CN118006803B - A molecular marker related to high altitude adaptation in sheep and its application - Google Patents
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Abstract
本发明属于遗传生物学技术领域，具体涉及一种与绵羊高海拔适应相关的分子标记及其应用。所述分子标记为SEQ ID NO.1～3所示的DNA片段组合和SEQ ID NO.4～6所示的DNA片段组合；SEQ ID NO.1～3所示的DNA片段组合对应绵羊β‑珠蛋白基因座A单倍型，该单倍型绵羊具有高海拔适应性；SEQ ID NO.4～6所示的DNA片段组合对应绵羊β‑珠蛋白基因座B单倍型，该单倍型绵羊不具有高海拔适应性。本发明还提供了用于检测该结构变异的特异性引物对和检测方法，本发明所述分子标记揭示了绵羊β‑珠蛋白基因座A单倍型与高海拔适应相关，可用于绵羊高海拔适应性引种和品种选育。
The present invention belongs to the field of genetic biology technology, and specifically relates to a molecular marker related to sheep high-altitude adaptation and its application. The molecular marker is a combination of DNA fragments shown in SEQ ID NO.1-3 and a combination of DNA fragments shown in SEQ ID NO.4-6; the combination of DNA fragments shown in SEQ ID NO.1-3 corresponds to the sheep β-globin locus A haplotype, and the haplotype sheep has high-altitude adaptability; the combination of DNA fragments shown in SEQ ID NO.4-6 corresponds to the sheep β-globin locus B haplotype, and the haplotype sheep does not have high-altitude adaptability. The present invention also provides a specific primer pair and a detection method for detecting the structural variation. The molecular marker of the present invention reveals that the sheep β-globin locus A haplotype is related to high-altitude adaptation, which can be used for the introduction of sheep high-altitude adaptability and variety breeding.
Description
Technical Field
The invention belongs to the technical field of genetic biology, and particularly relates to a molecular marker related to sheep high altitude adaptation and application thereof.
Background
The Qinghai-Tibet plateau is the highest elevation in the world, and the average elevation exceeds 4000 meters, and is called as the world ridge and the third pole, and is the origin of many large rivers in Asia. In Qinghai-Tibet plateau, livestock migration grazing is very common, and along with the migration of human beings, livestock are continuously adapted to various geographic environments and provide various livestock products necessary for living for local residents. Humans and animals exhibit convergent evolution in adapting to high altitude environments, i.e. to local environments through similar molecular genetic mechanisms.
Sheep is the most common livestock (> 2000 ten thousand) on Qinghai-Tibet plateau, is one of the oldest local sheep varieties in China, is a special livestock resource for the Qinghai-Tibet plateau, has the characteristics of cold resistance, coarse feeding resistance, strong disease resistance and the like, and can be well adapted to the anoxic environment of the Qinghai-Tibet plateau at high altitude. Various molecular mechanisms and related genetic variations related to the high altitude adaptability of Tibetan sheep, such as EPAS1, GNPAT, CRYAA, CDT, PPARG and MMP3 genes and the like, are mainly related to the functions of energy metabolism, oxygen transportation, hypoxia response, ultraviolet radiation resistance and the like, which are determined by utilizing a whole genome scanning technology at present.
Hemoglobin is the only oxygen transport protein in blood, and its oxygen affinity determines the oxygen transport capacity. Hemoglobin beta-chain gene (HBB) plays a vital role in regulating the transportation and exchange of oxygen and carbon dioxide, and a single nucleotide polymorphism site (single nucleotide polymorphism, SNP) of the gene may enhance the binding capacity of Hemoglobin and oxygen or inhibit the sensitivity of Hemoglobin to allosteric factors, which is an important molecular mechanism of high altitude vertebrates adapting to the plateau hypoxia environment. In addition, unknown genomic elements such as large fragment structural variations may also be involved in regulating the function of the key physiological phenotype of altitude adaptation, so analysis of genetic correlation between large fragment structural variations and the key physiological phenotype of sheep requires further investigation.
Disclosure of Invention
In order to solve the technical problems, the invention provides a molecular marker related to sheep high altitude adaptation and application thereof.
