CN102776180B - Sheep disease resistance related molecular marker of ISG15 gene and application thereof - Google Patents

Abstract

The invention relates to an ISG15 gene as a molecular marker related to sheep disease resistance and its application. A gene fragment related to sheep disease resistance used as a molecular marker is cloned from the sheep ISG15 gene. The nucleotide sequence is as follows: As shown in the sequence listing SEQ ID NO: 1, the gene has a base inversion of A235-C235 at the position of SEQ ID NO: 1. Beneficial effects of the present invention: three genotypes of wild homozygous AA, mutant heterozygous AC and mutant homozygous CC are found in the Hu sheep population. The invention also discloses primers used for amplifying part of the intron region of the ISG15 gene and a detection method for molecular markers. The invention provides a new molecular marker for marker-assisted selection of sheep.

Description

ISG15 gene as a molecular marker related to sheep disease resistance and its application

technical field

The invention relates to the technical field of molecular marker-assisted selection of sheep, in particular to an ISG15 gene as a molecular marker related to sheep disease resistance and its application.

Background technique

Disease is one of the important factors affecting the production efficiency of livestock and poultry. Modern industrialized feeding methods not only damage animal welfare, but also increase the susceptibility of animals to diseases; at the same time, due to the close contact between humans and livestock, the possibility of mutual infection of zoonotic diseases is increased; and the global trade circulation of livestock and poultry and products and The rapid and smooth traffic has increased the frequency of zoonotic infectious diseases and expanded the scope of infection. Although the application of biological products and antibiotics can alleviate animal epidemics to a certain extent, the use of vaccines and drugs can only prevent and control diseases but cannot eradicate them. The fundamental way to solve this problem is to cultivate livestock breeds with high disease resistance .

Interferon-stimulated gene 15 (IFN-stimulated gene 15, ISG15) is the first ubiquitin-like protein discovered so far, and its expression can be strongly induced by interferon stimulation and virus infection. Interferon binds to cell membrane receptors, activates the expression of ISG15 through the JAK-STAT signaling pathway, and then encodes antiviral proteins to achieve antiviral properties. ISG15 and its covalent modification of proteins can effectively stimulate cell proliferation and NK cell maturation, produce IFN-γ protein, activate neutrophils, promote dendritic cell maturation, and enhance cell antiviral effect, so in the interferon-regulated important role in the process.

Studies have found that ISG15 has a broad-spectrum antiviral effect. Respiratory syncytial virus, vaccinia virus, recombinant Sindbis virus, hepatitis C virus, Ebola VP40 virus, bovine viral diarrhea virus all have antiviral effects. At the same time, ISG15 also plays an important role in bacterial infection and acquired immunity.

As a unique and precious sheep breed resource in my country, Hu sheep integrates many excellent characteristics such as early sexual maturity, fast early growth, strong fecundity, four-season estrus, good lactation performance, year-round shedding, and strong disease resistance. In addition, Hu sheep have strong environmental adaptability, and can maintain excellent characteristics such as fast growth, high reproductive rate, and strong disease resistance in the hot and humid south of the Yangtze River and the northwest of the dry and cold desert. Therefore, the applicant developed sheep Cloning of ISG15 gene sequence, SNP screening and detection and its association analysis with sheep disease resistance.

Contents of the invention

The purpose of the present invention is to solve the deficiencies in the above-mentioned technologies, and to provide an ISG15 gene as a molecular marker related to sheep disease resistance and its application, as a molecular marker for sheep marker-assisted selection and related to sheep disease resistance. Cloning and application, the molecular marker of the present invention is related to ISG15 gene.

The purpose of the invention is to clone a molecular marker related to sheep disease resistance, and use the molecular marker as an application of sheep marker assisted breeding.

The technical scheme adopted by the present invention to solve its technical problems: this ISG15 gene is used as a molecular marker related to sheep disease resistance, and its nucleotide sequence is shown in SEQ ID NO: 1 in the sequence table, in the sequence table SEQ ID NO: 1 There is a base transversion of A235-C235 at 235bp.

Furthermore, the DNA sequences of the primer pair for cloning the sheep ISG15 gene are shown in SEQ ID NO: 2 and SEQ ID NO: 3.

The application of the molecular marker in sheep molecular marker assisted breeding.

Application of the primer pair in sheep molecular marker assisted breeding.

