CN114410800A - SNP marker locus related to bovine erythrocyte membrane structure and application thereof - Google Patents
SNP marker locus related to bovine erythrocyte membrane structure and application thereof Download PDFInfo
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Abstract
The invention relates to an SNP marker locus related to a bovine erythrocyte membrane structure and application thereof, belonging to the field of genetic engineering. The SNP marker is located on the No. 10 chromosome of cattleSPTBExon 19 of gene G108C (chr 10: 77230398) with nonsynonymous mutation (glutamic acid to aspartic acid, Glu1370Asp), and the whole blood high cut relative index of CC genotype individuals is very significantly lower than that of GG or GC genotype individuals: (P<0.01), indicating that the CC genotype has stronger erythrocyte deformability; the hypoxia tolerance and plateau adaptive capacity of CC genotype individuals are significantly higher than those of GG or GC genotype individuals. The invention provides a method for correlating bovine erythrocyte membrane structure and hypoxia toleranceSPTBSNP sites on the gene can be used as molecular markers for bovine hypoxia tolerance or breeding high altitude hypoxia environment-resistant cattle, and simultaneously, the characteristic genes of the cattle can be preserved and benefitedThe use is also of great significance.
Description
Technical Field
The invention belongs to the field of genetic engineering, and particularly relates to an SNP marker locus related to a bovine erythrocyte membrane structure and application thereof.
Background
Hereditary Spherocytosis (HS) is a group of genetic diseases characterized by the presence of spherical erythrocytes in peripheral blood smears, caused by mutations in genes regulating the proteins of the erythrocyte membrane. HS is characterized by anemia, jaundice, splenomegaly, and gallstones. The SPTB gene for coding the beta-spectrin subunit is mutated into a spherocytosis type 2, and the SPTB gene is a common pathogenic gene of HS except an ANKl gene. It is located on chromosome 14q23-q24.2 and consists of 32 exons. Beta-spectrin is a multifunctional protein consisting of two alpha and beta subunits, which, although somewhat similar, are structurally distinct and encoded by different genes (Speicheretal. 1984). The α and β spectrin chains intertwine with each other in an antiparallel fashion to form heterodimers, which in turn self-associate to form tetramers and oligomers. The molecular weight of the β subunit is 246kD, consisting of 106 amino acids (Ipsaro et al.2010). Research shows that the spectrin can form a net structure in the erythrocyte membrane, and the anchoring combination with the ankyrin plays a key role in maintaining the shape and the mechanical property of the erythrocyte membrane and the like. The mutation of spectrin causes the fragility of erythrocyte membrane to increase, and reduces the flexibility of erythrocyte to cause hereditary hemolytic anemia (Delaunay 2007). SPTB gene mutations include initiation codon disruptions, frameshifts, and nonsense mutations. The types and incidence rates of HS are different in different countries and regions worldwide, the content of the HS caused by the beta-spectrin defect is 15% -30% in North America, and the HS caused by the alpha-spectrin defect is more common in China (Li Zi infant et al 2005). Common types of mutations in the SPTB gene are the barri mutation (perrottaetal.2009) and the Guemene-Penfao mutation. In addition to HS, SPTB gene abnormalities can also lead to a variety of diseases, such as neonatal jaundice, oval polycythemia, etc. (Gallagheretal.1998).
The yaks and tibetan cattle are native livestock species distributed in Qinghai-Tibet plateau and adjacent areas thereof, and are rare genetic resources capable of adapting to high-cold and high-altitude climates of the cattle. Compared with the low-altitude cattle, the yak and the tibetan cattle can be well adapted to severe natural environments such as extreme hypoxia in the Qinghai-Tibet plateau and the like, and form a unique characteristic and mechanism which are stable and adapted to the plateau hypoxia in physiology, biochemistry and morphology, and are considered as typical animals for researching the plateau adaptability of mammals. In recent years, through researches on native residents in plateau and plateau animals, a plurality of hypoxia adaptation related genes are discovered, but at present, no relevant report is found on the effect of the SPTB gene on animal hypoxia adaptation in China.
Disclosure of Invention
Based on the background of the prior art, the invention provides SNP marker sites related to bovine erythrocyte membrane structures, and specific primers and application thereof.
