CN110241235B - ADSL gene and genetic marking method for its content of \28294u-goose inosinic acid - Google Patents
ADSL gene and genetic marking method for its content of \28294u-goose inosinic acid Download PDFInfo
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Abstract
The invention discloses an adenosine succinate lyase (ADSL) gene and a genetic marker for inosinic acid content in muscles of a pump goose, wherein the content of inosinic acid in the muscles of the pump goose is determined as 28294c, the content of inosinic acid in the breast muscles of the pump goose is high or low, the polymorphism of the ADSL gene of the pump goose is screened out, then the association analysis of the ADSL gene polymorphism and the inosinic acid character of the breast muscles is carried out, the fact that G/T mutation exists at 146bp, GG genotype and TT genotype 28294c exists in the ADSL gene of the pump goose as shown in a sequence, and the inosinic acid content of the breast muscles of the pump goose is obviously higher than GT genotype. The invention provides a molecular marker for marker-assisted selection of the properties of 28294u goose pectoralis inosinic acid.
Description
Technical Field
The invention belongs to the field of poultry molecular biotechnology and breeding, and mainly relates to a genetic marker for determining the inosinic acid content in muscle of \28294Whgoose, in particular to the determination of polymorphism in ADSL gene, namely the existence of mononucleotide polymorphism (SNP) of adenosine succinate lyase gene related to the inosinic acid character of \28294Whgoose.
Technical Field
\28294ThePhu goose is used as a local variety resource, has delicious meat quality, is rich in nutrients necessary for human bodies, and is very beneficial to human health. The goose meat is rich in nutrition, contains various amino acids, proteins, vitamins, sugar and trace elements necessary for human body, has low fat content and high unsaturated fatty acid content, and is easy to be digested and absorbed by human body. Goose meat is classified as one of green food which is mainly developed in the 21 st century by the food and agriculture organization of the united nations in 2002, and is popular with wide consumers.
Due to the fact that the Eremophilus geese lack of scientific systematic breeding, the individual difference of the inosinic acid content of goose meat is large, the industrial development of the geese is severely restricted, and the product quality and the economic benefit are affected. The conventional breeding is slower, with the development of a biomolecule technology, the major gene or molecular marker related to the inosinic acid character of the meat of the goose of \28294jpacan be searched from the DNA level, and the major gene or molecular marker is applied to marker-assisted selection in breeding, so that the breeding process is improved, and greater economic benefit is obtained.
Inosinic acid (IMP) is a major component of umami substances, and is currently internationally and generally used as an important index for measuring the umami of muscles. IMP is synthesized in vivo through a complex process including de novo synthesis and salvage synthesis of two pathways, requiring the participation of 10 enzymes, among which the adenylosuccinate lyase gene (ADSL) is the key enzyme catalyzing the purine step 8 reaction, and belongs to the aspartase/fumarase super family members, which can catalyze two non-consecutive reactions at the same activation site: (1) the method is used for catalyzing aminoimidazole succinyl carbamoyl nucleotide (SACIAR) to generate aminoimidazole carbamoyl nucleotide (AICAR), (2) catalyzing adenosine succinate monophosphate to generate adenosine monophosphate, and the two catalytic reactions have important effects on IMP generation. Since Jaeken and Berghe found the first ADSL-deficient patient, more than 50 pathogenic effect site mutations have been found in ADSL gene, so the research on ADSL, the rate-limiting enzyme for IMP synthesis, has mainly focused on the fields of physiology, pathology and immunity. At present, the research on Taihe black-bone chicken and recessive white feather broiler chickens by Zhang Yuan and the like preliminarily proves the correlation between ADSL gene and IMP content. The research of Jingjing et al shows that the nucleotide variation of exon 2 of ADSL gene is related to muscle IMP content. Shihao et al found SNP sites significantly related to IMP content on exon 9 of the MIYI chicken ADSL gene.
The study on DNA genetic markers, genetic diversity and related functional genes of the Pu goose is still in the initial stage, and no report on the genetic markers related to the Pu goose IMP is found in 28294um.
Disclosure of Invention
Aiming at the defects of the prior art, an ADSL gene and the application thereof as a character genetic marker of the breast muscle inosinic acid of the Jiu goose of 28294HJ are provided; simultaneously, SNP related to the properties of the breast muscle inosinic acid of the Erwinia hispida is screened to be applied to marker-assisted selection or marker-assisted introgression, and the application of the genetic marker in the Erwinia hispida marker-assisted selection is provided.
The embodiments of the present invention are implemented such that,
an ADSL gene, wherein the gene is related to the character of the inosinic acid content in breast muscle of the goose of \28294u.
Furthermore, the 146bp of the gene has a base mutation G → T.
The primer pair for detecting mutation at the 146bp position of ADSL gene sequence related to the inosinic acid character of v 28294u goose is as follows:
a forward primer: 5 '-CACCCATAAAGGGAACAG-3'
Reverse primer: 5 '-AGCACAGACAGCCAGATT-3'.
