CN106367502A - Gene SPP1 as molecular marker for ovine growth traits and application of molecular marker - Google Patents

Abstract

The invention belongs to the technical field of livestock molecular markers, and in particular relates to SPP1 gene as a molecular marker for sheep growth traits and its application. The molecular marker of the present invention is obtained by cloning the SPP1 gene, and its nucleotide sequence is described in SEQ ID NO: 2 in the sequence table. There is a base substitution of A359‑C359 at the 359th bp of SEQ ID NO: 2 in the sequence table, and the mutation results in a SmlI‑RFLP restriction polymorphism. The invention also discloses primers used for amplifying the complete CDS sequence and partial DNA sequence of the SPP1 gene and a method for polymorphism detection, providing a new molecular marker for marker-assisted selection of growth traits of sheep.

Description

SPP1 Gene as a Molecular Marker for Sheep Growth Traits and Its Application

technical field

The invention belongs to the technical field of preparation of sheep molecular markers, in particular to SPP1 gene fragments as molecular markers for sheep growth traits and applications thereof.

Background technique

The genetic improvement of livestock should focus on the genetic improvement of economic traits. Growth traits are one of the important economic traits of meat sheep and are the main selection traits. The number of sheep raised in my country increased from 107,562,100 in 1993 to 191,349,200 in 2013, an increase of 77.89%, while the output of sheep meat increased from 715,300 tons in 1993 to 2,079,900 tons in 2013, an increase of 190.79% (http ://www.fao.org/statistics/zh/). The traditional breeding methods and cross-breeding improvement based on phenotypic selection and progeny determination have played an important role in the genetic improvement of sheep growth traits.

Sheep growth traits are complex quantitative traits controlled by minor polygenes, including sheep body weight, body size, carcass weight, daily gain and weaning weight. The growth traits of sheep are mainly affected by factors such as environment, feeding conditions and genetics. In the production practice of mutton sheep, improving genetics is an effective way to improve production efficiency. Therefore, searching for candidate genes or effective QTLs related to growth traits is the fundamental way to improve growth traits and production performance of sheep.

Significant progress has been made in the study of candidate genes for sheep growth traits, among which GH gene and Callipyge gene have been determined to have significant effects on sheep growth traits.

(1) Growth hormone (Growth hormone, GH) is a species-specific single-chain protein hormone synthesized and secreted by the pituitary gland of animals. It consists of 186-191 amino acids and has a molecular weight of 21000-22000 Da. It is compatible with prolactin ( Prolactin, PRL) and placental lactogen (PL) belong to the same gene family and widely exist in various vertebrates (Shan Qiuzhi, Zhong Chonggang. Research progress on growth hormone (GH) gene. China Livestock and Poultry Breeding Industry .2008, 4(4):64-66). GH has a wide range of physiological effects, and the mechanism of action is complex. It affects the metabolism of protein, sugar and fat in animals, and is closely related to the growth, development and reproduction of animals. GH gene has biological functions such as regulating the growth and development of muscle, bone and cartilage tissue. GH can directly act on the tissue cells of the whole body to increase the volume and number of cells. It can also promote the deposition of calcium, phosphorus, etc. Promotes bone growth. The GH gene can promote the entry of amino acids into cells and DNA synthesis, stimulate the formation of RNA, increase the rate of protein synthesis from amino acids, and reduce the oxidative decomposition of amino acids. The GH gene mainly inhibits the use of glucose by muscle and adipose tissue, and at the same time promotes gluconeogenesis in the liver and decomposes glycogen, thereby increasing blood sugar. In addition, growth hormone can also promote lipolysis and increase plasma free fatty acids (Wang Peng. Hu sheep GH, MSTN genetic polymorphism, expression and analysis of their association with muscle growth traits. Yangzhou University. 2010). Studies have found that the GH gene can not only promote the growth and body weight of sheep, but also affect the growth rate and body size indicators (Gootwine E, Sise JA, Penty JM, et al. The duplicated gene copy of the ovine growth hormone gene contains a PvuII polymorphism in the second intron . Animal genetics. 1993, 24(4): 319-321).

