CN1279167C - Gene oder of directivity and differentiation factor 1 fat cell of pigs - Google Patents

Abstract

The present invention relates to a gene order of directional and differential factors of pig fat cells, and is used in the technical field of genetic engineering. The total length of the cDNA order and the amino acid order encoding protein of ADD1 genes enlarged in the total RNA of pig spleen cells is 3830 bp. An open reading frame is in the section of 11 bp to 3453 bp. 1151 amino acid protein is encoded. The length of non-encoded order at the end of 3' is 10 bp. The length of non-encoded order at the end of 5' is 366 bp. The core structure of helix-loop-helix is placed at 320 bp to 380 bp of protein order. On one hand, the present invention can be used for studying the action of the genes in the process of pig fat cell differentiation, cholesterol level regulation in vivo, etc. by preparing the protein of the genes; on the other hand, the present invention exerts guiding effect on production and propagation of muscle pigs and low-cholesterol pigs by marker assisted selection methods.

Description

The gene order of porcine adipocyte orientation and differentiation factor 1

Technical field

What the present invention relates to is a kind of albumen coded sequence that is used for gene engineering technology field, the gene order of particularly a kind of porcine adipocyte orientation and differentiation factor 1.

Technical background

Fatty character is that animal body grows and one of leading indicator of meat proterties, be related to animal in producing such as numerous economic characters such as lean ratio, the speed of growth, feed conversion rate, intramuscular fats.In the long-term production practice, people find on the different animal product price of lipid content very big difference is arranged already.People's consumption habit has been transferred to from happiness food fat meat and has been liked to eat lean meat, makes the economic benefit of fat individuality in the process that animal produces descend like this.Fat has become a kind of byproduct in producing for animal.It has reduced feed conversion rate, has reduced the economic benefit of animal production process simultaneously.Made significant headway in the fatty character breeding process in the past few decades.Along with the develop rapidly of molecular biology theory and technology, provide another method to improve the hereditary basis of animal to us.(obese, ob), (Melanocortin-4 receptor MC4R) has certain influence to fatty character to melanocyte cortical hormone receptor 4 genes of finding in 1998 to the ob gene of nineteen fifty discovery.

Excessive Intake cholesterol and heart trouble are contacted directly, and are to cause arteriosclerosis and cardiopathic main reason.Cardiovascular disordeies such as heart trouble, arteriosclerosis and apoplexy cause dead number annual nearly 1,000,000, have reached 2500 people average every day! And cholesterol level is very high in the animal food, eats the generation that too much animal food can cause above-mentioned disease.Egg healthy, low-cholesterol comes out, but how to produce the also not further research of pork of low-cholesterol.Undoubtedly, healthy, low-cholesterol pork all can bring huge economic benefit and social benefit.Directed and differentiation factor 1 (the Adipocyte Detetmination andDifferentiation factor-1 of adipocyte, ADD1) gene claims sterol regulatory element conjugated protein 1 (SterolRegulatory Element Binding proteins-1 SREBP1) gene again, directed and the differentiation factor 1 of coding adipocyte, ADD1 is a kind of important nuclear factor in the adipocyte atomization, strengthens the atomization that conjugated protein (C/EBPs) controls adipocyte jointly with peroxisome proliferation albumen activated receptor (PPAR γ) and CCAAT element.In addition, ADD1 can also regulate the intravital cholesterol levels of animal by the sterol regulatory element.

In analysis to the prior art document, though " Comparative Biochemistry and PhysiologyPart B; 1999; 28:307-318 " affirms fully the differentiation of DD1 gene pairs adipocyte and the function of directive action, particularly the ADD1 gene pairs regulate other and fatty deposits the genes involved function determine make the application of people aspect fatty deposits obtain rapid progress.But not finding as yet so far has polymorphism in the ADD1 gene order, can not carry out the genotype in mutational site and linkage analysis that proterties is said and carry out marker assisted selection.

Summary of the invention

The objective of the invention is to overcome deficiency of the prior art, the gene order of a kind of porcine adipocyte orientation and differentiation factor 1 is provided.The adipocyte orientation of pig disclosed in this invention and cDNA sequence, the aminoacid sequence of differentiation factor 1, the regulating effect of pig ADD1 gene protein encoding sequence in the adipocyte atomization made specified, make it can pass through the albumen of this gene of preparation on the one hand, be used for this gene in porcine adipocyte differentiation and control agent inner cholesterol level, the method that can pass through marker assisted selection (MAS) on the other hand acts on performance is guiding in the production of bacon hogs and low cholesterol type pig and the breeding.

The present invention is achieved by the following technical solutions, of the present invention is the ADD1 gene that amplifies from the total RNA of the spleen cell of pig, the aminoacid sequence (as follows) of its cDNA sequence and proteins encoded, this full length gene 3830bp, open reading frame 11bp-3453bp section, coding has 1151 amino acid whose protein, the long 10bp of its 3 ' end non-coding sequence, the long 366bp of 5 ' end non-coding sequence.Helix-loop-helix (Helix-Loop-Helix, core texture HLH) are arranged at the 320-380 place of protein sequence.

The search homologous sequence is 99.9% with the partial sequence homology of known two ADD1 genes in GenBank, it is GenBank AY307771 and 99.1%, be GenBank AF102873, sequence homology 82.9% with the people, be GenBank_NM 004176, homology with mouse is 72.8%, be GenBank U09103, homology with chicken is 65.4%, i.e. GenBank AY029224, AF103873 relatively, 1022 g-a at full length sequence, 1034 t-c, 108 a-g have sudden change, relative AY307771,532 a-g, 625 c-t have sudden change, and 600 c-t sudden changes cause aminoacid sequence to produce into the sudden change of Leu-Pro.Above mutational site all might be used in marker assisted selection.