The embodiment of the invention also aims to provide a method for using the ADSL gene as a genetic marker related to the character of 28294Whityas goose breast inosinic acid, which comprises the following steps:
(1) according to the sequence of goose ADSL gene in GenBank, using Primer5.0 to design Primer, covering all exon 9 to exon 10 sequences of said gene;
(2) PCR amplification, product purification and cloning are carried out by taking the genomic DNA of the aforementioned \28294Jiugoose as a template, so as to obtain a nucleotide sequence shown as SEQ ID NO.1 in a sequence table;
(3) and identifying whether the 146bp of the gene sequence has mutation.
Furthermore, the method selects \28294thePu goose as a research object, adopts a molecular biological method to screen the \28294thePu goose breast inosinic acid character related genes, and comprises the following steps:
(1) the same batch of 80-day-old healthy Ji28294; 60 goose are selected to carry out the feeding test;
(2) designing a primer according to the sequence of the ADSL gene, and amplifying and sequencing a sample;
(3) and screening SNP related to the inosinic acid character.
\28294screeningof Gene related to muscularis inosinate of Phoenix goose
Healthy \28294ugoose 60 of the same day age was selected for the gavage test. Slaughtering is carried out 28 days later, the inosinic acid content in the pectic muscle is detected through high performance liquid chromatography, and genome DNA of the samples with high inosinic acid content and low inosinic acid content is respectively extracted.
According to the sequence of goose ADSL gene in GenBank, designing primer with Primer5.0 to cover the whole sequence from exon 9 to exon 10 of the gene, the primer sequence is:
a forward primer: 5 '-CACCCATAAAGGGAACAG-3'
Reverse primer: 5 '-AGCACAGACAGCCAGATT-3'
The primers were synthesized by Hainan Okangke Biotechnology Ltd. The primers were used to amplify the genomic DNA of \28294J.though goose, respectively.
And (3) PCR: 20 μ l reaction: 2 × Master mix 12. mu.l, upstream and downstream primers (10. mu. mol/L)) 0.8. mu.l each, DNA template 1. mu.l, and ultrapure water to the desired volume.
PCR amplification procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 57.6 ℃ for 30s, and extension at 72 ℃ for 45s, for 35 cycles; prolonging for 10min at 72 deg.C, and storing at 4 deg.C.
Taking 5 mu l of PCR product to carry out gel electrophoresis, and detecting whether the size of the product fragment accords with the size of the target fragment, wherein the length of the PCR product is 1255 bp.
Taking 30 mu l of PCR amplification product, purifying the product, sending the purified product to Hunan Qin Biotechnology Limited company for sequencing, performing reverse sequencing for a reaction, and confirming that the PCR product is an ADSL gene according to a sequencing result, wherein the sequence is shown as SEQ ID NO:1 is shown. FIG. 1 is a graph of mutation sites and sequencing peaks.
The experimental result shows that the related gene extracted from the breast muscle of \28294ugoose is ADSL gene, and the SNP related to the inosinic acid content in the breast muscle is screened from the gene sequence, which is probably the reason for influencing the content of \28294uinosinic acid in the breast muscle.
The invention adopts a method of combining PCR and DNA sequencing to screen ADSL gene \28294u, Pu goose inosinic acid character related gene, then screen SNP related to character from \28294u, Pu goose ADSL gene, and confirm that ADSL gene is breast muscle inosinic acid content related gene through production practice verification, and screen SNP is related to SNP. The SNP of the ADSL gene of the Pu goose screened by the invention can be used as a genetic marker for auxiliary selection of the Pu goose, so that the < w > 28294C </w > goose with higher inosinic acid content in the pectoralis muscle and more fresh muscle quality can be cultured, and the Pu goose has extremely great application value and economic benefit.
Drawings
FIG. 1 is a graph of mutation sites and sequencing peaks.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1 < 28294; SNP screening of thymic inosinic acid related gene of Phone
Healthy \28294ugoose 60 of the same day age was selected for the gavage test. Slaughtering is carried out 28 days later, the inosinic acid content in the pectoralis muscle is detected through high performance liquid chromatography, and the genome DNA of the samples with high inosinic acid content and low inosinic acid content is respectively extracted.
According to the sequence of goose ADSL gene in GenBank, designing primer with Primer5.0 to cover the whole sequence from exon 9 to exon 10 of the gene, the primer sequence is:
a forward primer: 5 '-CACCCATAAAGGGAACAG-3'
Reverse primer: 5 '-AGCACAGACAGCCAGATT-3'
The primers were synthesized by Hainan Okangke Biotechnology Ltd. The primers were used to amplify the genomic DNA of \28294J.though goose, respectively.
And (3) PCR: 20 μ l reaction: 2 × Master mix 12. mu.l, upstream and downstream primers (10. mu. mol/L)) 0.8. mu.l each, DNA template 1. mu.l, and ultrapure water to the desired volume.