In a first aspect, the invention provides a molecular marker related to sheep high altitude adaptation, wherein the molecular marker is a DNA fragment combination shown in SEQ ID NO. 1-3 and a DNA fragment combination shown in SEQ ID NO. 4-6.
Further, the DNA fragment combinations shown in SEQ ID No. 1-3 correspond to sheep beta-globin locus A haplotypes, the haplotypes have high altitude adaptability, and the DNA fragment combinations shown in SEQ ID No. 4-6 correspond to sheep beta-globin locus B haplotypes, the haplotypes do not have high altitude adaptability.
In a second aspect, the invention provides a primer for detecting the molecular marker, wherein the primer for detecting the DNA fragment combination shown in SEQ ID NO. 1-3 is shown in SEQ ID NO. 7-12, and the primer for detecting the DNA fragment combination shown in SEQ ID NO. 4-6 is shown in SEQ ID NO. 13-18.
In a third aspect, the present invention provides a kit for detecting the molecular marker, the kit comprising the primer and a reagent for PCR amplification.
The reagents for PCR amplification were 2X RAPID TAQ MASTER Mix and ddH 2 O.
In a fourth aspect, the invention provides the use of the molecular marker, the primer or the kit for identifying high altitude adaptation of sheep.
In a fifth aspect, the invention provides the use of said molecular markers, said primers or said kit in sheep breeding.
Further, the breeding is to cultivate sheep breeds with high altitude adaptability.
In a sixth aspect, the invention provides a method of identifying sheep high altitude suitability comprising the steps of:
carrying out PCR amplification on sheep genome DNA by using the primer to obtain an amplification product;
Identifying the amplified product;
If the DNA fragments shown in SEQ ID NO. 1-3 are all present and the DNA fragments shown in SEQ ID NO. 4-6 are not present, judging that the sheep to be tested has high altitude adaptation characteristics;
if the DNA fragments shown in SEQ ID NO. 1-3 are not present and the DNA fragments shown in SEQ ID NO. 4-6 are not present, judging that the sheep to be tested does not have the high altitude adaptation characteristic.
Further, in the PCR amplification reaction, the amplification system was 10. Mu.L, which contained 50-100 ng/. Mu.L of the sheep blood genomic DNA template to be tested 1. Mu.L, 10. Mu.M forward and reverse primers each of 0.2. Mu.L, 2X RAPID TAQ MASTER Mix 5. Mu.L, ddH 2 O3.6. Mu.L.
Further, in the PCR amplification reaction, the amplification reaction was performed by pre-denaturing at 94℃for 3min, denaturing at 94℃for 10sec, annealing at 58℃for 20sec, extending at 72℃for 1min for 36 cycles, extending at 72℃for 5min, and preserving at 4 ℃.
The invention has the following beneficial effects:
(1) The invention utilizes PacBIO HiFi data from 13 sheep of different varieties, and combines the second generation sequencing data of 924 domestic sheep to carry out haplotype detection on a candidate region (chr15: 47,954,795-48,033,976), and finds that the coverage of B haplotype in the candidate region is lower due to the fact that a large fragment of about 40kb exists in the region, and a homozygote carrier has almost no coverage (figure 1), further analysis coverage shows that the frequency of A haplotype increases along with elevation, and the B haplotype can be used as a molecular marker for sheep high elevation adaptive breeding.
(2) The frequency of the structural variation A haplotype in the high-altitude sheep population is 0.93, and the frequency of the structural variation A haplotype in the low-altitude sheep population is only 0.24, which shows that the structural variation A haplotype has high genetic correlation with sheep high-altitude adaptation, and lays a foundation for molecular marker assisted selection of sheep high-altitude adaptation.
(3) The method for detecting the structural variation between the 47954795 th base and the 48033976 th base of the chromosome 15 in the sheep blood genome is simple to operate, accurate and reliable in result, and can further realize analysis of HBB haplotype functional differences.
Drawings
FIG. 1 shows a schematic diagram of the structure of the sheep beta-globulin gene locus (A), genomic arrangement of the beta-globulin gene regions of sheep, pan sheep, high altitude (Tibet) and low altitude (Tekesel) sheep (B). Red triangles indicate primer positions.