The cDNA sequence of ADD1 gene is as follows:

CGCCGGCGCC?ATGGACGAGC?CGCCCTTCAC?AGAGGCAGCC?TTGGAGCAGG?CGCTGGCCGA 60

GCCATGCGAG?CTGGACGCGG?CGCTGCTGAC?CGACATCGAA?GACATGCTTC?AGCTCATCAA 120

CAACCAAGAC?AGCGACTTCC?CCGGCCTGTT?CGACGCGCCC?TACGCTGGCG?TTGCAGGAGG 180

CACGGACCCC?ACCAGTCCTG?ATGCCAGCTC?CCCAGGCAGC?CCGACGCCGC?CTCCTTCCAC 240

CATGAGCTCC?CCGCTTGAAG?GCTTCCTGGG?GGGGGCCAGG?ACGCCGCCAC?CGCCCCCCGT 300

GTCCCCAACC?CAGCCTGCAC?CCACCCCGCT?GAAGATGTAC?CCGTCGGTGC?CTGCCTTCTC 360

GCCTGGGCCG?GGCATCAAGG?AGGAGCCAGT?GCCACTGACC?ATCCTGCAGC?CCCCCACCCC 420

ACAGCCCCTG?TCTGGGGCTC?TCCTGCCCCA?GAGCCTTCCA?GCCCTCGCCC?CTCCGCAGTT 480

GAGCCCTGCC?CCCGTGTTGG?GCTACCCCAG?CCCTCCTGGA?AGCTTCTCCT?CGGCGACCCC 540

TCCAGGGAGC?ACCTCACAGA?CGCTGCCTGG?CCTGCCGCTG?GCCTCTCTGC?CCGGGGTCCT 600

GCCCGTCTCC?GTGCACACCC?AGGTTCAAAG?TGCGGCCCCC?CAGCAGCTGT?TGACAGCCAC 660

GGCCACCCCC?GTGGTGTCCC?CTGGAACAAC?CACTGTGACC?TCGCAGATCC?AGCAGGTCCC 720

GGTCCTGCTG?CAGCCCCACT?TCATCAAGGC?GGACTCCCTG?CTCCTGACGA?CCATGAAAAC 780

AGACATGGGG?ACCCCTGTGA?AGGCGGCGGG?CATCGGCTCC?CTGGCCCCTG?GCACAGCCGT 840

GCAGGCAGCA?CCTTTGCAGA?CCCTGGTGAG?CGGCGGGACC?ATCCTGGCGA?CGGTGCCTCT 900

GGTAGTGGAC?ACTGACAAGC?TGCCCATCAA?CCGCCTGGCA?GCTGGGGGCA?AAGCCCTGAG 960

CTCAGGCCAG?AGCCGTGGTG?AGAAGCGGAC?GGCTCACAAT?GCCATCGAGA?AACGCTACCG 1020

CTCCTCCATC?AATGACAAGA?TCATCGAGCT?CAAGGACCTG?GTGGTGGGCA?CGGAGGCGAA 1080

GCTGAATAAA?TCCGCCGTCT?TGCGCAAGGC?CATCGACTAC?ATCCGCTTCC?TTCAGCAGAG 1140

CAACCAGAAG?CTCAAGCAGG?AGAACCTGAG?TCTGCGGACT?GCTGCCCACA?AAAGCAAGTC 1200

TCTGAAGGAC?CTGGTGTCCT?GCAGCAGCGG?AGGCCGCACC?GACGTGCCCA?TGGAGGGTGT 1260

GAAGCCGGAG?GTGGTAGACA?CCCTTAGCCC?GCCCCCCTCC?GACGCCGGCT?CGCCCTCCCA 1320

GAGCAGCCCC?TTGTCCCTCG?GCAGCAGGGG?CAGCAGCAGC?GGTGGCAGTG?GCAGTGACTC 1380

AGAGCCCGAC?AGCCCGGTTT?TTGAGGACAG?CCAGATGAAG?CCAGAGCAGC?TGCCGGCCCC 1440

CCACGGCCGG?GGCATGCTGG?ACCGCTCCCG?CCTGGCCCTG?TGCGTGCTCG?TCTTCCTCTG 1500

TCTCTCCTGC?AACCCCTTGG?CCTCACTGAT?GGGCAGCTGG?GCTCTCCCTG?GCCCCTCCGA 1560

TGCCACCAGC?GCCTACCACG?GGCCCTGGCG?CAGCGTACTG?GGCGCTGAGG?GCAGAGATGG 1620

CCCTGGCTGG?GTCCTGTGGC?TGCTGCCCCC?GCTGGTCTGG?CTGACGAATG?GGCTGCTGGT 1680

GCTGCTCTTC?TTGGCGCTTC?TCTTTGTCTA?TGGAGAGCCG?GTCACGCGGC?CCCACTCGGA 1740

TCCAGCTGTG?CGCTTCTGGA?GGCATCGCAA?GCAGGCCGAC?CTGGACCTGG?CCCGGGGGGA 1800

CTTCGCCCAG?GCTGCCCAGC?AGCTGTGGCT?GGCCCTGCGG?GCGCTGGGCC?GGCCCCTGCC 1860

CACCTCCCAC?CTGGACCTGG?CCTGCAGCCT?GCTCTGGAAC?CTCATCCGCC?ACCTGCTGCA 1920

