CN102586249A - microRNAs (micro Ribonucleic Acids) of mammary gland tissues of Jinhua pig - Google Patents

Abstract

The invention discloses microRNAs (micro Ribonucleic Acids) in the mammary gland tissues of the Jinhua pig. The invention utilizes the sequencing technique to carry out deep sequencing on the mammary gland tissues of the Jinhua pig to obtain the sequences of mammary gland smallRNAs, and utilizes the bioinformatics method to analyze the sequences to obtain the variety and copy number of Jinhua pig miRNAs, so that certain predicted miRNAs in mammals, which are not included, are obtained, and the nucleotide sequences of the miRNAs are shown as SEQ ID NO:1 to SEQ ID NO:90. Certain miRNAs provided by the invention can be chosen for the research of a series of activities including porcine mammary gland lactation, lactation suppression and the like, and the microRNAs can help to increase the lactation volume of sows and the growth speed of piglets, and can also be used along with the currently discovered miRNAs in the customization of chips for the research of mammary gland activities or mammary gland diseases.

Description

The microRNA of Jinhua pig mammary tissue

Technical field

The invention belongs to biology and agricultural technology field, relate in particular to a kind of Jinhua pig mammary tissue through the newfound microRNA sequence of sequencing analysis.

Background technology

MicroRNA (miRNA) is one type of little RNA of the non-coding of newfound endogenous strand, and length is about 21～25 Nucleotide.MiRNA came to light in beautiful nematode in 1993 at first, and Lee etc. found when research elegans development defective at that time Lin-4 genes are coded protein not, but can sequential regulate and control the gene that beautiful nematode late embryogenesis is grown.Yet Lin-4 discovery fails to cause too big concern, in nematode, has found the miRNA of the 2nd similar functions again until Reinhart in 2000 etc. Let-7 genes just start the research boom of one miRNA gradually.Result of study find miRNA through with the base pairing of target gene specific; Suppress the said target mrna translation or start the said target mrna degraded; Having multiple functions such as controlling gene expression and cell proliferation, differentiation and apoptosis, has been a kind of important function of gene expression regulation factors by a large amount of experiment confirms.

Pig is important meat provider; And pig is high at aspects such as anatomy, physiology, biological chemistry, pathology and pharmacology and human similarity; Therefore important effect is arranged in human health research; Be acknowledged as the important modular system of biomedical research in recent years gradually, exercise, the allosome organ transplantation that is widely used in transmissible disease research, drug test, new modus operandi attempted and some are like the exploration of the disease of life-style of cardiovascular disorder and obesity etc. etc.Now there has been a large amount of experiment proof miRNA in the biochemical activity of animal (comprising the mankind), to play a part multiple important.But the miRNA that on pig, finds is also fewer, in the miRBASE DB, includes people's 1424 of pre-miRNA (miRNA precursor), and mouse has 720, and rat has 408, and pig has only 228.MiRNA has species and tissue specificity, thus recent years each histioid miRNA of pig research report more and more, like Skelettmuskel, heart, liver, thymus gland, fat, intestines etc., report but rarely have with the research of the relevant miRNA of pig mammary gland.Research to pig mammary gland miRNA not only can replace human mammary to carry out mammary gland research, and is the ideal material of exploitation galactophore biological reactor.And can also help to disclose the molecule mechanism of mammary gland lactation, thereby, help to improve the economic benefit of pig industry for increasing the sow lactation amount and improving piglet growth speed and offer help to the research of pig mammary gland miRNA.The Jinhua pig has good reproductive performance, comprises early sexual maturity, and farrowing is many, farrowing year limit for length's (sustainable 8 ~ 9 years of good sow highly yielding ability), and number of nipples is many, and lactation power is strong, and maternal instinct is good etc.So the present invention is a research object with the Jinhua pig, and mammary tissue has been carried out the miRNA order-checking, and found new miRNA sequence.

Summary of the invention

The objective of the invention is to deficiency, the microRNA of a kind of Jinhua pig mammary tissue is provided to prior art.

The objective of the invention is to realize through following technical scheme: the present invention makes up the mammary tissue cDNA storehouse of Jinhua pig; Through sequencing technologies it being carried out the degree of depth checks order and obtains the sequence of mammary gland small RNA; Utilize bioinformatics method that it is analyzed, obtain kind and the copy number of Jinhua pig miRNA, compare with DB; Found some prediction miRNA that do not included Mammals, its nucleotide sequence is shown in SEQ ID NO:1-90.