A method for preparing and detecting the above-mentioned molecular markers according to the present invention, the steps of the method are as follows:

Primers were designed with the sheep ISG15 gene, and the target fragment was amplified; genomic DNA was extracted from sheep peripheral blood, primers were designed with the sheep ISG15 gene DNA sequence as a template, PCR amplification, PCR product purification and cloning and sequencing were obtained as shown in the sequence table SEQ ID The nucleotide sequence shown in NO: 1 was used to detect the polymorphism of sheep ISG15 gene by PCR-SSCP method, and the application of the association analysis between its genotype and sheep disease resistance was preliminarily carried out, which is a molecular marker for sheep Assisted selection provides a new molecular marker.

The beneficial effect of the invention is that three genotypes of wild homozygous AA, mutant heterozygous AC and mutant homozygous CC are found in the Hu sheep population. The invention also discloses primers used for amplifying part of the intron region of the ISG15 gene and a detection method for molecular markers. The invention provides a new molecular marker for marker-assisted selection of sheep. Cloning molecular markers related to sheep disease resistance, using the molecular markers as an application of sheep marker-assisted breeding.

Description of drawings

Fig. 1: is technical flowchart of the present invention.

Figure 2: is the electrophoretic pattern of the amplified sequence of the sheep ISG15 gene in the present invention, the fragment size is 313bp (the concentration of the agarose gel is 1.5%). M in the figure is DNA molecular weight marker (DL2000plus).

Figure 3: BLAST comparison results of individual sequences of Hu sheep ISG15 gene mutation homozygous BB genotype with the bovine whole genome database.

Figure 4: Sequencing of individuals homozygous for the mutation of the sheep ISG15 gene in the present invention found that there is a base transversion of A235-C235 at 235bp of SEQ ID NO: 1 in the sequence table.

Figure 5: Three band types of PCR-SSCP of the sheep ISG15 gene in the present invention (AA, AB and BB correspond to the three genotypes of AA, AC and CC at 235bp of SEQ ID NO: 1 in the sequence table.)

Detailed ways

Below in conjunction with accompanying drawing and embodiment the present invention will be further described:

Sequence listing SEQ ID NO: 1 is the DNA sequence of the sheep disease resistance-related gene ISG15 cloned and used for PCR-SSCP detection in the present invention.

Sequence Listing SEQ ID NO: 2 is the PCR-SSCP upstream primer used for cloning and PCR-SSCP detection of sheep disease resistance-related gene ISG15 gene DNA mutation in the present invention.

Sequence Listing SEQ ID NO: 3 is the PCR-SSCP downstream primer used for cloning and PCR-SSCP detection of sheep disease resistance-related gene ISG15 gene DNA mutation in the present invention.

Description of Figure 3: Bos taurus breed Hereford chromosome 16genomic scaffold, Bos_taurus_UMD_3.1,

whole genome shotgun sequence

Length=16624581

Features flanking this part of subject sequence:

646 bp at 5'side:ubiquitin-like protein ISG15

8248bp at 3'side:transcription factor HES-4 isoform 1

Score=492bits(266), Expect=1e-136

Identities=299/315(95%),Gaps=2/315(1%)

Strand=Plus/Minus

Query 1 CATAATTTCACAAtcccatcagcaatgtatgaagATTCCAtttttttgacatcttcacca 60

||| ||||||||||||||||||||||||||||||||||||||||||||||| |||||||||

Sbjct 6095038 CATCATTTCACAATCCCATCAGCAATGTATGAAGATTCCATTTTTTTGACATCTTCACCA 6094979

Query 61 acacttattattctttttttaattattattatattcatgCTAGTGGGTGTGACATAGTAc 120

|||||||||||||||||||||||||||||||||||||||||||||||||||| | |||||

Sbjct 6094978 ACACTTATTATTCACTTTTTAATTATTATTATATTCATGCTAGGGGGTGTGACACAGTAC 6094919

Query 121 atctggttttgatttgcatttccctggttcCCATCATTTTCCaatgatgttgaatatattt 180

|||||||||||||||||||||||||||||||||||||||||||||||||| || || |||

Sbjct 6094918 ATCTGGTTTTGATTTGCATTTCCCTGGTTCCCATCATTTCCAAAGATGTTGAACATCTTT 6094859

Query 181 tcatgtggCTGTTGgccattattgtattttctattttaaaatgtctatttaagtaatttg 240

||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||| |||||

Sbjct 6094858 TCATGTGGCTCTTGGCCATTTATGTATTTTCTATTTTAAAATGTCTATTTAAGTCATTTG 6094799

Query 241 cccaactTTTAATTGAGTTGCTTATCTTTTAGTTGTTGAGTTGTAAAAGTTCC--AGACG 298

||||||||||||||||||||||||||||||||||||||||||||||||||| || | | | |

Sbjct 6094798 CCCAACTTTTAATTGAGTTGCTTATCTTTAGTTGTTAAGTTGTAAAAGTTCCTTATATG 6094739

Query 299 ATGCAGATACTAGAC 313

|||||||||||||||

Sbjct 6094738 TTGCAGATACTAGAC 6094724

Example 1: Cloning of ISG15 gene sequence (preparation example)