GCGTCTCTGG?GTGGGCCGCT?GGCTGGCGGG?CCGGGCCGGG?GGCCTGCGGA?GGGACAGCGC 1980

CCTGGAGGCG?GACACCCGCA?CCAGCGCCTG?TGACGCGGCC?CTCGTCTACC?ACAAGCTGCA 2040

CCAGCTGCAC?ACCATGGGGA?AGTACCCAGG?CGGGCACCTC?GATGCCGCCA?ACCTGGCGCT 2100

GAGTGCCCTG?AACCTGGCGG?AGTGTGCGGG?TGATGCCCTG?TCCGTGGCCG?TGCTGGCGGA 2160

GGTCTATGTG?GCCGCTGCCC?TGCGAGTCAA?GACCAGTCTC?CCCCGGGCCT?TGCACTTTCT 2220

GACCCGCTTC?TTCCTGAGTA?GCGCCCGCCA?GGCCTGCCTG?GCACAAAGCG?GCTCGGTGCC 2280

TGTTGCCATG?CAGTGGCTCT?GCCACCCTGT?GGGCCACCGT?TTCTTCGTGG?ATGGGGACTG 2340

GGCCGTGTGC?GGTGCCCCCC?GGGACAGCTT?GTACAGCGTG?GCTGGGAACC?CAGTGGACCC 2400

CCTGGCCCAG?GTGACTCAGT?TGTTCCGTGA?ACACCTCTTG?GAGCAAGCCC?TGCATTGCGT 2460

GGCCCAGCCC?AGCCCCGGCC?CTGGATCAGC?TGATGGGGAC?AGGGAATTCT?CAGAGGCCCT 2420

CGGGTTCCTG?CAGCTGCTCA?ACAGCTGTTG?TGACACAGCC?GGGGCTCCTG?CCTGCAGCTT 2580

CTCCATTGCC?TCCAGCACAG?CCGCCACGGC?CGGCACAGAC?CCGGTCGCCA?AGTGGTGGGC 2640

CTCGCTGACG?GCTGTGGTGA?CCCACTGGCT?GGGGCGGGAC?GAGGAGGCGG?CCGAGCGGCT 2700

GTACCCGCTG?GTGGAGCACC?TGCCCCGCGC?CCTGCAGGAG?TCCGAGAGAC?CCCTGCCCAG 2760

GGCAGCTCTG?CACTCCTTCA?AGGCCGCCCG?GGCCCTGCTG?GGCCGCGGGA?AGGCAGAGTC 2820

TGGCTCAGCC?AGCCTGGCCA?TGTGTGAGAA?GGCCAGTGGG?TACCTGCAGG?ACAGCCTGGC 2880

CGCCACGCCA?GCCGGCAGCT?CCATTGACAA?GGCCATGCAG?CTGCTCCTGT?GTGACCTGCT 2940

CCTTGTGGCG?CGCACCAGCC?TGTGGCAGCG?GCAGAAGCCG?CCCCCACCCT?CCCAGGCCTC 3000

GCAGGGCTCG?AGCAGCGGGG?CCCAGGCCTC?TGCGCTCGAG?CTGCGCGGCT?TCCAGAGGGA 3060

CCTGAGTGGC?CTGAGGCGCC?TGGCGCAGAG?CTTCCGGCCT?GCCATGCGGA?GGGTGTTCCT 3120

CCATGAGGCC?ACCGCCAGGC?TGATGGCCGG?GGCCAGCCCC?GCGCGGACGC?ACCAGCTCCT 3180

GGACCGCAGT?CTGAGGAGGA?GGGCGGGCCC?CTGCGGCAGA?GGAGGCGCGG?CGGCCGCGGC 3240

GGCGGCGGAG?CTGGAACCGC?GGCCCACGCG?GCGGGAGCAG?GCCGAGGCCC?TGCTGCTCGC 3300

CTCCTGCTAC?CTGCCGCCCG?GCTTCCTGTC?GGCGCCGGGG?CAGCGCGTGG?GCATGCTGGC 3360

CGAGGCGGCG?CGCACGCTCG?AGAAGCTCGG?CGACCGCCGG?CTGCTGCACG?ACTGCCAGCA 3420

GATGCTCATG?CGCCTGGGCG?GCGGGACCAC?CGTGACCTCC?AGCTAGACCC?CTGCGGGCGG 3480

CCCAGCCCCA?GCGACCCCGT?CTCAGTCTCG?ACGGCCGAGG?GCAGCTCTGG?AGCCCCGGCC 3540

CCCGGCTCGC?CGGCGGAGTG?CCCGGTCCCT?GGGGGTATGG?AGACGCGGGG?CCCGGCGTTG 3600

ACGGGGCCGT?CCAGCCCGCA?CCCCGGGTCG?ACGAGCGGCC?GCCAGGGTGG?TGCTGGCCTC 3660

TGGTGGCCGG?CCGAGGGATT?GCCGGGGCCT?GGCGGCCGCC?AGGTGCCTCC?CATTCCACCC 3720

ACTGGCAGCC?ACTCCGCGGC?TTTCCCTCTC?TGTCCAGGGG?GAGGAAAGGG?GTGCGTTTCA 3780

CTGGGCCGAG?GGGCCGGCTT?GGCCTTCCTT?GCCGCCGGGC?GCCTCCTTCC 3830

The aminoacid sequence of ADD1 gene coded protein is as follows:

MDEPPFTEAA?LEQALAEPCE?LDAALLTDIE?DMLQLINNQD?SDFPGLFDAP?YAGVAGGTDP 60

TSPDASSPGS?PTPPPSTMSS?PLEGFLGGAR?TPPPPPVSPT?QPAPTPLKMY?PSVPAFSPGP 120

GIKEEPVPLT?ILQPPTPQPL?SGALLPQSLP?ALAPPQLSPA?PVLGYPSPPG?SFSSATPPGS 180

TSQTLPGLPL?ASLPGVLPVS?VHTQVQSAAP?QQLLTATATP?VVSPGTTTVT?SQIQQVPVLL 240

QPHFIKADSL?LLTTMKTDMG?TPVKAAGIGS?LAPGTAVQAA?PLQTLVSGGT?ILATVPLVVD 300

TDKLPINRLA?AGGKALSSGQ?SRGEKRTAHN?AIEKRYRSSI?NDKIIELKDL?VVGTEAKLNK 360

SAVLRKAIDY?IRFLQQSNQK?LKQENLSLRT?AAHKSKSLKD?LVSCSSGGRT?DVPMEGVKPE 420

VVDTLSPPPS?DAGSPSQSSP?LSLGSRGSSS?GGSGSDSEPD?SPVFEDSQMK?PEQLPAPHGR 480

GMLDRSRLAL?CVLVFLCLSC?NPLASLMGSW?ALPGPSDATS?AYHGPWRSVL?GAEGRDGPGW 540

VLWLLPPLVW?LTNGLLVLLF?LALLFVYGEP?VTRPHSDPAV?RFWRHRKQAD?LDLARGDFAQ 600

AAQQLWLALR?ALGRPLPTSH?LDLACSLLWN?LIRHLLQRLW?VGRWLAGRAG?GLRRDSALEA 660

DTRTSACDAA?LVYHKLHQLH?TMGKYPGGHL?DAANLALSAL?NLAECAGDAL?SVAVLAEVYV 720

AAALRVKTSL?PRALHFLTRF?FLSSARQACL?AQSGSVPVAM?QWLCHPVGHR?FFVDGDWAVC 780

GAPRDSLYSV?AGNPVDPLAQ?VTQLFREHLL?EQALHCVAQP?SPGPGSADGD?REFSEALGFL 840

QLLNSCCDTA?GAPACSFSIA?SSTAATAGTD?PVAKWWASLT?AVVTHWLGRD?EEAAERLYPL 900

VEHLPRALQE?SERPLPRAAL?HSFKAARALL?GRGKAESGSA?SLAMCEKASG?YLQDSLAATP 960

AGSSIDKAMQ?LLLCDLLLVA?RTSLWQRQKP?PPPSQASQGS?SSGAQASALE?LRGFQRDLSG 1020

LRRLAQSFRP?AMRRVFLHEA?TARLMAGASP?ARTHQLLDRS?LRRRAGPCGR?GGAAAAAAAE 1080

LEPRPTRREQ?AEALLLASCY?LPPGFLSAPG?QRVGMLAEAA?RTLEKLGDRR?LLHDCQQMLM 1140

RLGGGTTVTS?S 1151

The present invention has substantive distinguishing features and marked improvement, at present, has only the cDNA sequence (GenBank AY307771 and AF102873) of part in the research of pig ADD1 gene.Invention clones full length cDNA sequence first.An aspect of of the present present invention can be used for studying the effect of this gene in processes such as porcine adipocyte differentiation and control agent inner cholesterol levels by the albumen of this gene of preparation.The method that can pass through marker assisted selection (MAS) on the other hand is to performance directiveness effect in the production of bacon hogs and low cholesterol type pig and the breeding.

Description of drawings

Fig. 1 is pig spleen total tissue RNA agarose electrophoresis figure

The ratio of 28S and 18S RNA brightness is approximately 2 among the figure, illustrates that total tissue RNA is not degraded, and can carry out reverse transcription.

Fig. 2 is the agarose electrophoresis figure that template is carried out the PCR reaction product with the reverse transcription of the total RNA of spleen tissue

Through the gel imaging system analysis, the segment size is 750bp.

Fig. 3 pig is with the cluster analysis figure between encoding sequence between the ADD1 gene (SREBF1 gene) of other species and SREBF2 gene.

Fig. 4 is for to obtain helix-loop-helix (Helix-Loop-Helix, HLH) three-dimensional plot of core texture by the rpsBlast software analysis.

Embodiment

Below in conjunction with specific embodiment the present invention is further described: these embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone of pig ADD1 gene

1. separate tissue (Isolation)

Gather the fresh spleen tissue (kind is " a plum mountain pig ") of pig from the slaughterhouse and put into the freezing preservation of liquid nitrogen rapidly, and it is taken back the laboratory, put into-70 ℃ of preservations, prepare to extract total tissue RNA.

2. the separation (Total RNA isolation) of total RNA

(1) preparation work of extraction RNA

The NaOH soaked overnight of used in glass products 0.1M is washed repeatedly with tap water, uses distilled water flushing again 2 times, and 180 ℃ were toasted 4 hours.

Homogenizer, electrophoresis chamber be with 3% hydrogen peroxide dipping 20-30 minute, again with 0.1% DEPC water flushing.Because the DEPC of deactivation is not influential to the PCR reaction, available 0.5% SDS handles.

The DEPC water logging bubble of Tip head, EP effective 0.1% spends the night, and autoclaving makes the DEPC inactivation.

Distilled water and solution are handled with DEPC, and promptly every 100ml water or solution add 0.1mlDEPC liquid, and room temperature is placed and spent the night, and autoclaving made the DEPC inactivation in 30 minutes.