The invention has the beneficial effects as follows; MiRNA provided by the invention can get wherein several researchs of carrying out the lactation of pig mammary gland, moving back a series of activities such as breast separately; Offer help with raising piglet growth speed for increasing the sow lactation amount; Also can be used for custom chip, be used for the research of mammary gland activity or galactophore disease with the miRNA that has found at present.

Description of drawings

Fig. 1 is the total RNA gel electrophoresis figure of Jinhua pig mammary tissue;

Among the figure: Y1, Y2 are the Jinhua pigs, and Control is the total RNA of mouse.

Embodiment

One, the discovery of new miRNA

1, mammary tissue collection and total RNA extract and identify

Under identical raising condition; Select 3 of the Jinhua pigs (pig farm is planted in the Jinhua, Zhejiang Province city) in 21 days healthy postpartum and butcher, its breast is carried out disinfection, get each 3 pipe of mammary tissue with 75% alcohol; Drop into liquid nitrogen then rapidly, get back to-80 ℃ of preservations behind the laboratory.

According to Animal Tissue RNA Purification Kit (LC Sciences; USA) the total RNA of extraction is described; Detect its integrity (Fig. 1) with 1% sepharose, can see two rRNA bands of 28S and 18S, get the 2 μ L RNA aqueous solution and on ultraviolet-visible pectrophotometer, measure OD260 and OD280 value; Ratio explains that the RNA quality of extracting is better in 2.0 ~ 2.2 scopes.

2, little RNA cDNA library construction

According to Small RNA Sample Prep Kit explanation (Illumina; Protocol version 1004239_RevA; March 2008); The total RNA that gets 10 μ g mixes appearance and makes up the library, and process is specific as follows: from 15% TBE-urea-polyacrylamide gel, separate the gel fritter that contains 15-50 bp RNA, obtain the small RNA fragments of purifying again through elutriant wash-out and alcohol precipitation; Add SRA 5 ' adapter and SRA 3 ' adapter (Illumina) with T4 RNA ligase enzyme (Promega) respectively at the small RNA fragments 5 ' of the good 15-50 bp of purifying and 3 ' end, obtain the small RNA fragments of 64-99 bp again through wash-out and alcohol precipitation; The little RNA reverse transcription of using M-MLV (Invitrogen) reversed transcriptive enzyme will go up in the step is cDNA, uses then PfxArchaeal dna polymerase (Invitrogen) carries out 20 round-robin pcr amplifications with cDNA.Upstream primer is: AAT GAT ACG GCG ACC ACC GAC AGG TTC AGA GTT CTA CAG TCC GA, downstream primer is: CAA GCA GAA GAC GGC ATA CGA.Reaction system is totally 50 μ L, is specially: ddH ₂O 20.5 μ L, 5 * Phusion H-F buffer, 10 μ L, dNTP (each 2.5mM) 5.0 μ L, each 2.0 μ L of primer (25 uM stock), cDNA 10.0 μ L, Phusion enzyme (2U/ μ l) 0.5 μ L.Reaction conditions is: 98 ℃ of 30s, 98 ℃ of 10s, 60 ℃ of 30s, 72 ℃ of 15s, 72 ℃ of 7min.The PCR product is carried out electrophoresis; Separation of pure dissolves the pcr amplification product that length is 80-115 bp from 12% TBE-polyacrylamide gel; Again the PCR product is obtained the cDNA fragment of purifying through elutriant wash-out and alcohol precipitation, on Nanodrop (Thermo Scientific) and TBS-380 mini-fluorometer (Turner Biosystems) appearance, detect its quality with Picogreen dsDNA quantitative reagent (Invitrogen).Adjusting cDNA concentration at last is 10nM, uses Illumina Genome Analyzer IIx respectively the order-checking of the high-throughput degree of depth to be carried out in two cDNA libraries.

3, the bioinformatic analysis of miRNAs

Obtained initiation sequence 20 through the order-checking of the high-throughput degree of depth; 167; Article 190; According to the definition standard of 16.0 couples of Mammals miRNA of miRBase, these initiation sequences are screened, screen out the sequence joint, single plant nucleotide sequence surpass 80%, contain two and more than uncertain Nucleotide, length less than 15 Nucleotide, copy number less than 3 etc. ineligible; Also screen out known RNA sequence, like the non-coding RNA of other non-miRNA of including in the mRNA that includes in the ncbi database, the Rfam DB and the Tumor-necrosis factor glycoproteins fragment of in the Repbase DB, including etc.Through behind these standard screenings, 10,495,166 initiation sequences of Jinhua pig pig (52%) are screened out, and remain 9,672,024 (48%) aligned sequences.