In order to realize the purpose, the invention is realized by the following technical scheme:
the invention provides an SNP marker locus related to a bovine erythrocyte membrane structure, the SNP marker locus is positioned at the 19 th exon G108C locus (chr 10: 77230398) of a bovine, the locus generates non-synonymous mutation Glu1370Asp, and the base variation information is G > C; the whole blood high-cut relative index of the CC genotype individual is extremely lower than that of the GG or GC genotype individual, and the hypoxia tolerance and high adaptive capacity of the CC genotype individual are obviously higher than those of the GG or GC genotype individual.
The SNP marker loci are applied to detecting the plateau adaptive capacity of the cattle or breeding cattle resistant to the plateau hypoxia environment.
The SNP marker loci are applied to the preparation of a kit for detecting the adaptability of the plateau hypoxic environment of the cattle or breeding cattle resistant to the plateau hypoxic environment.
The invention also provides a method for detecting the adaptability of the cattle to the hypoxic environment, which comprises the following steps: detecting the SNP marker locus by using the whole genome DNA of a cattle to be detected as a template; if the locus is CC genotype, the hypoxia tolerance and plateau adaptability of the cattle to be detected are better, and the cattle to be detected is judged to be hypoxia tolerant dominant type; if the locus is GG or GC genotype, the hypoxia tolerance and plateau adaptability of the cattle to be detected are weaker, and the cattle to be detected is judged to be hypoxia tolerant disadvantaged.
The invention also provides a method for breeding cattle resistant to the high altitude hypoxia environment, which selects individuals with CC type gene type of the locus G108C of the SPTB gene as breeding parents by detecting the SNP marker locus.
PCR primers for detecting the SNP marker sites: the upstream primer SPTB-F is TCCGTCCCTGTCTTACC, and the downstream primer SPTB-R is CTGCCATCCATCATCCT.
Further, the primer PCR amplification reaction conditions are as follows: the primer PCR amplification reaction conditions were as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 45s, annealing at 59 ℃ for 45s, extension at 72 ℃ for 45s, 35 cycles; extension was carried out for 8min after 72 ℃.
The invention also provides a kit for assisting in breeding the cattle resistant to the high altitude low oxygen environment, which comprises the PCR primer.
The invention has the beneficial effects that:
the SNP marker provided by the invention is located at the 19 th exon G108C site (chr 10: 77230398) of the SPTB gene of the No. 10 chromosome of cattle, nonsynonymous mutation (Glu1370Asp) occurs at the site, the whole blood high-cut relative index of a CC genotype individual is extremely lower than that of a GG or GC genotype individual, and the hypoxia tolerance and high adaptive capacity of the CC genotype individual are obviously higher than those of the GG or GC genotype individual.
The SPTB gene G108C polymorphic site and the physiological index of the cattle blood are subjected to correlation analysis, and the result shows that the whole blood high-cut relative index of a high-landform CC individual is extremely lower than that of low-landform GG and heterozygote GC (P <0.01), which indicates that the CC genotype has stronger erythrocyte deformability. Through research, three genotypes are detected in middle-altitude yellow cattle, but only CC genotypes are detected in a yak group, the yaks generally live in high-altitude areas, and the anoxic environment can damage the deformability of red blood cells to a certain extent, so that the normal perfusion of the microcirculation of the body is influenced. Erythrocytes with low deformability are not easy to pass through capillaries, so that local blood vessels are blocked, and ischemia and hypoxia of local tissues are caused. In order to adapt to the plateau hypoxia environment, the yak fixes the CC genotype capable of enhancing the deformability of the red blood cells through generation selection, so that the damage of hypoxia to the deformability of the red blood cells is counteracted, and the normal microcirculation of an organism is not influenced.
The SNP locus on the SPTB gene related to the bovine erythrocyte membrane structure and hypoxia tolerance provided by the invention can be used as a molecular marker for bovine hypoxia tolerance and plateau adaptability and breeding plateau hypoxia-resistant environment, and has important significance for preservation and utilization of bovine characteristic genes.
Drawings
FIG. 1 is a Manhattan plot of MEMEA results top 1% broad-bar or higher; the abscissa is the chromosome number and the ordinate is the calculated score of the MEMA; all SNPs shown in the figure are front top 1% broad-over site; from the results, it can be seen that the SPTB gene is within the short range of the top 1%.
FIG. 2 shows 500mvs 4500mFSTA drawing; the abscissa is the chromosome number and the ordinate is the FST value for a population of cattle at an altitude of 500m and 4500 m.