(2) total tissue RNA is extracted

Getting 100mg organizes the sample adding that 1ml Trizol (trizal reagent, invitrogen BRL, USA) homogenate in homogenizer are arranged at liquid nitrogen cryopreservation.4 ℃ 12000 rev/mins centrifugal 10 minutes.Draw supernatant liquor, 15-30 ℃ of incubation 5 minutes.Add the 0.2ml chloroform, cover lid is used hand concuss 15 seconds.15-30 ℃ incubation 2-3 minute.4 ℃ 12000 rev/mins centrifugal 15 minutes.Draw supernatant liquor, add the 0.8ml Virahol, mix.15-30 ℃ of incubation 10 minutes, 4 ℃ 12000 rev/mins centrifugal 10 minutes, centrifuge tube bottom white precipitate is RNA.Outwell supernatant, add 1ml 75% washing with alcohol RNA, 4 ℃ 7500 rev/mins 10 minutes.Packing among seasoning or the vacuum-drying RNA. water-soluble (RNase free) ,-80 ℃ of preservations.

(3) evaluation of total tissue RNA

By measuring rna content on the spectrophotometer and with the quality of agarose electrophoretic analysis RNA, concrete grammar is as follows:

Electrophoresis chamber soaked 4-5 hour with RNA scavenging solution (100mM NaOH, 1mM EDTA), washed repeatedly with the DEPC treated water that to pour 1 * TBE into behind the several stand-by again.Sepharose is prepared with 1 * TBE.The 10ul sample-loading buffer (2 * TBE, 13% phenanthrene can, 0.1% bromjophenol blue, 7M urea) in add the above total tissue RNA of 1ul, 65 ℃ of sex change were put into after 10 minutes frozen water 2-3 minute immediately.Point sample is electrophoresis in sepharose.

3. Full Length cDNA Cloning (Cloning of Full-length cDNA)

According to cDNA (GenBank AY307771) the design reverse transcription primer of the known portions of pig (5 '-ACCGTCGCCAGGAT-3 ') and PCR downstream primer (5 '-CAGGGAGTCCGCCTTGAT-3 '), utilize homologous genes clone principle according to the amino acid conserved sequence of people and mouse design PCR upstream primer (5 '-CGCCGGCGCCATGGACGAGC-3 '), adopt RT-PCR to carry out the cDNA full-length clone, divide three phases to carry out with the method that 5 '-RACE (TaKaRa test kit) combines:

(1) cDNA's is synthetic

Utilize the total RNA 5ug of spleen tissue to dispose reaction solution and carry out the PCR reaction according to TaKaRa 5 '-RACE test kit.

(2) decomposition of heterozygosis chain RAN

Utilize the first step reaction solution according to TaKaRa 5 '-RACE test kit configuration reaction solution, with 30 ℃ of digestion of Rnase H 1 hour, behind the RNA in the digestion heterozygosis chain, with the single stranded DNA diluted for use.

(3) PCR reaction

Reaction solution with second step of diluting is that template is carried out the PCR reaction.PCR[BP001(SEQ?ID?NO.1)+BP002(SEQ?ID?NO.2)]。Its reaction parameter is: 95 ℃ of pre-sex change 5min, 95 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min, 35 circulations, 72 ℃ are extended 10min, 4 ℃ of 10min. obtain the dna segment of 760bp, wherein have 276bp with the known segment homology of pig up to 98.91%

(4) order-checking

The PCR product is reclaimed the back be connected, be transformed in the competent e. coli jm109 with PMD 18-T Vector (TaKaRa).In 37 ℃ of overnight incubation of LB substratum, deliver order-checking through the bacterial strain after bacterial strain PCR and the double digestion evaluation.Sequencing result is total to 760bp through the splicing back.

Embodiment 2

Pig ADD1 Gene Sequence Analysis and homology type are relatively

1, pig ADD1 Gene Sequence Analysis

On http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, carry out structure prediction after CDS in the cDNA sequence partly become aminoacid sequence with primer priemer 5.0 software translations with rpsBlast software.Discovery has helix-loop-helix (Helix-Loop-Helix, core texture HLH) (seeing accompanying drawing 4) at the 320-380 place of aminoacid sequence.

2, the homology analysis of pig ADD1 gene order

This aminopeptidase gene acid sequence and base sequence are carried out homology analysis with Blastn and Blastp software respectively on http://www.ncbi.nlm.nih.gov/BLAST/BLAST/Blast.cgi.Found that the base sequence of this gene and aminoacid sequence have high homology with the ADD1 gene of people and mouse.

3, the structure of phylogenetic tree

Come drawing system to set the ADD1 gene and SREBP2 gene (another one gene in the SREBPs family) sequence of different plant species with DNAMAN software.Found that homology is higher between pig ADD1 gene is with Mammals people, dog and musculus cdna, and have only 66% with the homology of chicken.Homology has 51% between two genes in the SREBPs gene family.(seeing accompanying drawing 3) result shows that the resulting sequence of experiment is a target sequence.

Finding mutational site, 6 place with the homology of the sequence of original pig in relatively.Find with AF103873 is relatively middle, in 1022 g-a of full length sequence, the sudden change of 1034 t-c, 108 a-g.With the relatively middle sudden change of finding 532 a-g, 625 c-t of AY307771,600 c-t sudden changes cause the sudden change of Leu-Pro.Above mutational site all might be used in marker assisted selection.