FIG. 2 shows agarose gel electrophoresis results of two haplotype structure variation products amplified by the PCR reaction, wherein P1-P6 are the 1 st-6 th primer pair, high is the High altitude sheep to be detected, and Low is the Low altitude sheep to be detected.
FIG. 3 is an oxygen dissociation curve of Hb solutions of sheep A and B haplotypes in the absence of Cl - and DPG (stripped) and in the presence of 0.1mol/L Cl - (0.1 mol/L KCl) and DPG (concentration 2 times that of Hb) at 37℃and pH=7.4.
Detailed Description
The present invention will now be described in detail with reference to the drawings and specific examples, which should not be construed as limiting the invention. Unless otherwise indicated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise indicated.
Example 1 Whole genome analysis of sheep beta-globin loci potential functional variation and high altitude adaptability
Structural variation of the β -globin locus was detected using PacBIO HiFi data from 13 different sheep, and the coverage of the candidate region (chr 15:47,954,795-48,033,976) divided by the average coverage of chromosome 15 to distinguish haplotypes A and B. As a result, it was found that Tibetan sheep, yunnan sheep, merino sheep, dongfoli sheep, wu Zhumu Qing sheep and Charland sheep carried the A haplotype, while Kazakhstan sheep, tasat sheep, romney sheep, SAFOK sheep, texel sheep, duPond sheep and Sunit sheep carried the B haplotype.
The distribution of the A haplotype in sheep populations at different altitudes in the world was studied using the second generation sequencing data of 924 domestic sheep, and coverage analysis results showed that the frequency of the A haplotype increased with increasing altitude. For example, in 98 high altitude sheep (habitat altitude about 5000 m), the frequency of the a haplotype is 0.93, whereas in 422 low altitude sheep (habitat altitude about 1000 m), the frequency of the a haplotype is only 0.24.
Therefore, the structural variation can be used for predicting the high altitude adaptation characteristics of sheep individuals, and provides an important guiding function for introduction and breeding of sheep in a plateau area. When the sheep carries the insertional structural variation (A haplotype), the sheep is judged to have strong high altitude adaptability, when the sheep carries the deletion structural variation (B haplotype),
It is determined that the sheep is less adaptable at high altitude. By crossing the excellent individuals carrying the deletion structural variation with the local variety, the high altitude adaptability gene can be introduced, and the overall adaptability of the filial generation can be improved. Meanwhile, by using a molecular marker assisted selection technology, individuals carrying the deletion type structural variation can be screened out, and the sheep breeding process in the plateau region is further accelerated.
Example 2 identification of type of structural variation of sheep beta-globin locus
1. Extracting genome DNA of sheep blood to be detected
Blood samples from 39 sheep living in the plateau region and 30 sheep living in the non-plateau region were collected from each place in China, and detailed information is shown in Table 1, and Genomic DNA in the Blood was extracted by using Blood Genomic DNA MINI KIT (CW 2087S).
Table 1 sheep sample collection table to be tested
Note that sheep living in the plateau region have high altitude adaptability by default.
2. PCR amplification of nucleotide fragments containing structural variation sites
Specific primers were designed for the structural mutation sites of the beta-globin Gene locus selected in example 1 based on the HBB Gene sequence (Gene ID: 100049064) recorded in NCBI website, and the nucleotide sequences of the primers were as follows:
TABLE 2 primer information
In the PCR amplification reaction, the amplification system is 10. Mu.L, which contains 50-100 ng/. Mu.L of sheep to be detected
Blood genomic DNA template 1. Mu.L, 10. Mu.M forward and reverse primers each 0.2. Mu.L, 2X RAPID TAQ MASTER Mix 5. Mu.L, ddH 2 O3.6. Mu.L.
The amplification reaction was performed by pre-denaturation at 94℃for 3min, denaturation at 94℃for 10sec, annealing at 58℃for 20sec, extension at 72℃for 1min for 36 cycles, and extension at 72℃for 5min for 4℃for storage.