FIG. 3 is a diagram showing the trend of SNP allele frequencies of the SPTB gene.
FIG. 4 shows the result of detecting the polymorphic site of exon G108C of SPTB gene 19.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, preferred embodiments of the present invention will be described in detail below to facilitate understanding of the skilled person.
1. Laboratory animal and blood sample Collection
The invention selects 100 Tibetan cattle and Yunnan local cattle with 5 altitude gradient distributions for whole genome re-sequencing, uses MEMEA and FSTTwo analysis methods are used for screening hypoxia adaptive SNPs and candidate genes of the plateau cattle. The altitude trend SNP of the SPTB gene related to the bovine erythrocyte membrane structure is found in the candidate genes. On the basis of screening hypoxia positive selection related genes and altitude trend sites thereof, 139 yaks and 296 cattle local at medium altitude are selectedThe blood physiological indexes are measured and SNP locus typing is carried out, and the sample information is detailed in Table 1.
TABLE 1 measurement of blood physiological indices and SNP site verification sample information
2. Experimental methods
2.1. Blood sample collection and physiological index determination
A total of 535 samples of 5ml blood were collected from the bovine jugular vein using a vacuum blood collection tube containing EDTA anticoagulant. After collection, the blood is quickly put into an ice box for storage, and the measurement of the physiological indexes of the blood is completed within 12 hours.
Fresh blood was measured using a fully automatic veterinary blood analyzer (BC-2800Vet) and an automatic hemorheology tester (ZL 6000). The measurement of the physiological indexes of blood comprises 11 indexes of blood routine and 13 indexes of blood viscosity, and the detailed information is shown in the following table.
TABLE 2 determination of physiological indices of blood
2.2. Extraction of genomic DNA
Tiangen blood genomic DNA extraction kit was used and the protocol was followed.
2.3. Detection of genomic DNA
DNA detection was performed using 1% agarose gel, which was prepared by adding nucleic acid dye and using a marker DL 2000. Electrophoresis was performed at 120V for about 30min in 1 XTAE buffer. And (5) observing and photographing by using a gel imaging system after electrophoresis is finished, and storing a DNA result picture.
3. Bovine whole genome re-sequencing, sequence alignment and SNP trapping
Breaking a qualified DNA sample into 300-500 bp fragments, then carrying out terminal modification, adding an Illumina sequencing joint, selecting a product of 400-500 bp through 2% gel electrophoresis, then carrying out LM-PCR amplification, and carrying out sequencing library preparation. Library sequencing adopts Illumina HiSeq 2000 to carry out double-end sequencing, and Illumina HiSeq Control Software controls the whole process. And (3) carrying out quality detection on the fragments obtained by sequencing, and filtering out the fragments with the end quality value of less than 20 and the length of less than 35. The remaining mass-detected fragments were aligned to the cattle reference genome (Bos taurus UMD 3.1) using BWA software with the parameters "mem-k 32-w 10-B3-O11-E4-t 20", followed by sorting and filtering of the aligned bam files using samtools software. In order to obtain high quality mutation information, SNP calling was performed using the GATK toolset. The HaplotypeCaller package of GATK is first utilized to obtain g.vcf files containing per-site alignment information for each individual. Then using genotypgvcfs package with parameter set "-stand _ call _ conf 30.0", merge all individuals' g.vcf and obtain the original variation information. Finally, the Variantfiltration is used for filtering low-quality SNP, the conditions are set as that QD is less than 2.0, MQRankSum is less than-12.5, ReadPosRankSum is less than-8.0, DP is less than 4, FS is more than 60.0 ", a python script is used for filtering missing values, and SNP data with the missing rate less than 0.1 are reserved. The cattle genome gff file was then downloaded from Ensemble and SNP annotated with ANNOVAR software.
4. Hypoxia adaptive selection signal analysis
MEAA selection Signal analysis
Based on a population genetics method, genetic loci subjected to environment adaptive selection among different populations are detected by an environment adaptive mixed effect model, the change of the allele frequency of SNPs loci caused by environment factors can be detected, and loci subjected to different environment selection pressures can be detected in a sliding window mode, wherein the specific model is shown in Wufuquan (2018). In this analysis, the correlation between allele frequency and altitude gradient was calculated using the altitude gradient of cattle life as an environmental variable. By using the MEMEA model, the window size of 10kb of physical positions before and after each SNP is taken as the center, a p value related to the environment is given to the interval, and genes near 10kb of upstream and downstream of the SNP are extracted as candidate genes. Sequencing the-log (P) values from large to small, selecting Top 1% as a candidate gene, and analyzing the result as shown in figure 1, wherein the result shows that the SPTB gene is within the threshold of the first 1%.