(2) The Callipyge gene was discovered in a Dorset ram in the United States in 1983 and named SoildGold. The sheep is named the Callipyge phenotype because of its muscular development, especially in the upper inner part of the leg. Studies have shown that the Callipyge phenotype is due to mutation of the wild-type allele N to C in one gene in sheep, resulting in a 35% increase in sheep-specific skeletal muscle (Koohmaraie M, Shackelford SD, Wheeler TL, et al. A musclehypertrophy condition in lamb (callipyge): characterization of effects on muscle growth and meat quality traits. Journal of animal science. 1995, 73(12): 3596-3607). The genetic mechanism of the Callipyge gene is quite special, called polar overdominance, which means that only when the heterozygote gets the C allele from the father, the sheep will have the Callipyge phenotype, otherwise, if the heterozygote is obtained from the mother If you get the C allele or form CC homozygosity, the sheep phenotype is normal and will not appear Callipyge phenotype (CharlierC.Towards the molecular understanding of the polar overdominance phenomenon associated with the callipyge phenotype in sheep. de Belgique. 2004, 159(10-12):490-496). Callipyge mutations lead to skeletal muscle hypertrophy and a reduction in the proportion of non-muscle organs after birth, and also increase the feed conversion rate of animals (White J, Vuocolo T, Grounds M, et al.Analysis of the callipyge phenotype through skeletal muscle development; association of Dlk1with muscle precursor cells.Differentiation; research in biological diversity.2008,76(3):283–298), the study found that the Callipyge gene was significantly correlated with the slaughter rate and carcass weight of sheep (Bidwell CA, Shay TL, GeorgesM, et al. Differential expression of the GTL2gene within the callipyge region of ovine chromosome 18. Animal genetics. 2001, 32(5): 248-256).

SPP1 secreted phosphorylated protein, also known as osteopontin, is a secreted protein located on sheep chromosome 6 (Ali FT, Ali MAM, Elgizawy MMA, et al. Secreted Phosphoprotein 1 Promoter Genetic Variants Are Associated with the Response to Pegylated Interferonalpha Plus Ribavirin Combination Therapy in Egyptian Patients with Chronic Hepatitis C Virus Infection. Gut Liver. 2015,9(4):516-524). SPP1 is expressed in multiple tissues of many species, such as lung, kidney, vascular smooth muscle cells, macrophages, lymphocytes, osteoblasts and skin epithelial cells of mice, chickens, humans, pigs and cattle (Denhardt DT, Guo XJ. Osteopontin-a Protein with Diverse Functions. Faseb J.1993,7(15):1475-1482). The SPP1 gene has the functions of mediating cell aggregation, adhesion, and proliferation, and participating in neural development, cell growth regulation, promoting angiogenesis, and promoting calcium deposition (Yuan Yunsheng. Gene expression profile analysis of liver regeneration and the regulation of SPP1 on liver regeneration. Shanghai Jiaotong University.2009). The study found that the SPP1 gene is related to tissue growth and embryonic development (Weintraub AS, Xinjie L, Itskovich VV, et al. Prenatal detection of embryo resorption in osteopontin-deficient mice using serialnoninvasive magnetic resonance microscopy. Pediatric Research. 2004, 55(3): 419–424). Some scholars have also found that the SPP1 gene is extremely significantly correlated with birth weight, weaning weight and yearling weight of cattle (Allan MF, Thallman RM, Cushman RA, et al. Association of a single nucleotide polymorphismin SPP1 with growth traits and twinning in a cattle population selected fortwinning rate. Journal of animal science. 2007, 85(2): 341-347).

Based on the above data, it can be concluded that the SPP1 gene plays an important role in the growth and development of animals. Finding the variation sites in genes and discovering the relationship between genes and traits through association analysis with traits is an important means of studying gene functions and the basis for marker-assisted selection. To this end, the cloning of sheep SPP1 gene, SNP screening, detection and association analysis with traits were carried out.

Contents of the invention

The purpose of the present invention is to provide a molecular marker for the detection of sheep growth traits in view of the above shortcomings.

Another object of the present invention is to provide a molecular marker method for detecting growth traits of sheep.

