Purification, precious biotechnology (Dalian) ltd) reclaims.
4, gene clone and recombinant screen
SLA-2 behind the purifying is connected with pMD-18-T (precious biotechnology (Dalian) ltd) carrier, and transforms host bacterium JM109 (precious biotechnology (Dalian) ltd).The reorganization bacterium contains the Amp resistance; The reorganization bacterium is through cultivating alkaline lysis upgrading grain; EcoR I (precious biotechnology (Dalian) ltd) and HindIII (precious biotechnology (Dalian) ltd) double digestion; 1% agarose gel electrophoresis screens positive reorganization bacterium according to the restriction enzyme digestion and electrophoresis result, be saved to-80 ℃ subsequent use.
5, order-checking
Reorganization bacterium positive colony is delivered to Shanghai and is given birth to worker's order-checking.
Embodiment 2: the discriminating of sequential analysis and kind
(Software Development co. Ltd) carries out genetic analysis to embodiment 1 gained gene order, and its aminoacid sequence of deriving through Gentyx9.0; Assemble DDBJ/EMBL/GenBank and go up other 44 SLA-2 allelotrope aminoacid sequences except that the pocket pig, DNAMAN Version5.2.2 (Lynnon Biosoft) is adopted in the aminoacid sequence contrast.
Analytical results is shown in accompanying drawing 1～4: pocket pig SLA-2 gene is characteristic amino acid, specifying information such as following table 47,48,67,68,74,97,119,138,240 of its coding region:
| The position | Amino acid | The amino acid English name | Amino acid abbreviations |
| 47 | Phenylalanine(Phe) | Phenylalanine | F or Phe |
| 48 | Isoleucine | Isoleucine | I or Ile |
| 67 | L-Ala | Alanine | A or Ala |
| 68 | Methionin | Lysine | K or Lys |
| 74 | Stimulina | Glutamine | Q or Gln |
| 97 | L-asparagine | Asparagine | N or Asn |
| 119 | Isoleucine | Isoleucine | I or Ile |
| 138 | L-arginine | Arginine | R or Arg |
| 240 | Seryl | Serine | S or Ser |
According to the amino acid information of these site characteristics, can carry out the discriminating of pocket pig variety.
With reference to the allelic characterization of molecules of pocket pig SLA-2-HB; Liaoning Province pocket pig seed farm is protected kind of center personnel and from the local strain pig of producing region county 40 bulls such as Jianchang, the Green Dragon, Nai Man, enclosed pasture, has been screened pocket pig among the people that 14 headbands have pocket pig SLA-2-HB gene expression characteristics with additional pocket swinery body; And the pig resemblance that screens and performance all meet typical pocket pig characteristic; Breed through 3 generations, the characteristic feature of pocket pig still exists.With before only through the pig external appearance characteristic judge whether into the method for pocket pig relatively, present method has more science.The researchist can distinguish pocket pig among the people and other strain pig very easily on gene level at present, helps the protection of pocket pig resource.
Embodiment 3: the drafting of molecular evolution collection of illustrative plates and the application in optimizing genetic breeding thereof
The molecular evolution collection of illustrative plates adopts DNAMAN and Mega2 software, and (Mega software Co., neighbor-joining method Ltd) is drawn.Molecular evolution collection of illustrative plates such as accompanying drawing 5.Through the molecular evolution collection of illustrative plates, all SLA-2 allelotrope clusters are 3 big types, i.e. I class, II class and III class, and pocket pig SLA-2 allelotrope belongs to B III district.Can very clearly judge thus pocket pig strain and U.S. Hanford AF464039, U.S. Da Bai EF125047, and the genetic affinity of clone 10sk21 EU432083 approaching; And it is far away with genetic affinities such as China hybridization strain AF205147, America NI H miniature pig EU867814 and U.S. CMS miniature pig EU431219.According to the principle of genetic breeding, under advantage proterties equal conditions, select genetic affinity strain far away as far as possible, could embody heterosis, hybrid vigor; And, should select the nearer strain of genetic affinity again in order to protect kind of a seed selection as far as possible.
Based on above information and breeding principle, Liaoning Province's herding research institute pocket pig seed farm is formulated reasonably apolegamy plan, avoids close-inbreeding (avoiding genetic connection within the three generations), takes the homogeneity apolegamy.Outstanding boar is strengthened breeding intensity, and advantageously combined can repeat apolegamy.In the selection that focuses on proterties such as build appearance, production performance, hereditary defect of the kind of selecting and remain.Through the seed selection of three generations, each item index is specially: grow up boar body weight 91kg, sow body weight 76kg, boar height 64cm, sow height 57cm, the long 111cm of boar body, the long 104cm of sow body, boar age at sexual maturity 135d, sow 90d.9 of first farrowing sow litter sizes, 10 of multiparity number born of sow; The stage of fattening day weight gain 410 gram, feedstuff-meat ratio 3.9: 1; Dressing percentage 74%, lean ratio 48%, intramuscular fat content 5.1%; Compare from generation to generation with zero: the first farrowing sow litter size improves 0.4, and the multiparity sow is improved 0.5; The stage of fattening, day weight gain improved 52 grams; The material anharmonic ratio reduces by 0.4 ratio point; Lean ratio improves 0.9 percentage point; Boar body chi, body weight, build appearance reach breeding target.
Claims (4)
1. pocket pig SLA-2 gene is characterized in that it being 4 SLA-2 allelotrope, and its base sequence is shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.
2. the described pocket pig of claim 1 SLA-2 gene is characterized in that utilizing RT-PCR amplification pocket pig SLA-2 gene, increases used upstream and downstream primer sequence respectively shown in SEQ ID NO:5 and SEQ ID NO:6; Amplification PCR program is:
94℃?5min；
94 ℃ of 45s, 64 ℃ of 30s, 72 ℃ of 1min, 3 circulations;
94 ℃ of 45s, 62 ℃ of 30s, 72 ℃ of 1min, 3 circulations;
94 ℃ of 45s, 60 ℃ of 30s, 72 ℃ of 1min, 31 circulations;
72℃?10min。
3. the application of the described pocket pig of claim 1 SLA-2 gene on the pocket pig variety is differentiated; Be to differentiate, it is characterized in that described characteristic amino acid sites is positioned at 47 F of the coded aminoacid sequence of pocket pig SLA-2 gene coding region, 48 I, 67 A, 68 K, 74 Q, 97 N, 119 I, 138 R and 240 S according to the characteristic amino acid sites of the coded aminoacid sequence of pocket pig SLA-2 gene coding region.
4. the described pocket pig of claim 1 SLA-2 gene is optimized the application on the genetic breeding the pocket pig.
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| CN105518134A (en) * | 2015-06-11 | 2016-04-20 | 深圳市第二人民医院 | Method for pig SLA-2 gene specific knockout through CRISPR-Cas9 and sgRNA for specially targeting SLA-2 gene |
| CN105461811B (en) * | 2015-12-18 | 2018-12-18 | 大连大学 | A kind of method of O-shaped foot-and-mouth disease virus polypeptide and SLA-2 heavy chain, the crystallization of light chain β 2m renaturation |
| CN112480238B (en) * | 2020-12-03 | 2023-12-26 | 大连大学 | Construction and expression of smoke table black pig SLA-2 gene eukaryotic expression cell line |
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