Table 1

Same AF103873

Query：994 tcacaatgccatcgagaaacgctaccgctcctccatcaatgacaagatcatcgagctcaa?1053

||||||||||||||||||?|||||||||||?|||||||||||||||||||||||||||||

AF103873：301 tcacaatgccatcgagaagcgctaccgctcttccatcaatgacaagatcatcgagctcaa?360

Query：1054?ggacctggtggtgggcacggaggcgaagctgaataaatccgccgtcttgcgcaaggccat?1113

|||||||||||||||||||||||||||?||||||||||||||||||||||||||||||||

AF103873：361 ggacctggtggtgggcacggaggcgaaactgaataaatccgccgtcttgcgcaaggccat?420

Same AY307771

Query：485 cctgcccccgtgttgggctaccccagccctcctggaagcttctcctcggcgacccctcca?544

|||||||||||||||||||||||||||||||||||||||||||||||?||||||||||||

AY307771 1 cctgcccccgtgttgggctaccccagccctcctggaagcttctcctcagcgacccctcca?60

Query：545 gggagcacctcacagacgctgcctggcctgccgctggcctctctgcccggggtcctgccc?604

|||||||||||||||||||||||||||||||||||||||||||||||||||||||?||||

AY307771：61 gggagcacctcacagacgctgcctggcctgccgctggcctctctgcccggggtcccgccc?120

Query：605 gtctccgtgcacacccaggttcaaagtgcggccccccagcagctgttgacagccacggcc?664

||||||||||||||||||||?|||||||||||||||||||||||||||||||||||||||

AY307771：121 gtctccgtgcacacccaggtccaaagtgcggccccccagcagctgttgacagccacggcc?180

Query: the nucleotide sequence of the porcine adipocyte differentiation and the directed factor 1

Sequence that the present invention relates to and mark apportion are as follows:

(1) information of SEQ ID NO.1

(i) sequence signature:

(A) length: 14bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii). molecule type: oligonucleotide

(iii). sequence description: SEQ ID NO.1

ACCGTCGCCAGGAT

(2) information of SEQ ID NO.2

(i) sequence signature:

(A) length: 20bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii). molecule type: oligonucleotide

(iii). sequence description: SEQ ID NO.2

CGCCGGCGCCATGGACGAGC

(3) information of SEQ ID NO.3

(i) sequence signature:

(A) length: 18bp

(B) type: Nucleotide

(C) chain: strand

(D) topological framework: linearity

(ii). molecule type: oligonucleotide

(iii). sequence description: SEQ ID NO.3

CAGGGAGTCCGCCTTGAT

(4) information of SEQ ID NO.4

＜110〉Shanghai Communications University

＜120〉pig ADD1 gene protein encoding sequence

<140>

<141>2004-01-6

<160>3

<170>PatentIn?version?3.1

<210>1

<211>3830

<212>DNA

＜213〉pig Sus scrofa ADD1 albumen

<400>1

CGCCGGCGCC?ATGGACGAGC?CGCCCTTCAC?AGAGGCAGCC?TTGGAGCAGG?CGCTGGCCGA 60

GCCATGCGAG?CTGGACGCGG?CGCTGCTGAC?CGACATCGAA?GACATGCTTC?AGCTCATCAA 120

CAACCAAGAC?AGCGACTTCC?CCGGCCTGTT?CGACGCGCCC?TACGCTGGCG?TTGCAGGAGG 180

CACGGACCCC?ACCAGTCCTG?ATGCCAGCTC?CCCAGGCAGC?CCGACGCCGC?CTCCTTCCAC 240

CATGAGCTCC?CCGCTTGAAG?GCTTCCTGGG?GGGGGCCAGG?ACGCCGCCAC?CGCCCCCCGT 300

GTCCCCAACC?CAGCCTGCAC?CCACCCCGCT?GAAGATGTAC?CCGTCGGTGC?CTGCCTTCTC 360

GCCTGGGCCG?GGCATCAAGG?AGGAGCCAGT?GCCACTGACC?ATCCTGCAGC?CCCCCACCCC 420

ACAGCCCCTG?TCTGGGGCTC?TCCTGCCCCA?GAGCCTTCCA?GCCCTCGCCC?CTCCGCAGTT 480

GAGCCCTGCC?CCCGTGTTGG?GCTACCCCAG?CCCTCCTGGA?AGCTTCTCCT?CGGCGACCCC 540

TCCAGGGAGC?ACCTCACAGA?CGCTGCCTGG?CCTGCCGCTG?GCCTCTCTGC?CCGGGGTCCT 600

GCCCGTCTCC?GTGCACACCC?AGGTTCAAAG?TGCGGCCCCC?CAGCAGCTGT?TGACAGCCAC 660

GGCCACCCCC?GTGGTGTCCC?CTGGAACAAC?CACTGTGACC?TCGCAGATCC?AGCAGGTCCC 720

GGTCCTGCTG?CAGCCCCACT?TCATCAAGGC?GGACTCCCTG?CTCCTGACGA?CCATGAAAAC 780

AGACATGGGG?ACCCCTGTGA?AGGCGGCGGG?CATCGGCTCC?CTGGCCCCTG?GCACAGCCGT 840

GCAGGCAGCA?CCTTTGCAGA?CCCTGGTGAG?CGGCGGGACC?ATCCTGGCGA?CGGTGCCTCT 900

GGTAGTGGAC?ACTGACAAGC?TGCCCATCAA?CCGCCTGGCA?GCTGGGGGCA?AAGCCCTGAG 960

CTCAGGCCAG?AGCCGTGGTG?AGAAGCGGAC?GGCTCACAAT?GCCATCGAGA?AACGCTACCG 1020

CTCCTCCATC?AATGACAAGA?TCATCGAGCT?CAAGGACCTG?GTGGTGGGCA?CGGAGGCGAA 1080

GCTGAATAAA?TCCGCCGTCT?TGCGCAAGGC?CATCGACTAC?ATCCGCTTCC?TTCAGCAGAG 1140

CAACCAGAAG?CTCAAGCAGG?AGAACCTGAG?TCTGCGGACT?GCTGCCCACA?AAAGCAAGTC 1200

TCTGAAGGAC?CTGGTGTCCT?GCAGCAGCGG?AGGCCGCACC?GACGTGCCCA?TGGAGGGTGT 1260

GAAGCCGGAG?GTGGTAGACA?CCCTTAGCCC?GCCCCCCTCC?GACGCCGGCT?CGCCCTCCCA 1320

GAGCAGCCCC?TTGTCCCTCG?GCAGCAGGGG?CAGCAGCAGC?GGTGGCAGTG?GCAGTGACTC 1380

AGAGCCCGAC?AGCCCGGTTT?TTGAGGACAG?CCAGATGAAG?CCAGAGCAGC?TGCCGGCCCC 1440

CCACGGCCGG?GGCATGCTGG?ACCGCTCCCG?CCTGGCCCTG?TGCGTGCTCG?TCTTCCTCTG 1500

TCTCTCCTGC?AACCCCTTGG?CCTCACTGAT?GGGCAGCTGG?GCTCTCCCTG?GCCCCTCCGA 1560

TGCCACCAGC?GCCTACCACG?GGCCCTGGCG?CAGCGTACTG?GGCGCTGAGG?GCAGAGATGG 1620

CCCTGGCTGG?GTCCTGTGGC?TGCTGCCCCC?GCTGGTCTGG?CTGACGAATG?GGCTGCTGGT 1680

GCTGCTCTTC?TTGGCGCTTC?TCTTTGTCTA?TGGAGAGCCG?GTCACGCGGC?CCCACTCGGA 1740

TCCAGCTGTG?CGCTTCTGGA?GGCATCGCAA?GCAGGCCGAC?CTGGACCTGG?CCCGGGGGGA 1800

CTTCGCCCAG?GCTGCCCAGC?AGCTGTGGCT?GGCCCTGCGG?GCGCTGGGCC?GGCCCCTGCC 1860

CACCTCCCAC?CTGGACCTGG?CCTGCAGCCT?GCTCTGGAAC?CTCATCCGCC?ACCTGCTGCA 1920

GCGTCTCTGG?GTGGGCCGCT?GGCTGGCGGG?CCGGGCCGGG?GGCCTGCGGA?GGGACAGCGC 1980

CCTGGAGGCG?GACACCCGCA?CCAGCGCCTG?TGACGCGGCC?CTCGTCTACC?ACAAGCTGCA 2040

CCAGCTGCAC?ACCATGGGGA?AGTACCCAGG?CGGGCACCTC?GATGCCGCCA?ACCTGGCGCT 2100

GAGTGCCCTG?AACCTGGCGG?AGTGTGCGGG?TGATGCCCTG?TCCGTGGCCG?TGCTGGCGGA 2160

GGTCTATGTG?GCCGCTGCCC?TGCGAGTCAA?GACCAGTCTC?CCCCGGGCCT?TGCACTTTCT 2220

GACCCGCTTC?TTCCTGAGTA?GCGCCCGCCA?GGCCTGCCTG?GCACAAAGCG?GCTCGGTGCC 2280

TGTTGCCATG?CAGTGGCTCT?GCCACCCTGT?GGGCCACCGT?TTCTTCGTGG?ATGGGGACTG 2340

GGCCGTGTGC?GGTGCCCCCC?GGGACAGCTT?GTACAGCGTG?GCTGGGAACC?CAGTGGACCC 2400

CCTGGCCCAG?GTGACTCAGT?TGTTCCGTGA?ACACCTCTTG?GAGCAAGCCC?TGCATTGCGT 2460

GGCCCAGCCC?AGCCCCGGCC?CTGGATCAGC?TGATGGGGAC?AGGGAATTCT?CAGAGGCCCT 2420

CGGGTTCCTG?CAGCTGCTCA?ACAGCTGTTG?TGACACAGCC?GGGGCTCCTG?CCTGCAGCTT 2580