4.2.FSTSelection signal analysis
FSTThe analysis mainly used software of Vcftools (Danecek et al, 2011), the window was set to 10kb, the step size was set to 5kb, high-low population cattle were compared, the top 1% of the window was set as a potential candidate window, and Ensemble website (http:// asia. Ensemble. org/bionomirt/martview /) was used to extract window-related genes as candidate genes. Extracting the genes with the Top 1% of the whole genome to perform enrichment analysis function annotation, and finally selecting the gene candidate sites with population differentiation, wherein the analysis result is shown in figure 2.
4.3. Candidate gene
Taking intersection of candidate genes respectively obtained by the two analysis methods, searching bovine hypoxia adaptation related genes, and finally screening 10000 altitude related loci, wherein 114 SNP loci exist in SPTB genes detected on chromosome 10, 101 SNPs allele frequencies obviously increase along with the rise of altitude, and chr 10: the elevation trend at position 77230398 was significant and was a non-synonymous mutation (Glu1370Asp) (FIG. 3).
Genotype detection of SNP marker loci
Design and Synthesis of SPTB Gene primers
To amplify bovine chr 10: 77230398 site, according to the cattle genome data published in the UCSC database (version number jun.2014(Bos-taurus _ UMD _3.1.1/bosTau8)), in chr 10: 77230398 site as center, downloading about 1000bp base sequences, introducing the downloaded DNA sequences into Premier5.0 for primer design, the primers were synthesized by Kunming Shuozhi Biotech, Inc., and the primer information is shown in Table 3.
TABLE 3 SPTB Gene primer information
PCR amplification procedure
In order to ensure the PCR amplification efficiency, the PCR reaction system of each pair of primers needs to be pre-tested before amplification, generally, on the premise of ensuring the quality of template DNA, the annealing temperature is mainly adjusted, and the reaction time and the cycle number of each amplified program can be properly adjusted according to the length of an amplified fragment so as to optimize the PCR reaction system. The optimal PCR amplification system and optimal PCR amplification program of the test are shown in Table 4 and Table 5 respectively.
TABLE 4 genomic DNAPCR amplification System
TABLE 5 genomic DNAPCR amplification program
Detection of PCR amplification products
The PCR amplification product was detected using 1% agarose gel, mixed with anthocyanins and buffer, and then applied to the gel well using a pipette gun, using 5. mu. LDNAMaker (DL2000) as a reference. Electrophoresis is carried out in 1 XTAE buffer solution at a voltage of 120V for about 30min, and after the electrophoresis is finished, the gel imaging system is used for observation and photographing for preservation.
5.4 recovery, purification and sequencing of PCR products
And purifying and sequencing the PCR product by Kunming Optimalaceae biotechnology, and introducing a sequencing result into BioEdit for analysis and comparison to search SNP sites.
6. Data analysis
All data are subjected to primary processing by using Excel according to standard deviation rejection of the mean +/-3 times, then SAS software and SPSS software are used for data analysis, data in a table are represented by the mean +/-standard deviation, and whether the indexes have significant difference or not is analyzed.
6.1. Calculation of allele frequency and genotype frequency
Allele frequency (allelic frequency) is the ratio of the number of one allele to the number of all alleles at the same locus in a population, and ranges from 0 to 1 (spanaran 2001), and is calculated as:
Pi＝(2Nii+Nij)/2N
wherein Pi is the frequency of allele i, NiiNumber of individuals of genotype ii, NijThe number of individuals of genotype ij and N is the number of samples.
Genotype frequency (genotypic frequency) means the proportion of a specific genotype of a locus in the diploid organism population in all genotypes, and ranges from 0 to 1 (spanish 2001), and the sum of the frequencies of all genotypes of the same locus is 1. The calculation formula is as follows:
Pij＝Nij/N
wherein, PijFrequency of genotype ij, NijThe number of individuals of genotype ij and N is the number of samples.
