Pouch pig SLA-2 genes and application thereof
A technology of SLA-2 and purse pig, which is applied in application, genetic engineering, plant gene improvement, etc., can solve the problems of commercial breeding of purse pig, lack of scientific and effective guidance for breed identification and artificial breeding, etc.
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Embodiment 1
[0028] Example 1: Cloning of SLA-2 alleles of piglets
[0029] 1. Primer design
[0030] Utilize RT-PCR to amplify the above-mentioned pouch pig SLA-2 allele, the primer sequence used for amplification is:
[0031] Upstream primer S1: 5'-AGATGCGGGTCAGGGGCCCTCAAG-3' (SEQ ID NO: 5);
[0032] Downstream primer S2: 5'-CAGTCCCCACAAGGC AGCTGTCTC-3' (SEQ ID NO: 6);
[0033] 2. Extraction of total RNA from porcini
[0034] The spleen of the purse pig was collected, and the total RNA of the nucleic acid was extracted using the TRIZOL kit (BS409, Canada Bio Basic Inc.), and the method was carried out according to the kit instructions.
[0035] 3. RT-PCR
[0036] Using Oligo dT as a reverse transcription primer, reverse-transcribe the nucleic acid into cDNA according to the instructions of the reverse transcription kit (M-MLV, RNase H-, Treasure Bioengineering (Dalian) Co., Ltd.); then use PCR to amplify primer S1 / S2 amplifies SLA-2.
[0037] The amplification RT-PCR program is: ...
Embodiment 2
[0048] Embodiment 2: Sequence analysis and identification of varieties
[0049] The gene sequence obtained in Example 1 was analyzed through Gentyx9.0 (Software Development co., Ltd), and its amino acid sequence was deduced; the amino acid sequences of 44 SLA-2 alleles except the purse pig were mobilized on DDBJ / EMBL / GenBank , amino acid sequence comparison using DNAMAN Version5.2.2 (Lynnon Biosoft).
[0050]The analysis results are attached Figure 1~4 Shown: The 47th, 48th, 67th, 68th, 74th, 97th, 119th, 138th, and 240th positions of the SLA-2 gene in the coding region of the purse pig are characteristic amino acids, and the specific information is as follows :
[0051] Location
amino acid
English name of amino acid
amino acid abbreviation
47
F or Phe
48
I or Ile
67
A or Ala
68
K or Lys
...
Embodiment 3
[0054] Example 3: Drawing of Molecular Evolution Atlas and Its Application in Optimizing Genetic Breeding
[0055] Molecular evolution maps were drawn using the neighbor-joining method of DNAMAN and Mega2 software (Mega software Co., Ltd). The molecular evolution map is attached Figure 5 . According to the molecular evolution map, all SLA-2 alleles were clustered into three categories, namely, I, II and III, and the SLA-2 alleles of the purse pig belonged to the B III region. From this, it can be clearly judged that the purse pig strain is close to the American Hanford AF464039, the American Large White EF125047, and the cell line 10sk21 EU432083; while it is genetically related to the Chinese hybrid strain AF205147, the American NIH miniature pig EU867814, and the American CMS miniature pig EU431219. The relationship is far away. According to the principle of genetic breeding, under the condition of the same dominant traits, we should try our best to select the strains wit...
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