The nucleotide sequence of the amplified product of the primer SEQ ID NO. 7-8 is as follows:
SEQ ID NO.1：ATCTTGGCGCTACTGTCTCGGGGAAAATGTCTTACCTT CTAGGCCTCTGATCAGCAGCCACGAAGTGCTGTCACTCTCCATGTCACTTTGGCTAAAACTTGAATGTGTGAACATGCTCCAGAGATATTTTCTGATTTTCCTACCTACCCATCCAGCATCTTCATTTTATTTCATTTATTTAATTTTGGCTGTCCTAGTCCTTCACTGATGCATGTAGACTTTCTCTAGTAGCGGCAAGCAAGGCTACTTTCCAGCTGTGGTGCTTAGGCCTCTCACTGTGATTTCTCTGGCTGTGGAGCACGGACTCCAGGGTACATAGACTTCAGCAGCTGTGGTGCTTAGGCTCAGCAGTTGTGGCCCACGGGCTTAATTGCTCAGTGGCGTGGAAGTATCCCCTACATTGGCAGGTGCTTTCTTAACCACAGGACCACCAGGGAAGTACCACTATCCTCATTTAAAAGCAAGAAATCTGAGGCAGTTAATGTGATCAACAAAGAAATCAACGAAACTCAATGTAAAGACTCTCTTTGTCTGACTTTCATATCCTTCCACACAGATACTCACATTCAGAAATTAGCACGTGCAAGAGCATGTGCATGCATGCACGCTCATACACACACCAACAGCTCACAAATTCCACATCCTGTTAGAGAATCCACTTGGCGTTAAATATTTATTTACAGGTTGACCCCTTGCTACCACCTCCCTGCTTTAAGGCAATATAATCTCCACCTGGATGCTTACAGCAGATTCTTAACTAGTCTGTCTACCACTGCTCCTGACAGTGTTTTCCCAACATGGCAGTCAAAGC.
The nucleotide sequence of the amplified product of the primer SEQ ID NO. 9-10 is as follows:
Primer(s) SEQ ID NO.2：TTTCTGCATCCAGACGCACTGGATCAGCCAATCA CAGATGAAGGGCACTGAGGAAGCACAGTGCATCTTACATCCCCCCAAACCAATGAACTTGTGTTATGCCCTGGGTTAATCTACTCTCAGAGGCAGAGAGGGCAGGAGGCTCATGGGGCTCACAAGGAAGACCAGGGCCCCTGCTGCTTACACCTGCTTTTGACACAACTTGCAGCTGGGTAAACACACATCATGGTGCATCTGACTCTAGAGGAGAAGGCTACTTGTCACTGCCCTGCGGAGCAAGATGAGGGTGGCTGAAGTTGGTGTTGAAACCCTAGGCAGGCAGGTATTCAGCTTACAAGGCAGGCTGAAGGAGAGTGAATGTCAGCTGGGTGTGTGGGGACAGAGCCATTGCCTGAGACTGAGGCACTGATCCCTCTGTTCTTATGCTGTTTTCACCCCCTAGGTTGTTGGTTGTCTACCCATGGACTCAGAGGTTCTTTGAGTCCTTTGGGAACTTGCCCTCTGCTGATGATATTATGGGCAACGCTAAGGTGAAGGCCCGTGACAATA.
The nucleotide sequence of the amplified product of the primer SEQ ID NO. 11-12 is as follows:
Primer(s) SEQ ID NO.3：AACCATGCTAGCAGCATCCATGTTTAAAAGAAAA TTGAATTGCTATGTACATCTGGAATCAACAAGTTAAAGAAAAGTTTCTCGTTTTTAAGTCAAAGGTTTTAGGGGGGATCAAATCAGAGATGAAATTGTGCTCTTCACTGTAGTGTGTGGACTTCTCATTGCAGTGTCTTCTCCTGGTGGAGCACATGCTCCTGGGTGCATGGGCTTAAGTAGTAACAGAGTGTGGGCTTCAGTAGTCTGGTGCAGCCTCAGTAGCCAGGGTGCTCAGGTTTAGTTGCTTTGCAGCATGTGGAATATTCCCAGACCAGGGTCAAACCCATGTTCCTTGCTTTCGCAGAAAATTCTTATCTACTATACACCAAGCAAGTCCTAGGTCTCACGATTTTAAGATTCCACATAGTGCACATCCTCTTTGCAGGGTTCTGTGGTTATGGACTGAGATATAAGGATGAAAGGGGGGGGGGCATGAAACAGGAACAGGAAAACATTGTTACCCATTTTAGGGTACCCACGGCATCTGCTGACAAAGGGCCTGTGACATGCTGGGCAGATCTGACTGGGGATGACACCAGGTAATGCTTGACTTAAGGAACCATCGCAGGCCCGTCTTGGCGCTGCTGCTCTCAGGGGAAAATGTCTTACCTTCTAGGCCTCTGATCAGCAGCCACAAAGTGCTGTCACTG.