Navigate to the sequence after the screening on the pig genome through NCBI Local BLAST, position fixing process mainly comprises following step: the pig miRNA among sequence and the miRBASE 16.0 and pre-miRNA (miRNA precursor) and other 22 kinds of mammiferous miRNA and pre-miRNA are compared in (1); (2) with the sequence that can compare in the first step and the comparison of Ensembl pig genome database; (3) use UNAFold the sequence that can not compare in the first step is carried out the hairpin structure prediction.

Finally obtain 90 and do not include as yet, called after SEQ ID NO.1-SEQ ID NO.90 in the miRNA of the prediction of miRBASE DB.

Two, the application of newly discovered miRNA

The Jinhua pig goes up newfound miRNA can make up the miRNA chip with the miRNA of the pig of having found; The expression of studying miRNA under pig growth, growth or the morbid state changes; Help to understand its molecule mechanism behind, thereby be the economic worth of pig, saving aquaculture cost etc. is offered help.For detected miRNA is all arranged on people and pig, can study these miRNA in the intravital effect of people with pig as model animal, thereby contribute for human health.The miRNA of prediction detects through RAKE just can turn out to be new miRNA, and uploads the miRBASE DB, for follow-up study lays the foundation.Further explain the present invention with regard to the following embodiment that will combine, but embodiment does not do any type of qualification to the present invention.

The target prediction of embodiment 1, SEQ ID NO 90

With TargetScan software this sequence is carried out the target gene prediction; Owing to do not contain the information of pig in this software; So the mutual work with between human mRNA and the miRNA is predicted corresponding target gene, the result has found 3 target spots, is distributed on 3 target genes; Be respectively IQSEC1 (IQ motif and Sec7 domain 1), EPHB2 (EPH receptor B2) and SHANK3 (SH3 and multiple ankyrin repeat domains 3).Find that in KEGG retrieval back IQSEC1 is human and the common gene of pig, its encoded protein is the ArfGEF family protein, in the cell endocytic process, plays a role; EPHB2 also is human and the total gene of pig, and encoding one contains 668 amino acid whose albumen, belongs to EPHB family, to its effect of the axon guidance in allelotaxis's process; SHANK3 is present in human genome, but on pig, does not find, its encoded protein is participated in the variation that the neural system signal transmits glutamate receptor in the transmittance process.Therefore, can be with pig as laboratory animal, the effect of research miRNA SEQ ID NO 90 in human cell's endocytosis and allelotaxis's process, thus be human health service.

SEQUENCE?LISTING

 

< 110>Zhejiang University

 

< 120>microRNA in the Jinhua pig mammary tissue

 

<160> 90

 

<170> PatentIn?version?3.3

 

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aggcatggct?gaatactgt 19

 

 

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tgggttggct?gggggcggg 19

 

 

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tccacccgcc?ctccgtcc 18

 

 

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<212> DNA

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tctagaggaa?gtcaaag 17

 

 

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<212> DNA

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tgtatatctt?tggagaa 17

 

 

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<212> DNA

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tggcatggga?agtctgattt?a 21

 

 

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agtggtgaat?gatagcagtt?agact 25

 

 

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<400> 13

aatcggagct?gtagct 16

 

 

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gttacttagg?tgttcccc 18

 

 

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ggtttcatga?gtgtagagac?aataa 25

 

 

<210> 16

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< 213>Jinhua pig

 

<400> 16

cataatcatc?cctcttacc 19

 

 

<210> 17

<211> 21

<212> DNA

< 213>Jinhua pig

 

<400> 17

ctctcccagc?ccaacttcca?g 21

 

 

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<212> DNA

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gatttgatat?ctgattctg 19

 

 

<210> 19

<211> 22

<212> DNA

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<400> 19

accctacttc?cttctacccc?ag 22

 

 

<210> 20

<211> 18

<212> DNA

< 213>Jinhua pig

 

<400> 20

acagagactg?tgttcttg 18

 

 

<210> 21

<211> 17

<212> DNA

< 213>Jinhua pig

 

<400> 21

cgggaggggt?gcggtgc 17

 

 

<210> 22

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<212> DNA

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<400> 22

ggagaggcct?aggagaacc 19

 

 

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<212> DNA

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<400> 23

ctcccgggtt?tcggcacca 19

 

 

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gtttgactgg?ggcggca 17

 

 

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<212> DNA

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<400> 25

ctggactgaa?ggaagaag 18

 

 

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<212> DNA

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<400> 26

tgagggtaga?ttgtatagtt 20

 

