CN108130363B - Method for identifying Hexi black pig by using mutation site of 12SrRNA gene - Google Patents

Abstract

The invention relates to the field of animal genetics, in particular to a method for identifying Hexi black pigs by using mutation sites of 12S rRNA genes. The method comprises the following steps: downloading 12S rRNA genes in a pig reference sequence in NCBI; designing a PCR primer at the upstream and the downstream of the 314 th base of the 12S rRNA gene respectively; extracting DNA of a sample to be detected, and performing PCR amplification by taking the DNA of the sample to be detected as a template and the PCR primer in the step (2) as a primer; carrying out electrophoresis gel running (agarose gel electrophoresis) identification on the PCR product, and recovering a target product; and (3) comparing the sequenced target product with the 12S rRNA gene of the Hexi black pig to complete the identification of the Hexi black pig. The invention not only solves the distinguishing among black pigs in the same region, but also solves the problems that the existing pork market is disordered, defective pork is used as high-quality pork, and an effective detection method is not available.

Description

Method for identifying Hexi black pig by using mutation site of 12SrRNA gene

Technical Field

The invention relates to the field of animal genetics, in particular to a method for identifying Hexi black pigs by using mutation sites of 12S rRNA genes.

Background

The black pigs in Hexi are mainly distributed around Lu county in the three gorges region in Henan province, and through long-term natural selection, local mountain area masses carry out artificial breeding according to self feeding habits and living consumption habits, so that the local pig breeding method is suitable for local feeding environment conditions. It is resistant to rough feeding, has strong adaptability and disease resistance, has good meat quality, has great market influence in the western Henan region, and is habitually called black pigs in the Western Henan by the industry.

The mitochondrial genome is a segment of relatively conservative circular DNA sequence in animal genome, has the characteristics of strong conservation and high evolution speed, and is an important material for researching genetic evolution. The species identification technology is mainly focused on several regions such as D-Loop Loop, rRNA gene, CO oxidase subunit gene and the like. PCR technology is widely used in food, feed, and animal species identification because of its simple and easy-to-operate method, species specificity, and high sensitivity.

The pig mitochondrial 12S ribosomalRNA (12S rRNA) gene has a total length of 960 bp (NCBI Reference Sequence: NC-000845.1), does not contain introns, and is located between 1246 bp and 2205 bp of the pig mitochondrial Reference genome (Sscofa 11.1). The secondary structure of the 12S rRNA gene is functionally divided into four functional regions, namely a region I, a region II, a region III and a region IV, wherein each functional region is a single standard region, and the region III and the region IV are highly conserved. Structurally, four types of structures are involved, respectively: helix or stem region, lobe, loop region, unpaired region not within a helix. Wherein the stem region is a region in which bases are perfectly complementarily paired, and this region has a great influence on the stability of the secondary structure of 12S rRNA.

Disclosure of Invention

The invention provides a method for identifying Hexi black pigs by utilizing mitochondrial 12S ribosomal RNA (12S rRNA) genes, which solves the problems that the existing pork market is disordered, defective pork serves as high-quality pork, and an effective detection method does not exist.

The technical scheme of the invention is realized as follows:

the method for identifying Hexi black pigs by using mutation sites of 12S rRNA genes comprises the following steps:

(1) selecting 12S rRNA gene of Hexi black pig in NCBI;

(2) designing a PCR primer at the upstream and the downstream of the 314 th base of the 12S rRNA gene respectively;

(3) extracting DNA of a sample to be detected, and carrying out PCR by taking the DNA of the sample to be detected as a template and the PCR primer in the step (2) as a primer;

(4) carrying out agarose gel electrophoresis detection on the PCR product, and recovering a target product;

(5) and (3) comparing the sequenced target product with 12S rRNA gene of Hexi black pig (reference sequence on NCBI) to complete the identification of the Hexi black pig.

And (4) determining that the 314 th base of the 12S rRNA gene is C according to the sequencing result in the step (4) as a Hexi black pig breed.

The method can also be used to identify Duroc pigs.

When the method is used for identifying Duroc pigs, Duroc pigs with A inserted in the 314 th base of 12S rRNA gene are sequenced.