The object of the present invention is also to provide the application of the above molecular marker.

The present invention clones a molecular marker related to growth traits as a sheep marker-assisted selection from the sheep SPP1 gene fragment. The complete coding region sequence of the marker is shown in the sequence table SEQ ID NO: 1, and its partial DNA sequence is shown in Described in SEQ ID NO: 2 in the sequence listing. There is an A/C base mutation at the 195th base of the sequence shown in SEQ ID NO: 1, that is, at the 359th base of the sequence shown in SEQ ID NO: 2 in the sequence table as the molecular marker of the present invention There is a base mutation of A359-C359 at the base, which is located in the third exon, and there is a base mutation of G803-A803 at the 803rd base, which is located in the intron. The above two mutations (A359-C359 and G803-A803) both lead to SmlI-RFLP polymorphisms. The results of trait association analysis showed that the G803-A803 loci had no significant correlation with sheep growth traits, while the A359-C359 loci had no significant correlation with sheep growth traits. The traits are significantly correlated, therefore, the present invention mainly describes the A359-C359 site of the SPP1 gene as a molecular marker for the growth traits of sheep and its application.

Based on this, technical scheme of the present invention is as follows:

A cloned molecular marker related to sheep growth traits, its nucleotide sequence is shown in the sequence table SEQ ID No.2, the M at the 359th bp of the sequence is A or C, and the mutation leads to PCR-SmlI-RFLP polymorphism sex.

A pair of PCR primers for the above-mentioned molecular marker, the primer pair at least specifically amplifies the DNA fragment containing the SNP site of the molecular marker.

In a preferred embodiment of the present invention, the DNA sequence of the PCR primer pair is as follows:

Forward primer: GGACAGAGGCTGAAGGAATA,

Reverse primer: CCATCCAAAGCAGGTCTTAT.

A molecular marker method for detecting growth traits of sheep, the method uses the primers to specifically amplify the genomic DNA of sheep samples, and detects the SNP sites of the amplified products.

Further, the method uses the above-mentioned primers to specifically amplify the genomic DNA of the sheep sample, and performs SmlI enzyme cleavage typing and detection on the fragments obtained by PCR amplification.

The source of the sheep sample genomic DNA includes but not limited to sheep tissues, organs or blood. In one example of the present invention, the sample genomic DNA is the genomic DNA extracted from the whole blood of sheep Hu sheep.

A method for preparing the molecular marker, the method uses the above-mentioned primer pair to amplify the sheep genome DNA by PCR to obtain an amplified product.

Specifically, the total RNA extracted from adult Hu sheep rumen, kidney, lung, spleen and other tissue samples can be used as a template for RT-PCR amplification, PCR product purification and cloning and sequencing, and sequence analysis is performed to obtain the sequence table SEQ ID NO: The cDNA sequence (including all CDS) described in 1; DNA was extracted from the sheep blood genome, primers were designed, PCR amplification, PCR product purification and sequencing were carried out to obtain the nucleotide sequence shown in the sequence table SEQ ID NO:2.

The conventional PCR-RFLP method was used to detect the 359th base mutation of SEQ ID NO: 2 in the sequence table, and the application of the correlation analysis between its genotype and the growth traits of sheep was initially carried out, which is a molecular marker for sheep Assisted selection provides a new molecular marker. It provides good technical means for sheep breed breeding, reduces cost, improves efficiency, and has broad application prospects.

Description of drawings

Figure 1: is the gel electrophoresis of the CDS fragment of the sheep SPP1 gene used for cloning in the present invention. Explanation of reference numerals: lanes 1-10: SPP1; lane M: DL 2000 Marker.

Fig. 2: is the gel electrophoresis picture of the DNA fragments of the sheep SPP1 gene used in PCR-RFLP detection in the present invention. Among them: Lane 1~8: SPP1; Lane M: DL 2000Marker

Figure 3: Sequencing peak map of the mutation site of the sheep SPP1 gene.