6.2. Calculating polymorphic information content
Polymorphic Information Content (PIC) for estimating polymorphism of marker gene; wherein PIC >0.5 indicates high polymorphism of gene, PIC 0.25. ltoreq.0.5 indicates moderate polymorphism of gene, and PIC 0.25. ltoreq.0.25 indicates low polymorphism of gene (Zhang Yuan 2001). The PIC is calculated as follows:
wherein, PiAnd PjRespectively, the frequency of the ith and jth alleles in a population, and n the allele number.
6.3. Calculating the degree of heterozygosity
Heterozygosity (H) of a population represents the proportion of marker genes as heterozygotes in a population, and genetic diversity in a population can be seen by heterozygosity (guanar 2001). The calculation formula is as follows:
wherein n is the allelic factor of a marker locus in a population; piIndicating the frequency of the ith allele in a population.
Hardy-Weinberg equilibrium test
Firstly, supposing that the population studied by us is in Hardy-Weinberg balance, then calculating the theoretical number of individuals of each genotype according to the genotype frequency of the population theoretically, and finally calculating the χ according to the actual number of individuals and the theoretical number of individuals of each genotype2The value:
wherein k denotes the presence of k genotypes in the population, OiActual number of individuals of i-th genotype, EiThe theoretical number of individuals of the ith genotype. Will actually calculate χ2Critical χ with df ═ k-12The values are compared and corresponding statistical inferences are made accordingly.
6.5. Correlation analysis of genotype and blood physiological index
Through preliminary examination, no significant interaction effect is found among genotypes, sexes and ages, so that the significance of the difference of the blood physiological indexes of different genotypes, sexes and ages is analyzed by adopting a three-factor non-interaction least square analysis model (Rushao male and Ling Sheng 2003), and the specific model is as follows:
Yijk＝μ+Gi+Hj+Sk+eijkl
wherein, YijkIs the observed value of the physiological index, mu is the population mean, GiIs the i genotype effect of the SPTB gene, HjFor age effect j, SkFor the k sex effect, eijklFor random errors, a normal distribution is followed.
According to the model, a least square mean value of corresponding physiological indexes of each genotype of the SNP locus is calculated by adopting a GLM process of SAS (Ver.9.4) statistics, and the difference significance test is carried out.
7. Results and analysis
SPTB Gene polymorphic site analysis
Amplifying target fragments of SPTB genes of yaks and medium-altitude cattle by using the amplified samples, introducing sequencing results into BioEdit software, respectively comparing and analyzing the sequencing results with SPTB gene sequences of the cattle downloaded from an NCBI database, and verifying whether single nucleotide polymorphism exists in target sites.
SPTB Gene polymorphic site sequencing results
The sequencing results were aligned with the published bovine SPTB gene sequence (NC _037337), and the mutation site alignment results are shown in fig. 4. The alignment shows that the SPTB gene 19 has a mutation at exon G108C, and that the mutation is a non-synonymous mutation, resulting in a change from glutamate to aspartate (Glu1370 Asp).
Gene frequency and genotype frequency of the SPTB gene 19 exon G108C mutation site
And (3) calculating the genotype frequency and allele frequency of the SPTB 19 exon G108C locus of the yaks and medium-altitude yellow cattle to determine whether the locus is the dominant SNP locus, and the result is shown in Table 6. Three genotypes, namely homozygous low-land type GG, heterozygous type GC and homozygous high-land type CC, are detected in the middle-altitude cattle, and the genotype frequencies are respectively 0.52, 0.38 and 0.10 through experimental result analysis, so that the allele G (0.71) is more dominant than C (0.29) in the middle-altitude cattle group, and the GG genotype is a dominant genotype as can be seen from Table 6. In a yak population, only the CC genotype is detected, and the frequency of the allele C is 1, which indicates that the locus is fixed in yaks due to selection.
The mutation sites of two groups of medium altitude cattle belong to medium polymorphism (0.25< PIC <0.5), the genetic diversity is rich, and the heterozygosity (H) is 0.41. Hardy-Weinberg equilibrium test was performed on both populations, and from the results of the chi-square test, Lijiang local cattle were in equilibrium (P > 0.05).
TABLE 6 SPTB Gene 19 exon G108C mutation site genotype frequencies and Gene frequencies
Note: ns represents P > 0.05.