The invention has the beneficial effects that:

(1) the pig mitochondrial 12S rRNA gene has a total length of 960 bp (NCBI Reference Sequence: NC-000845.1), and structurally comprises four types of structures, which are respectively: a helix or stem region, a bulge, a loop region, an unpaired region not within a helix; wherein the stem region is a region in which bases are completely complementary and paired, and this region has a great influence on the stability of the secondary structure of 12S rRNA, and a base mutation at position 314 of the 12S rRNA gene occurs in the stem region, as shown in FIG. 1.

(2) By amplifying mitochondrial genomes of black pigs in western Henan and comparing with breeds in other parts of Henan province (black pigs in Town, Huainan and Nanyang), breeds in other parts of China (Min, Laiwu, Erhualian, Bama, Banna, Wuzhi, Tibetan and Taiwan blue), major commercial breeds in West (Duroc, Changbai and Dabai), and local breeds in southeast Asia (Moncai and JNP), the existence of a unique mutation of black pigs in western Henan on is found on the 12S ribosomal RNA gene as shown in FIG. 4; the mutation can be used in related research of variety identification.

(3) The method can not only identify the Hexi black pig and the Western commercial pig breed to standardize the high-quality meat market, but also can quickly and accurately identify the high-quality black pig breed of the same region; as is well known, the meat quality of western pig species sold in the market is not as good as that of local black pig, and the price is low, so that part of vendors are introduced to be full; another common problem is that local black pigs in the same region are easily confused in appearance, so that the conservation and utilization effects of the subsequent local black pigs are affected.

(4) According to the invention, through simultaneously amplifying the 12S rRNA gene 314 upstream and downstream sequences of Henan black pigs, three Henan local varieties (the Jianshan black pigs, the Huainan pigs and the Nanyang black pigs) and Western pig breeds (the Duroc pigs, the Changbai pigs, the Dabai pigs, the Vietnam Moncai pigs and the JNP pigs), the mutation of the Henan black pigs at the 314 locus is found to have specificity by comparing the Henan black pigs with other pig breeds; the method is proved to be capable of distinguishing the Hexi black pig and the Western pig species and accurately distinguishing other black pig species in Henan province belonging to the same region; compared with a large-scale sequencing method, the method is simple to operate, rapid, efficient and low in cost, and lays a foundation for the subsequent variety utilization of Hexi black pigs.

Drawings

FIG. 1 is a schematic diagram of the secondary structure of the pig mitochondrial 12S rRNA gene.

FIG. 2 is an electrophoretogram of the amplification product in example 1.

FIG. 3 is a graph showing the sequencing results of the PCR products recovered in example 1.

FIG. 4 is a graph showing a comparison of 12S rRNA genes of a pig breed in southeast Asia (and a commercial pig in the West) with a black pig in the West of Henan.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

The principle is as follows: the mitochondrial DNA mutation speed of eukaryotes is very high, and the eukaryotic DNA mutation speed is commonly used for analyzing and identifying genetic evolution relations. According to the invention, mitochondrial genomes of four regional pigs (namely the black pig in Henan province, the Huainan pig and the black pig in Nanyang) in Henan province are amplified and compared, and the result shows that a special mutation g.314T mutation is C in the black pig in Hexi province at position 314 of a 12S rRNA gene, as shown in figure 4. In order to verify the specificity and reliability of the mutation site, the invention further downloads eight other local pig breeds (Min pig, Laiwu black pig, Erhualian pig, Bama miniature pig, Banna pig, Wuzhishan pig, Tibetan pig, Taiwan Lanyu pig) with regional representativeness, three main western commercial pig breeds (Duroc pig, Changbai pig and Dabai pig), and two local pig breeds in southeast Asia (Vietnam Moncai pig and JNP pig) from NCBI, and finds that 12S rRNA g.314T > C really has breed specificity and can be used for identifying the black pig in West Henan.