Figure 4: five genotypes (AAGG, ACGG, CCGG, AAGA, ACGA) and electrophoresis results of the sheep SPP1 gene SmlI-RFLP in the present invention. Explanation of reference numerals: M in the figure: DNA molecular weight standard (DL2000ladder).

detailed description

The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. On the premise of not departing from the spirit and essence of the present invention, any modifications or replacements made to the present invention belong to the scope of the present invention.

Embodiment 1, the cloning of SPP1 gene

(1) Primer design

Using the sheep SPP1 gene mRNA (GenBank accession number: NM_001009224.1) as a template, a pair of primers M-F and M-R were designed using Primer5.0 software. The primer sequences are as follows

SPP1: M-F: 5'-CATCAGCATCACAGGGGACT-3',

M-R: 5'-GGAAAGAACATAGACTAAACCCT-3'.

(2), Cloning and sequencing of PCR products

Ligate the purified PCR product with the pMD-18T vector (purchased from Treasure Bioengineering Dalian Co., Ltd.) in a water bath at 4°C overnight; take 100-120 μl of competent cells in a 1.5 mL Ependorff tube under sterile conditions, and connect 5 μL of the The product was added and mixed, placed on ice for 30 minutes, heat-shocked at 42°C for 90s, then ice-bathed for 3-4 minutes, added 400 μL of LB liquid medium without antibiotics, and cultured with shaking at 37°C for 45 minutes. Take 100 μL and spread it on the agar plate of isopropylthio-β-D-galactoside (IPTG) X-gal, place it flat at 37°C for 1 hour, and then culture it upside down. Pick a single colony on the plate, inoculate in 2-3mL LB, and culture overnight at 37°C and 300r/min. Use a 1.5mL EP tube to centrifuge at 12000r/min for a few seconds to collect the bacteria to prepare a small amount of plasmid. The verified recombinant plasmid was sequenced on an automatic DNA sequencer by the dideoxy terminal termination method. The sequence determination was completed by Shanghai Yingjun Biotechnology Co., Ltd., and a cDNA sequence with a length of 1122bp was obtained (see SEQ ID NO: 1) . The gel electrophoresis of PCR products is shown in Figure 1.

DNA sequence homology search and identification:

Through the BLAST (Basic Local Alignment Search Tool) software of the National Center for Biotechnology Information (NCBI, National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) website, the DNA sequence obtained after sequencing was compared with the GenBank database Sequence homology comparison of the known physiological function genes published in to identify and obtain the functional information of the DNA sequence. The search results showed that the sequence homology between the measured sequence and sheep SPP1 gene DNA (GenBank accession number: NC_019463.2) reached 99%.

Embodiment 2, establishment of PCR-RFLP diagnostic method

(1), primer sequence design

SPP1: M-F: 5'-GGACAGAGGCTGAAGGAATA-3'

M-R: 5'-CCATCCAAAGCAGGTCTTAT-3'

The total volume of the PCR reaction is 20 μL, including about 100 ng of sheep genomic DNA, containing 1 times the buffer solution (purchased from Promega), 1.5 mmol/L MgCl ₂ , the final concentration of dNTPs is 150 μmol/L, and the final concentration of primers is 0.4 μmol/L. 2U Taq DNA polymerase (Promega). The PCR amplification program was: 94°C for 3min, 35 cycles of 94°C for 30s, 58°C for 30s, then 72°C for 60s, and finally 72°C for 10min. PCR reaction products were detected by 2% agarose gel electrophoresis. A 885bp specific amplified fragment was obtained, which was located in the third exon (as shown in Figure 2). As a result of sequencing, it was found that there were two SmlI restriction sites (C↓TYRAG) in the 885bp fragment, of which the 359bp site was the polymorphic site, located in the third exon, and the 803bp site was the polymorphic site point, located on the intron (Figure 3).