Correlation between different genotypes of SPTB gene 19 at exon G108C mutation site and blood physiological indexes
The analysis of the significant difference between the SPTB gene 19 exon G108C mutation site and the blood physiological indices is shown in table 7. Only significant difference of whole blood high-cut relative indexes is detected in a middle-altitude cattle population, and the difference of whole blood high-cut relative indexes of a GG genotype and a GC genotype is not significant (P >0.05), but both are significantly higher than a CC genotype (P < 0.01); the differences among the three genotypes of the other indexes are not obvious.
TABLE 7 SPTB gene exon 19 genotype and blood physiological index of medium altitude cattle
Note: the data in the table are very significant in comparison to the row with capital letters (P <0.01) and insignificant in comparison to the row with non-capital letters (P > 0.05).
Adaptation of the SPTB Gene to hypoxia
Mature mammalian red blood cells are enucleated and, in a quiescent state, have the shape of a biconcave disk. The shape of the red blood cells has a large relative surface area, and the characteristic enables the red blood cells to realize various deformations under the condition of constant volume without additionally increasing the surface area. The main function of erythrocytes is to transport O2And CO2The microcirculation between the micro-artery and the micro-vein can realize the function of exchanging substances between blood and tissue fluid, so that the physiological function of each organ in the organism can be normally operated. Hemorheological abnormalities are one of the important factors affecting the normal perfusion of the microcirculation, in which the altered deformability of erythrocytes has an important influence on the microcirculation (Linlin et al, 2008).
Hypoxia promotes the generation of red blood cells in the body, and also damages the deformability of red blood cells, resulting in increased blood viscosity, microcirculation stasis, and impairment of the functions of various organs of the body. Because the number of red blood cells in blood is the largest, the degree of aggregation and deformability of red blood cells are important factors affecting blood viscosity, and the high cut relative index of whole blood reflects the deformability of red blood cells, and the higher the high cut relative index of whole blood, the lower the deformability of red blood cells. The invention performs correlation analysis on the SPTB gene G108C polymorphic site and the physiological index of cattle blood, and the result shows that the whole blood high-cut relative index of a high-land CC individual is extremely lower than that of low-land GG and heterozygote GC (P <0.01), which indicates that the CC genotype has stronger erythrocyte deformability. The rats such as Liangtaiyi and the like are used as research objects to research the influence of hypoxia on the deformability of the red blood cells, and the fact that the deformability of the red blood cells of the rats in the hypoxia group is reduced is found, which indicates that the hypoxia can damage the deformability of the red blood cells. Three genotypes are detected in the middle-altitude yellow cattle in the experiment, but only the CC genotype is detected in a yak group, the yaks generally live in high-altitude areas, and the anoxic environment can cause certain damage to the deformability of the red blood cells, so that the normal perfusion of the microcirculation of the body is influenced. Erythrocytes with low deformability are not easy to pass through capillaries, so that local blood vessels are blocked, and ischemia and hypoxia of local tissues are caused. In order to adapt to the plateau hypoxia environment, the yak fixes the CC genotype capable of enhancing the deformability of the red blood cells through generation selection, so that the damage of hypoxia to the deformability of the red blood cells is counteracted, and the normal microcirculation of an organism is not influenced.
The SPTB gene is a hypoxia positive selection gene discovered by scanning a cattle whole genome, mainly encodes beta-spectrin, and the beta-spectrin is an important erythrocyte membrane skeleton protein and plays an important role in maintaining the stability, structure and shape of a cell membrane. Spectrin interacts with actin, ankyrin, adducin, etc. to create a complex intracellular two-dimensional network that maintains the elasticity of the cell membrane surface and the shape of the erythrocyte membrane, and the intrinsic elasticity of spectrin is considered to be a major factor in maintaining the cellular elasticity of erythrocytes in the circulatory system. The loss of these cytoskeletal components leads to fragile and fragile cells and clinically manifest hemolytic anemia such as hereditary elliptocytosis and hereditary thermophilic polycythemia. The common characteristics of the genetic anemia diseases are represented by a defect of a spectrin-actin-4.1R complex, loss of relative surface area of an erythrocyte membrane, spherical shape and reduced deformability of the erythrocyte, and the damage of the abnormal erythrocyte by a spleen is a main reason for hemolysis of a patient. Mutations in both SPTA1 and SPTB and EL1 gene encoding 4.1R cause hereditary anemic disease, but most commonly result from mutations in the SPTB gene, which are often manifested as autosomal dominant mutations.