Selecting a pig 12S rRNA gene in NCBI, and respectively designing a PCR primer at the upstream and the downstream of the 314 th base of the 12S rRNA gene, wherein the sequence of the upstream primer is 5'-GCTCATAACGCCTTGCTC-3', and the sequence of the downstream primer is 5'-ATTCTTTCATCTTTCCCTT-3';

extracting DNA of each sample of the pig ear tissue to be detected by a phenol chloroform method, carrying out PCR amplification by using the DNA of the sample to be detected as a template and the PCR primers as primers, and adopting a 25 mu L Polymerase Chain Reaction (PCR) system comprising 40ng of DNA, 12.5 mu L of 2 × ES Tap MasterMix (kang century CW 2214M), 10pmol of forward and reverse primers respectively and 8.5 mu L of water. The primer amplification conditions are 94 ℃ and 5 min; 30s at 94 ℃; 30s at 50.1 ℃; at 72 ℃ for 25 cycles, 50s, and finally at 72 ℃ for 10 min. The PCR products were identified by running gel electrophoresis (agarose gel electrophoresis), and the amplification products are shown in FIG. 2. Recovering the target product, sending the PCR product to a sequencing company, and performing Sanger bidirectional alignment sequencing (the reference sequence is shown as SEQ ID NO: 1), wherein the sequencing result is shown as figure 3; the base at position 314 of the 12S rRNA gene is mutated from T to C, namely the Hexi black pig.

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, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

<110> Henan university of agriculture

<120> method for identifying Hexi black pig by using mutation site of 12S rRNA gene

<160> 1

<210> 1

<211> 960

<212> DNA

<213>Pig(Sus)

<220>

<221> RNA

<222> (1)...(960)

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cacaggtttg gtcctggcct ttctattaat tcttaataaa attacacatg caagtatccg 60

cgccccggtg agaatgccct ccagatctta aagatcaaaa ggagcaggta tcaagcacac 120

ctataacggt agctcataac gccttgctca accacacccc cacgggaaac agcagtgata 180

aaaattaagc catgaacgaa agtttgacta agttatatta attagagttg gtaaatctcg 240

tgccagccac cgcggtcata cgattaaccc aaattaatag atccacggcg taaagagtgt 300

ttaagaaaaa aaatcacaat agagttaaat tataactaag ctgtaaaaag ccctagttaa 360

aataaaataa cccacgaaag tgactctaat aatcctgaca cacgatagct aggacccaaa 420

ctgggattag ataccccact atgcctagcc ctaaacccaa atagttacat aacaaaacta 480

ttcgccagag tactactcgc aactgcctaa aactcaaagg acttggcggt gcttcacatc 540

cacctagagg agcctgttct ataatcgata aaccccgata gaccttacca acccttgcca 600

attcagccta tataccgcca tcttcagcaa accctaaaaa ggaacaatag taagcacaat 660

catagcacat aaaaacgtta ggtcaaggtg tagcttatgg gttggaaaga aatgggctac 720

attttctaca tgagtatatc caccacacga aagtttttat gaaactaaaa acccaaggag 780

gatttagcag taaatcgaga atagagtgct tgattgaata aggccatgaa gcacgcacac 840

accgcccgtc accctcctca agcatgtagt aataaaaata acctatattc aattacacaa 900

ccatgcaaga agagacaagt cgtaacaagg taagcatact ggaaagtgtg cttggattac 960

Claims (1)

1. The method for identifying Hexi black pigs by using the mutation sites of 12S rRNA genes is characterized by comprising the following steps:

(1) selecting a pig 12S rRNA gene sequence from NCBI as shown in SEQ ID No. 1;

(2) designing a PCR primer at the upstream and the downstream of the 314 th base of the 12S rRNA gene respectively;

(3) extracting DNA of a sample to be detected, and carrying out PCR by taking the DNA of the sample to be detected as a template and the PCR primer in the step (2) as a primer;

(4) performing electrophoresis gel running identification on the PCR product, and recovering a target product;

(5) after sequencing is carried out on the target product, comparing the target product with 12S rRNA genes of the Hexi black pig to complete the identification of the Hexi black pig;

and (4) determining that the 314 th base of the 12S rRNA gene is C according to the sequencing result in the step (4) as a Hexi black pig breed.

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