1. Primer Design

Using the DNA sequence of bovine ISG15 gene (GenBank accession number: AC_000173.1), a pair of primers for amplifying the DNA sequence of sheep ISG15 gene was designed. The primers were synthesized by the Shanghai Office of Invitrogen Life Technologies Co., Ltd., the United States. The DNA sequences of the primer pairs are as follows

Forward primer: 5'-CATAATTTCACAATCCCATCAGCAA-3' (corresponding to SEQ ID NO: 2),

Reverse primer: 5'-GTCTAGTATCTGCATCGTCTGGAA-3' (corresponding to SEQ ID NO: 3).

2. Amplification, purification and sequencing of PCR products

(1) PCR amplification

Add 2 μL of 50ng/μL DNA template, 2.5 μL of 10×PCR Buffer, 1 μL of 2.5 mM dNTP, 0.1 μL of 10 mM forward and reverse primers, 1 U of Taq enzyme, and double distilled water to 25 μL of the 25 μL reaction system. The PCR reaction program was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 10 sec, renaturation at 58°C for 10 sec, extension at 72°C for 20 sec, 35 cycles; 10 min at 72°C; storage at 4°C. PCR products were detected by 1.5% agarose gel electrophoresis (Figure 2).

(2) Purification and sequencing of PCR products

After a sufficient amount of PCR products are separated by 1.5% agarose gel electrophoresis, the target fragment with a molecular weight of 313bp is cut in the agarose gel, and EZN is used to After purification with the Gel Recovery Kit (Omega), it was sent to the Shanghai Office of Invitrogen Life Technologies Co., Ltd., USA for sequencing.

(3) DNA sequence homology search and identification and determination of mutation sites

Using the NCBI-BLAST (Basic Local Alignment Search Tool) tool, the sequence obtained after sequencing was compared with the data published in the GenBank cattle genome database, and the results showed that the cloned sequence was located in the upstream 5′ flanking region of the bovine ISG15 gene (Fig. 3 ). And this gene has a base transversion of A235-C235 at the place of SEQ ID NO: 1 (Fig. 4). Three genotypes, wild homozygous AA, mutant heterozygous AC and mutant homozygous CC, were found in the Hu sheep population.

Embodiment 2: The establishment of ISG15PCR-SSCP genotype detection method

Take 5 μL of the PCR amplification products of the forward and reverse primers, mix them with 10 μL of loading buffer (98% formamide, 0.025% xylene cyanol, 2% glycerol, 0.01mmol·L-1EDTA), centrifuge briefly and put them in PCR Denature in the instrument at 99°C for 10min, and immediately place on ice, after 30min in ice bath, load the sample on 15% non-denaturing polyacrylamide gel, electrophoresis at 4°C, 180V, 200mA for 12-15h. After electrophoresis, the gel was taken for silver staining to determine the genotype.

SSCP analysis of PCR amplification products found three band types: AA, AB, and BB, among which AA type was wild homozygous type, corresponding to AA genotype; BB type was mutation homozygous type, corresponding to CC genotype; AB was mutation heterozygous type type, corresponding to the AC genotype (Figure 5).

Example 3: Detection of the distribution of ISG15PCR-SSCP polymorphism in Hu sheep

The genotype and allele frequency of ISG15 mutation in Hu sheep were detected by PCR-SSCP. The results showed that allele C (i.e. mutant 235C) was the main mutation detected in the 5′ flanking region of the ISG15 gene in the Hu sheep population, reaching 0.5306, and the allele frequency of allele A (i.e. wild type 235A) was 0.4694 (Table 1). The information content of SNP polymorphism in the 5'flanking region of ISG15 gene was 0.3741, which belonged to moderate polymorphism, indicating that the genetic variation of SNP in the 5'flanking region of ISG15 gene was relatively large, and more selection effects were expected to be obtained. The results of SNPsχ2 fitness test showed that the Hu sheep population had not yet reached the Hardy-Weinberg equilibrium state at this site (P<0.05).

Table 1 Genotypes and allele frequencies of mutations in the 5′ flanking region of ISG15 gene in Hu sheep

A represents adenosine; C represents cytosine.