 

<210> 27

<211> 21

<212> DNA

< 213>Jinhua pig

 

<400> 27

gtgcgtcgcg?ggggtggcgg?c 21

 

 

<210> 28

<211> 23

<212> DNA

< 213>Jinhua pig

 

<400> 28

tattggccta?cagaaatgac?aga 23

 

 

<210> 29

<211> 18

<212> DNA

< 213>Jinhua pig

 

<400> 29

gaggcgccgc?cgccaccc 18

 

 

<210> 30

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 30

tagttcatct?ttgcttttt 19

 

 

<210> 31

<211> 24

<212> DNA

< 213>Jinhua pig

 

<400> 31

gaggaaggga?ggaggctgaa?tggg 24

 

 

<210> 32

<211> 21

<212> DNA

< 213>Jinhua pig

 

<400> 32

gggaccaaga?gaaagaagca?g 21

 

 

<210> 33

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 33

gtgaggactg?caacccagg 19

 

 

<210> 34

<211> 24

<212> DNA

< 213>Jinhua pig

 

<400> 34

cgccgttgtg?ctggaggcct?ctgg 24

 

 

<210> 35

<211> 17

<212> DNA

< 213>Jinhua pig

 

<400> 35

ggtgcggcca?cttgttt 17

 

 

<210> 36

<211> 20

<212> DNA

< 213>Jinhua pig

 

<400> 36

cacacctgtg?caagtcttga 20

 

 

<210> 37

<211> 23

<212> DNA

< 213>Jinhua pig

 

<400> 37

gtggggttga?tggattgtat?ggt 23

 

 

<210> 38

<211> 17

<212> DNA

< 213>Jinhua pig

 

<400> 38

atttctagtg?atcggtc 17

 

 

<210> 39

<211> 21

<212> DNA

< 213>Jinhua pig

 

<400> 39

atgatgaaac?caaacactcc?t 21

 

 

<210> 40

<211> 18

<212> DNA

< 213>Jinhua pig

 

<400> 40

gcgggcgccg?gcgcgccc 18

 

 

<210> 41

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 41

gagcaaggaa?ctttgagtg 19

 

 

<210> 42

<211> 16

<212> DNA

< 213>Jinhua pig

 

<400> 42

ctggtgagac?tgtgga 16

 

 

<210> 43

<211> 18

<212> DNA

< 213>Jinhua pig

 

<400> 43

cggcccccgg?cgcgccgg 18

 

 

<210> 44

<211> 17

<212> DNA

< 213>Jinhua pig

 

<400> 44

cggggtcccg?cgggccc 17

 

 

<210> 45

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 45

ccttatcttg?ctcaactgc 19

 

 

<210> 46

<211> 22

<212> DNA

< 213>Jinhua pig

 

<400> 46

aagagaaaac?ttgagtctag?gg 22

 

 

<210> 47

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 47

gggtttttct?tggtggggg 19

 

 

<210> 48

<211> 17

<212> DNA

< 213>Jinhua pig

 

<400> 48

ctcggcgttg?cttctac 17

 

 

<210> 49

<211> 17

<212> DNA

< 213>Jinhua pig

 

<400> 49

cgaatcagag?ctgtagc 17

 

 

<210> 50

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 50

tcaatcccta?cctacaaac 19

 

 

<210> 51

<211> 17

<212> DNA

< 213>Jinhua pig

 

<400> 51

aagtaaaatt?tctgagt 17

 

 

<210> 52

<211> 26

<212> DNA

< 213>Jinhua pig

 

<400> 52

agcttttgat?ctaagggcct?ttgagt 26

 

 

<210> 53

<211> 18

<212> DNA

< 213>Jinhua pig

 

<400> 53

atgttctcgg?atggattt 18

 

 

<210> 54

<211> 21

<212> DNA

< 213>Jinhua pig

 

<400> 54

gttggagatc?gagctgtttg?a 21

 

 

<210> 55

<211> 21

<212> DNA

< 213>Jinhua pig

 

<400> 55

tttggcttgt?ttctgtagag?g 21

 

 

<210> 56

<211> 20

<212> DNA

< 213>Jinhua pig

 

<400> 56

ataaataaaa?ttataccgta 20

 

 

<210> 57

<211> 21

<212> DNA

< 213>Jinhua pig

 

<400> 57

aacgggttgt?ctcagttgtc?a 21

 

 

<210> 58

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 58

actatctagg?aagtaaaag 19

 

 

<210> 59

<211> 23

<212> DNA

< 213>Jinhua pig

 