The invention detects the polymorphic site of the SPTB gene, and after sequence comparison, the mutation of the 19 th exon G108C site of the SPTB gene is found, so that glutamic acid is changed into aspartic acid (Glu1370 Asp). Glutamic acid and aspartic acid (Glu/Asp) are major acidic amino acids in the body, which account for 12.1% of the total amino acids in the protein system, and play an important role in maintaining the structure and function of proteins. There are studies showing that glutamate at position 6 of the beta chain of hemoglobin is an amino acid residue necessary to maintain the normal structure of the protein. The well-known cause of sickle cell anemia is abnormal sickle-like hemoglobin (Hb S) produced by a single amino acid mutation, differing from normal adult hemoglobin (HbA) only in that the glutamic acid is replaced by valine at position 6 of the beta chain (E6V). The acidic amino acid residues play a key role in maintaining the structure and function of the protein in organisms, but the molecular mechanism that the glutamic acid → aspartic acid substitution affects the structural function of spectrin needs to be studied intensively.
The SNP marker provided by the invention is located at the non-synonymous mutation (glutamic acid is changed into aspartic acid, Glu1370Asp) of the exon G108C of the SPTB gene 19 of the No. 10 cow chromosome, the whole blood high-cut relative index of a CC genotype individual is extremely lower than that of a GG or GC genotype individual, and the hypoxia tolerance and high adaptive capacity of the CC genotype individual are obviously higher than that of the GG or GC genotype individual. The SNP locus on the SPTB gene related to the bovine erythrocyte membrane structure and hypoxia tolerance provided by the invention can be used as a molecular marker for bovine hypoxia tolerance and plateau adaptability and breeding plateau hypoxia-resistant environment, and has important significance for preservation and utilization of bovine characteristic genes.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (8)
1. An SNP marker locus associated with a bovine red blood cell membrane structure, characterized by: the SNP marker locus is positioned on the No. 10 chromosome of the cattleSPTBThe 19 th exon G108C site (chr 10: 77230398) of the gene generates nonsynonymous mutation Glu1370Asp, and the base variation information is G>C; the whole blood high-cut relative index of the CC genotype individual is extremely lower than that of the GG or GC genotype individual, and the hypoxia tolerance and high adaptive capacity of the CC genotype individual are obviously higher than those of the GG or GC genotype individual.
2. The application of the SNP marker loci of claim 1 in detecting plateau adaptive capacity of cattle or breeding cattle resistant to plateau hypoxia environment.
3. The application of the SNP marker loci of claim 1 in the preparation of kits for detecting the adaptability of high altitude hypoxia environment of cattle or breeding cattle resistant to high altitude hypoxia environment.
4. A method for detecting the adaptability of a cattle to a low-oxygen environment is characterized by comprising the following steps: detecting the SNP marker locus according to claim 1 by using the whole genome DNA of a cattle to be detected as a template; if the locus is CC genotype, the hypoxia tolerance and plateau adaptability of the cattle to be detected are better, and the cattle to be detected is judged to be hypoxia tolerant dominant type; if the locus is GG or GC genotype, the hypoxia tolerance and plateau adaptability of the cattle to be detected are weaker, and the cattle to be detected is judged to be hypoxia tolerant disadvantaged.
5. A method for breeding cattle resistant to high altitude and low oxygen environment is characterized in that: by detecting the SNP marker loci according to claim 1SelectingSPTBIndividuals with CC-type genotype at the gene G108C locus are used as breeding parents.
6. PCR primers for detecting SNP marker sites according to claim 1, characterized in that: the upstream primer SPTB-F is TCCGTCCCTGTCTTACC, and the downstream primer SPTB-R is CTGCCATCCATCATCCT.
7. The PCR primer of claim 6, wherein: the primer PCR amplification reaction conditions were as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 45s, annealing at 59 ℃ for 45s, extension at 72 ℃ for 45s, 35 cycles; extension was carried out for 8min after 72 ℃.
8. The utility model provides a supplementary kit of breeding resistant high altitude low oxygen environment ox which characterized in that: comprising the PCR primer of claim 6 or 7.
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| CN109994153A (en) * | 2019-04-09 | 2019-07-09 | 山东省农业科学院奶牛研究中心 | A kind of method and its application for screening ox high altitude hypoxia adaptation molecular labeling |
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