Example 4: Application of molecular markers cloned by the present invention in association analysis of sheep disease resistance (application example)

(1) Genotype scan

The ear tissue samples of 30 Hu sheep used for the analysis of disease resistance traits were all collected from slaughterhouses around Hangzhou City, Zhejiang Province. Use a razor to gently shave the hair on the surface of the fresh Hu sheep ear tissue, keep the inner ear tissue with more muscle tissue, and cut it into 1cm ³ muscle pieces with sterilized large scissors. The cut tissue pieces were picked up with sterilized ophthalmic forceps and washed once in 75% alcohol, then transferred to PBS with pH 8 and washed once. Put the cleaned tissue pieces into a sterilized glass bottle, and use sterilized ophthalmic scissors to trim the ear tissue into small pieces of about 1 mm ³ . During the cutting process, 1-2 drops of culture solution can be appropriately added to the ear tissue to keep the ear tissue moist during the trimming process. Use ophthalmic scissors to place the cut small pieces of tissue in a petri dish, and then use a dental probe or an elbow pipette to evenly divide the tissue pieces on the petri dish at a distance of about 0.5 cm until the entire petri dish is covered. After the tissue blocks are placed, gently turn the Petri dish over and place it with the bottom up for 30 minutes. Afterwards, add a small amount of culture medium to the culture dish to cover the tissue block, cover the culture dish, and place it in a CO ₂ incubator at 37°C for cultivation. After 2 to 4 hours, after the tissue pieces adhered to the wall, add 5 mL of medium to the culture dish, and place it in a CO ₂ incubator at 37°C for cultivation, paying attention to changing the fresh medium every 24 hours.

When the fibroblasts cover the bottom of the culture dish, the primary cells can be subcultured, and the fibroblasts can be purified by taking advantage of the different resistance of the fibroblasts and epithelioid cells to trypsin. Aspirate the culture medium in the culture dish with a sterile pipette, add 2~3mL of PBS pre-warmed to 37°C with a pH of 8, gently shake the culture dish to make the PBS flow over the surface of all cells, and then remove the added PBS. Add 2~3mL of 0.125% trypsin pre-warmed to 37°C to the washed cells, shake the culture dish gently to make the digestion solution flow over all the cell surfaces, and observe under the inverted microscope that the cytoplasm of fibroblasts retracts, When the intercellular space increases, the culture dish can be turned over to make the digestive juice leave the cells, and the digestive juice can be sucked away with a straw. Take a sterile pipette tip to suck up the culture medium and blow the cells in the dish to make the cells detach to form a cell suspension, and transfer the cell suspension to a new culture dish, add the culture solution in the dish to 5mL, and use a pipette to Pipette evenly, inoculate in four sterilized 15mL petri dishes, and culture in a 37°C, 5% _CO2 incubator with saturated humidity. Observe the original culture dish under a phase-contrast microscope, there should be epithelioid cells and a small amount of fibroblast-like cells in the dish. Repeat the above operation 2 to 3 times to separate and purify the fibroblasts.

Dilute the Poly I:C powder at a ratio of 1:100 with a pH of 8. After the diluted Poly I:C is filtered with a 0.22 μm filter, it is dispensed into sterilized 1.5mL centrifuge tubes. After the cells in the 4 ear tissue fibroblast culture dishes aliquoted from the same individual cover the bottom of the dish, take 3 plates of cells and add Poly I:C at the ratio of 50 μL Poly I:C/5mL cell culture medium Fibroblasts were stimulated to express interferon-α and interferon-β proteins, which in turn stimulated the expression of ISG15 mRNA. At 3h, 12h and 24h after Poly I:C stimulation, the culture dishes added with Poly I:C were taken out in turn, and the medium was aspirated to extract RNA from fibroblasts.

Add the following reagents sequentially to a nuclease-free 200 µL PCR tube:

0.8 μg total RNA sample 16 μL

Oligo (dT) 25 (20 μM) 1 μL

dNTP Mix (10mM ofeach dNTP) 2μL

Pipette and mix the above solution and centrifuge briefly, so that the solution on the cap and inner wall of the PCR tube is concentrated at the bottom of the PCR tube, and then place the PCR tube in an air bath at 70°C or incubate on a PCR instrument for 5 minutes, then immediately put it in an ice bath for 5 minutes; after a brief centrifugation Add the following components:

5×First-Strand Reaction Buffer 5μL

RevertAid ^™ H Minus Reverse Transcriptase 1μL

RNase Inhibitor (TaKaRa) 1 μL

Mix by pipetting, briefly centrifuge, and incubate the PCR tube in an air bath at 42°C or on a PCR machine for 90 minutes; incubate at 72°C for 15 minutes, cool down to 4°C, the product is the first-strand cDNA, and store at -20°C.