The nucleotide sequence of the amplified product of the primer SEQ ID NO. 13-14 is as follows:
primer(s) SEQ ID NO.4：AGGAAAGCACTGGATCTGCCTACCTCTGTTAATT GAAAACCAAACAAAATAAAATAAATTGCCATCCCCAGCAAAGTCCCAGAAAACAAGTGAACACATAGAAGGAGAAGGAGATGGAGGAGGAGAATGAGACAGAAGAGAAGGAGAAGGAGAGGGGTAGAGGAGGAGAAAGAAGAAGGGGAAGAAGAAAAGGAAGGGAAAGAAAAACAAAACAAAACAAAAAAACAGAACAAGTGCAGGAAATAATCGGTGACATTAATGCCTGAAGGGAGCTGGAGTAGAGAGAAGGGCTATAGTAGCTTGTTGGTCTAGGAACCTAAGAAGTTTTCCTTTCCCTCAGAGACACCTCAGATTCAGTAAGATAGAAGTTGTGAAAATTTGGTTTCAGAAAGAGCCCTCTGGGGCTACAATTAGCAAAGGTTGCCAGGGGCTTGGGGCTTCCCATGTGGCACTAGAGGTAAAAGAACCTGCCTGCCAATGCAGGGGACTTAAGAGATATGGGTTCTATCTCTGGGTTGGGAAAATCCCCTGGAGAAGGGCATGGCAACTCACTCCATTATTCTTGCCTGGAGAATCCCACGGACAGCTGAGCCTGGCGGGCTACAGTCCAATGGGTCTCATAGAGTCACAGACAACTAAAGAGACAGCATGGCACGGCACAGTATGGAAAGGAACTTGGGAGAGGAGCAA.
The nucleotide sequence of the amplified product of the primer SEQ ID NO. 15-16 is as follows:
Primer(s) SEQ ID NO.5：CTGTTACCCTCGCAGTCTTGTAGTCTAAAATTAG CCAGCCAGGGCTTGGCGACATGCATGAAAATGGCTTTCTGGAAGTGGAAGAGAGAGATATAGATAGAGGATTAGGAAAGACTAGATAACATAAAGCTTTGAACATAAAAATAAAAAGTGTTACTATGATAGTAGCAAGAGTAAATAATAAGTATTCTAAGAACTTTAAATGACTCAAGGAATAGGATTTGGAGGGTGGAAGTAATCTGCTTAATTCACACAAAATGAGTTGAAAATTGCAGAGACTAGACATGGATACTAATTGAAGAGAACAGACATAATGGCTTCAATCCAAGAAAAGCAGTGTGTTAGTCTGTGTTTGACTTTTTGTGACCCTATGGACTGAAGAGGAACTAAAGAGCCTCTTGATGAAAGTGAAAGAGGAGAGTGAAAAAGTTGACTTAAAGCTCAACATTCAGAAAACTAAGATTATGGCATCTGGTCCCATCACTTCATGGCAAATAGATGGGGAAAAAGTGGAAACAATGTCAGACTTTATTTTGGGGGGCTCTAAAATCACTGCAGATGGTGATTGCAGCCATGAAATTAAAAGACGCTTACTCCTTGGAAGGAAAGTTATGACCAAACTAGATAGCATATTGAAAAGCAGAGATATTACTTTGCCAACAAAGGTTTGTCTAGTCAAGGCTATATTTTTCCAGTGGTCATGTAAGGCTGTGAGAGTTGGACTGTGAAGAAGGCTGAGTGCCGAAGAATTGATGCTTTGAACTGTGGTGTTGGAGAAGACTCTTGAGAGTCCCTTGGACTGCAAGGAGATCCAACCAGTCCATCCTAAAGGAGATCAGTCCTGAGTGTTCTTTGGAAGGACTGATGCTAAATCTGAAACTCCAGTACTTTGGCCACCTCATGTGAAGAGTTGACTCATTGGAAAAGACTTTGATGCTGGGAGGGATTGGGGGCATGAGGAGAAGGGGATGACAGAGGATGAGATGGCTGGATGGCATCACTGACTCAATGGACGTGAGTTTGGGTGAACTCTGGGAGTTGGTGATGGACAGGGAGCCCTGGCGTGCTGTGATTCATGGGGTTGCAAGAGTCGAACACGACTGAGCGACTGAACTGATGGACTGTAGTAGACCCAGCTCCTCTATCCATGGGATTTTCCTGGCAAAAATGCTGGAGCTGGTTGCCATTTCCTCCTCCAGGGGATCTTCCTGACCCAGTGATTGAACCTGTGTCTCTTGTTTGGCGGGCAGATTCTTTACCACTGAGCCACCAGGGAAGCCCTGGAAGAAGGTCAAGGAGGTCTGCAAAAAGTGAAGGATGAAGAAGGGAAGAGTGAAGAGGACGTGTTTTCTGATTGGCTAGGGTGGATGAGAAGG.