PCR amplification procedure: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 57.6 ℃ for 30s, and extension at 72 ℃ for 45s for 35 cycles; prolonging for 10min at 72 deg.C, and storing at 4 deg.C.
Taking 5 mul of PCR product to carry out gel electrophoresis, and detecting whether the size of the product fragment accords with the size of the target fragment, wherein the length of the PCR product is 1255 bp.
Taking 30 mu l of PCR amplification product, purifying the product, sending the purified product to Hunan Qin Biotechnology Limited company for sequencing, performing reverse sequencing for a reaction, and confirming that the PCR product is an ADSL gene according to a sequencing result, wherein the sequence is shown as SEQ ID NO:1 is shown. FIG. 1 is a graph of mutation sites and sequencing peaks.
The experimental results show that the related gene extracted from the breast muscle of \28294; pump goose is ADSL gene, and SNP related to the inosinic acid content in the breast muscle is screened in the gene sequence, which is probably the reason for influencing the inosinic acid content in \28294; pump goose.
Example 2 validation of SNPs related to the inosinic acid content in breast muscle of Erwinia
The 80-day-old goose under the same feeding and management conditions was selected and tested at 28294U, 20 geese. After 28 days of gavage, all tested \28294; the goslings were slaughtered and the inosinic acid content in the pectorals was examined.
Extracted 20 breast muscle tissue-like genomic DNAs of \28294Hepsgoose were amplified using the primers in example 1, and SNPs thereof were detected according to the method of example 1.
TABLE 1 PCR reaction System
The PCR cycle parameters were as follows:
pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 57.6 ℃ for 30s, and extension at 72 ℃ for 45s for 35 cycles; prolonging for 10min at 72 deg.C, and storing at 4 deg.C.
ADSL gene SNP locus genotype frequency analysis
The genotype frequency and allele frequency of the mutation site were examined by chi-square method, and the results are shown in Table 2, and the site is in Hardy-Weinberg equilibrium (P > 0.05).
TABLE 2SNP site genotype and Gene frequency
ADSL gene and its 28294Hps goose fat liver character correlation analysis
According to the table 3, the influence of different SNP genotypes on the inosinic acid content of the breast muscle of the Ph goose is analyzed, the influence of different SNP genotypes on the inosinic acid content of the breast muscle of the Ph goose is obvious, wherein the different SNP genotypes have obvious influence on the inosinic acid content of the breast muscle of the Ph goose, the GG genotype and the TT genotype have obvious influence, the inosinic acid content of the breast muscle of the Ph goose is higher than that of the breast muscle of the GT type individual by 31.65 percent and 25.23 percent, and the difference reaches an obvious level (P < 0.05).
TABLE 3 comparison of inosinic acid content in breast muscle of Phi goose for different genotypes \28294c
Note: the difference is significant when different lower case letters are annotated on the same column (P < 0.05).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Hunan agriculture university
<120> ADSL gene and genetic marking method for using as \28294u, Pu goose inosinic acid content
<130>
<160> 1
<170> PatentIn version 3.1
<210> 1
<211> 445
<212> DNA
<213> goose (goose)
<220>
<221> mutation
<222> (146)
<223> k = t or g
<400> 1
tttgcagcac aagtgtaata ttaataactc tgtgtgagtg ggaagagaac aaagaaagat 60
gggatattca ggacaagaac ccagaagctt ttgtttctca ggagtgaaac atcaggaagt 120
gaaaaaaggt atttttgaat tcattkgtac ctgacgattg cctcctgctt tcaccatcgc 180
catgattata ttctctgtgg ccatgaatgg aagctcctgc cggatccttc tgtcaatcac 240
cttaaaaaaa aacaaaacaa aacaacaaac cagaaaaaaa gagaaacagc attacaggaa 300
ttcttgtaga caaccgtgat tcctggtgaa gttttcctct ccttttgaat ccccaggact 360
gtgcagtgat gaaaaggtaa ttactgtttt tgagcttagc tcaaaatcta ttgaagctga 420
taataaaaac tgaactttct atgga 445
Claims (1)
1. A method for using ADSL gene shown in SEQ ID NO.1 as a genetic marker related to the character of \28294jpsgoose inosinic acid is characterized by comprising the following steps:
(1) downloading a goose ADSL gene sequence in GenBank, designing a primer by using a Primer5.0, and covering the whole sequence from exon 9 to exon 10 of the gene;
(2) using \28294ugoose genome DNA as a template, performing PCR amplification, product purification and reverse sequencing to obtain a nucleotide sequence shown in a sequence table SEQ ID NO. 1;
(3) and identifying whether G-T mutation exists in 146bp of the gene sequence, and GG and TT genotypes are \28294c, and the inosinic acid content of the thoracic muscle of the Phu goose is higher than that of GT type individuals.
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