Add the following components to the real-time quantitative PCR tube:

Pipette and mix well, and perform real-time quantitative PCR amplification after brief centrifugation. The PCR amplification conditions are 94°C pre-denaturation for 3 minutes; then 94°C denaturation for 30 sec, 55.3°C refolding for 30 sec, 72°C extension for 20 sec, 40 cycles; 72°C Extend for 10 min and store at 10°C.

The genotype frequency, allele frequency and polymorphic information content of ISG15 mutation were calculated by PopGen32 software. The Generalized Linear Models in the statistical software package SPSS17.0 was used to construct the general linear model between the Hu sheep ISG15 mutation site and the relative expression of ISG15 gene in fibroblasts induced by Poly I:C:

Yij=μi+Mj+eijk①

Where Yij is the measured value of the relative expression of ISG15 gene in Hu sheep fibroblasts induced by Poly I:C; μi is the population least squares mean; Mj is the fixed genotype (3 levels: AA, AC and CC) effect; eijk is the residual effect. Since the individuals used for genotype effect analysis were all from the same breed, the breed effect was not included in the model.

Model ① was used to analyze the correlation analysis between the mutation of the 5′ flanking region of the ISG15 gene intron of the Hu sheep population and the relative expression of the ISG15 gene in fibroblasts induced by Poly I:C. The relative expression of ISG15 gene in sheep fibroblasts showed a significant correlation (P<0.05)

Table 2 Mutation genotype pairs of ISG15 gene 5'flanking region

Effect of poly I:C on the relative expression of ISG15 gene in Hu sheep fibroblasts

Note: Different lowercase letters marked after the data in the same column indicate P<0.05, different capital letters indicate P<0.01, and the same marked letters indicate no significant difference (P>0.05).

In addition to the above-mentioned embodiments, the present invention can also have other implementations. All technical solutions formed by equivalent replacement or equivalent transformation fall within the scope of protection required by the present invention.

<110> Zhejiang Academy of Agricultural Sciences

<120> ISG15 gene as a molecular marker related to sheep disease resistance and its application

<160>3

<170>PatentIn version 3.1

<210>1

<211>313

<212>DNA

<213> Sheep (Ovis aries)

<220>

<221> gene

<222>(1)..(313)

cataatttca caatcccatc agcaatgtat gaagattcca tttttttgac atcttcacca 60

acacttatta ttcttttttt aattattatt atattcatgc tagtgggtgt gacatagtac 120

atctggtttt gatttgcatt tccctggttc ccatcatttc caatgatgtt gaatatattt 180

tcatgtggct gttggccatt tatgtatttt ctattttaaa atgtctattt aagtaatttg 240

cccaactttt aattgagttg cttatctttt agttgttgag ttgtaaaagt tccagacgat 300

gcagatacta gac

<223>

<220>

<221>mutation

<222>(235)..(235)

<223>

<210> (2)

<211>25

<212>DNA

<213> Artificial sequence

<220>

<223> was designed with reference to the known sequence, synthesized by the Shanghai Office of Invitrogen Life Technology Co., Ltd., and used as the upstream primer for ISG15 mutation detection

<400> (2)

cataatttca caatcccatc agcaa 25

<210> (3)

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> was designed with reference to the known sequence, synthesized by the Shanghai Office of Invitrogen Life Technologies Co., Ltd., and used as a downstream primer for ISG15 mutation detection

<400> (3)

gtctagtatc tgcatcgtct ggaa 24

Claims (4)

1. A molecular marker of a sheep disease resistance-related gene ISG15, characterized in that: its nucleotide sequence is as shown in the sequence table SEQ ID NO: 1, and there is an A235 at the 235th bp of the sequence table SEQ ID NO: 1 -Base transversion at C235. 2. The molecular marker of the sheep disease resistance-related gene ISG15 according to claim 1, characterized in that: the primer pair for amplifying the molecular marker, its DNA sequence is as shown in SEQ ID NO: 2 and SEQ ID NO: 3 Show. 3. The molecular marker of sheep disease resistance-related gene ISG15 according to claim 1, characterized in that: the application of said molecular marker in molecular marker-assisted breeding of sheep. 4. The molecular marker of sheep disease resistance related gene ISG15 according to claim 2, characterized in that: the application of said primer pair in sheep molecular marker assisted breeding.

Images