The nucleotide sequence of the amplified product of the primer SEQ ID NO. 17-18 is as follows:
primer(s) SEQ ID NO.6：GGGGACTTGTCCAATGCTGATGCTGTTATGAACA ACCCTAAGGTGAAGGCCCATGGCAAGAAGGTGCTAGACTCCTTCACTAAAGGCCTGAAGCATCTCCACCGTCTCAAGCGTTTCTTTGCTGCACTGAGTGAGCTGCACTGTGAGAAGCTGCATGTGAACCCTGAGAATTTCAAGGTGAGTCTATAGGACCCTTAAAGTTTTCCTGTTTTTGGTGGTCAAACTCCCATCATGGGAGAGGGCTGAGCAGCAGGACGCAGTTCAGAATGGAGAAGAGGTATTCTGATTATATCACCATGAACTCCTCAGGACCATTTAGCTTCTTTTACCTTCTTTGTTCTCAGCCATCATTTCTTCTTATTCATTCTTGTTTTTCTCTGTTTGTTCTCTGCAGTATCTTTTTTATATTTAAGCATTTTGAGGGTTTGAGTGTTAAGACTTTCTTCTTTTAAGTCACTTAAAAATTTTGTCTCATGTTTTTCCCGTATCTCTTCCTTTCAAAGCAAGGAAGACAAAATGATGTATTGCTTCTTGAAATAGTTCAAAAGAATAAAAAATGATAAGTTATGAATTAAGACAGAAAGAAACATTTCTCAGTACAAATTCAGGCTGATATGGATGGCTTCACATCAGTAGTAACAACTACACTTCAGCCATCTTTCTCCTTATATTCTAGGAGCACAGCTTGGGATGAGACTGAAATACTCTAAGTACAAATTGGGTGCTTCTGCTAATTGTGTTCTTGTTTTTTTATCTCTACCACACAGCTCCTGGGCAACATACTGGTGATTACACTGGCTCGACACTTTGGCAAGGAATTCACCCCAGAGTTGCAAGCTGCCTGTCAGAAGGTGGTGGCTGTTGTGGTTAATGCCCTCACCGA.
3. Analysis of structural variation type of target site of sheep to be detected
The PCR amplified products in step 2 are detected by 1% agarose gel electrophoresis, and the result is shown in figure 2. As can be seen from figure 2, the PCR amplified products of each pair of primers have uniform and bright bands, conform to the expected band size, and the structural variation type of the sheep individual to be detected can be obviously judged.
When the DNA fragments shown in SEQ ID NO. 1-3 are all present and the DNA fragments shown in SEQ ID NO. 4-6 are all absent, the structural variation carried by the sheep to be tested is judged to be the A haplotype and has the high altitude adaptation characteristic, and when the DNA fragments shown in SEQ ID NO. 1-3 are all absent and the DNA fragments shown in SEQ ID NO. 4-6 are all present, the structural variation carried by the sheep to be tested is judged to be the B haplotype and does not have the high altitude adaptation characteristic.