<400> 59

ttccactgtc?tctgctgctc?aga 23

 

 

<210> 60

<211> 24

<212> DNA

< 213>Jinhua pig

 

<400> 60

gctttctgtt?ttattgacct?acca 24

 

 

<210> 61

<211> 21

<212> DNA

< 213>Jinhua pig

 

<400> 61

cggctcctgg?ctggctcgcc?c 21

 

 

<210> 62

<211> 23

<212> DNA

< 213>Jinhua pig

 

<400> 62

aatctgcgta?gtgtctctca?agt 23

 

 

<210> 63

<211> 17

<212> DNA

< 213>Jinhua pig

 

<400> 63

cattgctgtg?gccgtga 17

 

 

<210> 64

<211> 18

<212> DNA

< 213>Jinhua pig

 

<400> 64

gccgcggctg?cctgagcg 18

 

 

<210> 65

<211> 21

<212> DNA

< 213>Jinhua pig

 

<400> 65

aatttgtccc?atggcctgtt?c 21

 

 

<210> 66

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 66

accgggctga?ggcggcagg 19

 

 

<210> 67

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 67

aaaactgtac?ctggtgtga 19

 

 

<210> 68

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 68

cacactgccg?gcctctgag 19

 

 

<210> 69

<211> 22

<212> DNA

< 213>Jinhua pig

 

<400> 69

acaggaactc?tgaggatgtg?gt 22

 

 

<210> 70

<211> 20

<212> DNA

< 213>Jinhua pig

 

<400> 70

gggcaccccc?ggccgggccc 20

 

 

<210> 71

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 71

gagaggctgc?tctggggga 19

 

 

<210> 72

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 72

tatgcattgg?tggttcagt 19

 

 

<210> 73

<211> 17

<212> DNA

< 213>Jinhua pig

 

<400> 73

cgttaaccac?tgcgcca 17

 

 

<210> 74

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 74

atgcgcatcg?tctgagaca 19

 

 

<210> 75

<211> 23

<212> DNA

< 213>Jinhua pig

 

<400> 75

gaaagtacca?gagagtgaga?gat 23

 

 

<210> 76

<211> 16

<212> DNA

< 213>Jinhua pig

 

<400> 76

ccacagcaac?gtcaga 16

 

 

<210> 77

<211> 18

<212> DNA

< 213>Jinhua pig

 

<400> 77

aggacggagg?cctgagag 18

 

 

<210> 78

<211> 24

<212> DNA

< 213>Jinhua pig

 

<400> 78

tgtccctcgc?gggggcgcgc?cggg 24

 

 

<210> 79

<211> 21

<212> DNA

< 213>Jinhua pig

 

<400> 79

ataagttttc?aggcctttgg?a 21

 

 

<210> 80

<211> 23

<212> DNA

< 213>Jinhua pig

 

<400> 80

gggtttgact?gtctagtgac?ggt 23

 

 

<210> 81

<211> 21

<212> DNA

< 213>Jinhua pig

 

<400> 81

tatcaagtat?tggtgaggat?c 21

 

 

<210> 82

<211> 25

<212> DNA

< 213>Jinhua pig

 

<400> 82

ttcttttgct?aaacttcctc?ttaca 25

 

 

<210> 83

<211> 21

<212> DNA

< 213>Jinhua pig

 

<400> 83

ggttggatat?gcaagtttct?t 21

 

 

<210> 84

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 84

atcggtgtcg?tcggtggcg 19

 

 

<210> 85

<211> 20

<212> DNA

< 213>Jinhua pig

 

<400> 85

gcagcctgtg?cctgtgggat 20

 

 

<210> 86

<211> 20

<212> DNA

< 213>Jinhua pig

 

<400> 86

ttaagctgtg?ctatttagac 20

 

 

<210> 87

<211> 18

<212> DNA

< 213>Jinhua pig

 

<400> 87

ctgggaaggc?cggtgggg 18

 

 

<210> 88

<211> 18

<212> DNA

< 213>Jinhua pig

 

<400> 88

gactgtggtt?cctgctcc 18

 

 

<210> 89

<211> 19

<212> DNA

< 213>Jinhua pig

 

<400> 89

aaggctggag?tagcaggag 19

 

 

<210> 90

<211> 22

<212> DNA

< 213>Jinhua pig

 

<400> 90

acgcccttcc?cccccttctt?ca 22

Claims (1)

1. the microRNA of a Jinhua pig mammary tissue is characterized in that, its nucleotides sequence is classified the sequence shown in any among the SEQ ID NO.1-SEQ ID NO.90 as.