CTCCATTGCC?TCCAGCACAG?CCGCCACGGC?CGGCACAGAC?CCGGTCGCCA?AGTGGTGGGC 2640

CTCGCTGACG?GCTGTGGTGA?CCCACTGGCT?GGGGCGGGAC?GAGGAGGCGG?CCGAGCGGCT 2700

GTACCCGCTG?GTGGAGCACC?TGCCCCGCGC?CCTGCAGGAG?TCCGAGAGAC?CCCTGCCCAG 2760

GGCAGCTCTG?CACTCCTTCA?AGGCCGCCCG?GGCCCTGCTG?GGCCGCGGGA?AGGCAGAGTC 2820

TGGCTCAGCC?AGCCTGGCCA?TGTGTGAGAA?GGCCAGTGGG?TACCTGCAGG?ACAGCCTGGC 2880

CGCCACGCCA?GCCGGCAGCT?CCATTGACAA?GGCCATGCAG?CTGCTCCTGT?GTGACCTGCT 2940

CCTTGTGGCG?CGCACCAGCC?TGTGGCAGCG?GCAGAAGCCG?CCCCCACCCT?CCCAGGCCTC 3000

GCAGGGCTCG?AGCAGCGGGG?CCCAGGCCTC?TGCGCTCGAG?CTGCGCGGCT?TCCAGAGGGA 3060

CCTGAGTGGC?CTGAGGCGCC?TGGCGCAGAG?CTTCCGGCCT?GCCATGCGGA?GGGTGTTCCT 3120

CCATGAGGCC?ACCGCCAGGC?TGATGGCCGG?GGCCAGCCCC?GCGCGGACGC?ACCAGCTCCT 3180

GGACCGCAGT?CTGAGGAGGA?GGGCGGGCCC?CTGCGGCAGA?GGAGGCGCGG?CGGCCGCGGC 3240

GGCGGCGGAG?CTGGAACCGC?GGCCCACGCG?GCGGGAGCAG?GCCGAGGCCC?TGCTGCTCGC 3300

CTCCTGCTAC?CTGCCGCCCG?GCTTCCTGTC?GGCGCCGGGG?CAGCGCGTGG?GCATGCTGGC 3360

CGAGGCGGCG?CGCACGCTCG?AGAAGCTCGG?CGACCGCCGG?CTGCTGCACG?ACTGCCAGCA 3420

GATGCTCATG?CGCCTGGGCG?GCGGGACCAC?CGTGACCTCC?AGCTAGACCC?CTGCGGGCGG 3480

CCCAGCCCCA?GCGACCCCGT?CTCAGTCTCG?ACGGCCGAGG?GCAGCTCTGG?AGCCCCGGCC 3540

CCCGGCTCGC?CGGCGGAGTG?CCCGGTCCCT?GGGGGTATGG?AGACGCGGGG?CCCGGCGTTG 3600

ACGGGGCCGT?CCAGCCCGCA?CCCCGGGTCG?ACGAGCGGCC?GCCAGGGTGG?TGCTGGCCTC 3660

TGGTGGCCGG?CCGAGGGATT?GCCGGGGCCT?GGCGGCCGCC?AGGTGCCTCC?CATTCCACCC 3720

ACTGGCAGCC?ACTCCGCGGC?TTTCCCTCTC?TGTCCAGGGG?GAGGAAAGGG?GTGCGTTTCA 3780

CTGGGCCGAG?GGGCCGGCTT?GGCCTTCCTT?GCCGCCGGGC?GCCTCCTTCC 3830

<210>2

<211>1151

<212>PRT

＜213〉pig Sus scrofa ADD1 albumen

<400>2

MDEPPFTEAA?LEQALAEPCE?LDAALLTDIE?DMLQLINNQD?SDFPGLFDAP?YAGVAGGTDP 60

TSPDASSPGS?PTPPPSTMSS?PLEGFLGGAR?TPPPPPVSPT?QPAPTPLKMY?PSVPAFSPGP 120

GIKEEPVPLT?ILQPPTPQPL?SGALLPQSLP?ALAPPQLSPA?PVLGYPSPPG?SFSSATPPGS 180

TSQTLPGLPL?ASLPGVLPVS?VHTQVQSAAP?QQLLTATATP?VVSPGTTTVT?SQIQQVPVLL 240

QPHFIKADSL?LLTTMKTDMG?TPVKAAGIGS?LAPGTAVQAA?PLQTLVSGGT?ILATVPLVVD 300

TDKLPINRLA?AGGKALSSGQ?SRGEKRTAHN?AIEKRYRSSI?NDKIIELKDL?VVGTEAKLNK 360

SAVLRKAIDY?IRFLQQSNQK?LKQENLSLRT?AAHKSKSLKD?LVSCSSGGRT?DVPMEGVKPE 420

VVDTLSPPPS?DAGSPSQSSP?LSLGSRGSSS?GGSGSDSEPD?SPVFEDSQMK?PEQLPAPHGR 480

GMLDRSRLAL?CVLVFLCLSC?NPLASLMGSW?ALPGPSDATS?AYHGPWRSVL?GAEGRDGPGW 540

VLWLLPPLVW?LTNGLLVLLF?LALLFVYGEP?VTRPHSDPAV?RFWRHRKQAD?LDLARGDFAQ 600

AAQQLWLALR?ALGRPLPTSH?LDLACSLLWN?LIRHLLQRLW?VGRWLAGRAG?GLRRDSALEA 660

DTRTSACDAA?LVYHKLHQLH?TMGKYPGGHL?DAANLALSAL?NLAECAGDAL?SVAVLAEVYV 720

AAALRVKTSL?PRALHFLTRF?FLSSARQACL?AQSGSVPVAM?QWLCHPVGHR?FFVDGDWAVC 780

GAPRDSLYSV?AGNPVDPLAQ?VTQLFREHLL?EQALHCVAQP?SPGPGSADGD?REFSEALGFL 840

QLLNSCCDTA?GAPACSFSIA?SSTAATAGTD?PVAKWWASLT?AVVTHWLGRD?EEAAERLYPL 900

VEHLPRALQE?SERPLPRAAL?HSFKAARALL?GRGKAESGSA?SLAMCEKASG?YLQDSLAATP 960

AGSSIDKAMQ?LLLCDLLLVA?RTSLWQRQKP?PPPSQASQGS?SSGAQASALE?LRGFQRDLSG 1020

LRRLAQSFRP?AMRRVFLHEA?TARLMAGASP?ARTHQLLDRS?LRRRAGPCGR?GGAAAAAAAE 1080

LEPRPTRREQ?AEALLLASCY?LPPGFLSAPG?QRVGMLAEAA?RTLEKLGDRR?LLHDCQQMLM 1140

RLGGGTTVTS?S 1151

Claims (2)

1, the gene order of a kind of porcine adipocyte orientation and differentiation factor 1 is characterized in that, the nucleotide sequence of nucleotide sequence 1-3830 position constitutes among the SEQ ID NO.4.

2, the aminoacid sequence of described porcine adipocyte orientation of a kind of claim 1 of encoding and differentiation factor 1 is characterized in that, the aminoacid sequence of 1-1151 position constitutes among the SEQ ID NO.4.

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