(3) PCR-RFLP detection conditions

The volume of PCR product digestion reaction is 10 μL, including 1 μL of 1× buffer, 3-5 μL of PCR product, 0.3 μL (10 U) of restriction endonuclease SmlI, make up 10 μL with H ₂ O, mix the sample and centrifuge at 37°C Bath in water for 4 hours, use 2% agarose gel electrophoresis to detect the digestion result, record the genotype, and take pictures under ultraviolet light. The sequencing results of the two homozygotes at this site showed that when the 359bp position was A, the detection result after SmlI digestion was only 1 fragment with a length of 885bp (designated as allele A), and when the 803bp position was G , the detection result after SmlI digestion was only 1 fragment with a length of 885 bp (determined as allele G); but when A359→C359 existed, the result resulted in the generation of SmlI restriction site, and two fragments with lengths of 526bp and 359bp (determined as allele C), when G803→A803, the polymorphism at this site does not exist, and the above two sites form three genotypes of AA, AC, and CC and two genotypes of GG and GA, respectively. Genotype; the above two mutation sites can be recognized by the restriction enzyme SmlI, thereby forming five genotypes (AAGG, ACGG, CCGG, AAGA, ACGA) as shown in Figure 4.

(4) Application of the molecular markers of the present invention in the correlation analysis of sheep growth trait marker traits

A total of 204 Hu sheep and 85 Hu-Du-hu hybrid sheep (Hu ♂×(Dorper ♂×Hu sheep ♀)♀) were tested for polymorphism, their genotypes were determined, and the genotypes were correlated with growth traits. analyze. Build a least squares model as follows:

Y _ijlk ＝μ+Genotype _i +Breed _i +Sex _l +P _k +Combination _m +ε _ijlkm

Among them, Y _ijkl is the observed value of traits, μ is the overall mean, Genotype _i is the genotype effect, Breed _j is the variety effect, Sex _l is the gender effect, P _k is the batch effect, Combination _m is the combined effect, ε _ijlmk As a random error, it is assumed that ε _ijlmk is independent of each other and obeys N(0, σ ² ) distribution. The results of genotype detection showed that in 289 individuals, there were 143 individuals with AA genotype, 102 individuals with AC genotype, and 44 individuals with CC genotype. The results of the association analysis between genotype and traits showed that the g.803G>A mutation site genotype was not significantly correlated with body weight, but the g.359A>C mutation site genotype was significantly correlated with birth weight and year-old weight (P <0.05). It can be seen from Table 1 that the g.359A>C site of SPP1 gene has a significant effect on birth weight and one-year weight (P<0.05).

Table 1 Association analysis of g.359A>C polymorphism of SPP1 gene and body weight in sheep

Table 1 Theresult for the association analysis of ovine SPP1g.359A>Cin experimental population

Continued Table 1

Note: Different uppercase letters in the shoulder corners of the data in the same column indicate extremely significant differences (P<0.01), different lowercase letters indicate significant differences (P<0.05), and the same letters or no letters indicate no significant differences (P>05).

Claims (9)

1. the molecular marker related to ovine growth character of a kind of clone, its nucleotide sequence such as sequence table seq id no.2 Shown, the m at this sequence 359bp is a or c, and this mutation leads to pcr-smli-rflp polymorphism.

2. a kind of pcr primer pair for molecular marker described in claim 1, this primer pair at least specific amplification comprises described The dna fragment in molecular marker snp site.

3. pcr primer pair according to claim 2 is it is characterised in that the dna sequence of this primer pair is as follows:

Forward primer: ggacagaggctgaaggaata,

Reverse primer: ccatccaaagcaggtcttat.

4. a kind of molecule labelling method for the detection of ovine growth character, the primer described in the method Claims 2 or 3 is special Specific amplification sheep sample gene group dna, the snp site of detection amplified production.

5. molecule labelling method according to claim 4 is it is characterised in that the method usage right requires drawing described in 3 Thing specific amplification sheep sample gene group dna, the fragment that pcr amplification is obtained carries out spp1 enzyme action typing and detection.

6. molecule labelling method according to claim 5 is it is characterised in that described sheep sample gene group dna is from silk floss The genome dna extracting in sheep sheep whole blood.

7. a kind of method preparing molecular marker described in claim 1, the method is to silk floss with the primer pair described in claim 3 Sheep genome dna carries out pcr amplification and obtains amplified production.

8. application in the detection of ovine growth character for the molecular marker described in claim 1.

9. application in the detection of ovine growth character for the pcr primer pair described in Claims 2 or 3.