4. Statistical analysis of structural variation types of 69 sheep beta-globin loci
Statistical analysis was performed on the types of structural variation carried by 69 sheep individuals living at different altitudes according to the determination method described in 3, and the results are shown in table 3, with a frequency of 1 for the a haplotype in the high-altitude sheep population, 0.4 for the low-altitude sheep population, 0.28 for the b haplotype in the high-altitude sheep population, and 0.9 for the low-altitude sheep population.
Table 3 structural variation of the beta-globin locus haplotype numbers in sheep in two different living environments
Example 3 analysis of different haplotype functional differences of sheep beta-globin
1. Sheep blood collection and hemoglobin lysate preparation
The total jugular vein of sheep was collected into heparin sodium anticoagulation tubes and the beta-globin haplotype in sheep blood was identified as in example 2. 200. Mu.L of each of the two identified haplotypes was placed in a 2mL centrifuge tube, 1mL of ddH 2 O was added, incubated on ice for 30min, mixed on a shaking mixer, and centrifuged at 8000rpm for 15min at 133.4. Mu.L with 1M Tris-HCl solution at 4℃to remove cell membranes and cell debris. The resulting Hb lysate was placed in a dialysis bag and dialyzed overnight with 0.01M Tris-HCl solution added at a ratio of Hb lysate: dialysate=1:100, changing the dialysate every 4 h. The next day Hb solution was collected, naCl was removed using a PD-10 desalting column (GE HEALTHCARE, 17-0851-01), hb concentration was determined using a cyanidation high-iron Hb assay, calculated as hemoglobin (mmol/L) =measurement tube absorbance×5.7.
2. Determination of the oxygen dissociation curves of hemoglobin under different conditions
The oxygen dissociation curves of Hb were determined using the "modified diffusion chamber method", all at 37 ℃, ph=7.4, in a 0.1mol/L HEPES buffer system with 0.3mmol/L Hb in the system, in the absence (stripped) and in the presence of the allosteric effectors Cl - (0.1 mol/L KCl added) and DPG (double the Hb concentration). The results are shown in figure 3, where the P50 value for the a haplotype is significantly less than that for the B haplotype (P <1.50 x 10 -4), indicating that the a haplotype has higher intrinsic oxygen affinity. Further, it is verified that sheep carrying the A haplotype structural variation better adapt to high altitude environment by enhancing the blood hemoglobin oxygen binding capacity.
It should be noted that, when the claims refer to numerical ranges, it should be understood that two endpoints of each numerical range and any numerical value between the two endpoints are optional, and the present invention describes the preferred embodiments for preventing redundancy.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
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| CN101824414A (en) * | 2010-03-29 | 2010-09-08 | 云南农业大学 | Alpha D globin gene as chicken molecular mark with high oxygen carrying capability as well as detection method and application thereof |
| CN105886642A (en) * | 2016-05-23 | 2016-08-24 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker associated with Chinese sheep cadual type characters |
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| CN107988379B (en) * | 2017-11-08 | 2021-04-30 | 甘肃农业大学 | Genetic marker related to Tibetan sheep plateau hypoxia adaptability and application thereof |
| CN109371146B (en) * | 2018-12-24 | 2021-09-21 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker, primer pair, detection kit and application of SNP molecular marker and primer pair for sheep multi-thoracic vertebra number character |
| CN114438225B (en) * | 2022-02-16 | 2023-01-24 | 西北农林科技大学 | A molecular marker of structural variation associated with adaptation to hypoxia in goats and its application |
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| CN101824414A (en) * | 2010-03-29 | 2010-09-08 | 云南农业大学 | Alpha D globin gene as chicken molecular mark with high oxygen carrying capability as well as detection method and application thereof |
| CN105886642A (en) * | 2016-05-23 | 2016-08-24 | 中国农业科学院北京畜牧兽医研究所 | SNP molecular marker associated with Chinese sheep cadual type characters |
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