Application of TMPRSS2 gene in goat breeding work

Abstract

The invention discloses application of a TMPRSS2 gene in goat breeding work, which is characterized in that a Guizhou black goat TMPRSS2 gene reference region Chr1: 141288001-141291000 is taken as a locus, copy number variation of the locus in a Guizhou black goat group is detected through a real-time fluorescent quantitative PCR (polymerase chain reaction) technology, CNV (CNV type of the TMPRSS2 gene is detected, and type frequency statistics is performed; carrying out correlation analysis on the locus and the growth traits of the goats; results show that the detection site has the highest frequency of the normal CNV in the Guizhou black goats, the normal CNV of the site has a remarkable positive effect on the tube circumference of the goats, and the TMPRSS2 gene can be used as a candidate gene for improving the growth traits of the Guizhou black goats and can be used for detecting the growth traits of the Guizhou black goats. The copy number variation site can be used as a candidate molecular genetic marker site for improving the growth traits of the Guizhou black goats, and the breeding process can be accelerated.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular genetics research, and in particular relates to application of a TMPRSS2 gene in goat breeding. Background Art

[0002] Goat growth rate and meat quality are two major factors restricting the development of the goat industry. Research on the molecular mechanisms regulating goat growth and muscle development can provide a theoretical basis for the development of China's goat industry. With the rapid development of biotechnology, the study of copy number variations (CNVs) has entered a period of rapid development.

[0003] Copy number variation (CNV) is a source of genetic diversity. It refers to insertions or deletions of genomic sequences greater than 50 base pairs between two individuals of a species and is a type of genomic structural variation. CNVs were initially discovered in the genomes of patients, but subsequent studies have shown that they are also common across species, indicating that CNVs represent a group of genomic structural alterations with benign, pathogenic, or unknown clinical significance. CNVs may affect gene expression and remodel genomic structure, leading to significant phenotypic differences. Research has shown that five types of CNV exist in the genome: simultaneous deletion of both homologous chromosomes; deletion of one homologous chromosome while the other is normal; duplication of one homologous chromosome while the other is normal; deletion of one homologous chromosome while the other is normal; and simultaneous duplication of both homologous chromosomes. CNVs can influence gene function and individual phenotypes through dosage effects, position effects, disruption of functional genes, fusion genes, exposure of recessive alleles, and potential transition effects. Most loss of heterozygosity (LOH) CNVs are typically recessive, and in many cases, upregulation and downregulation of dosage-sensitive CNVs coincide with copy number gains and losses.

[0004] Numerous studies have shown that growth factors play a vital role in regulating the growth and development of the body, and transmembrane serine protease 2 (TMPRSS2) plays such a role. This family of proteins is involved in a variety of physiological processes, such as embryonic development, cell growth, morphogenesis, and tissue repair. As an important member of the TMPRSS family, TMPRSS2 is located on chromosome 1. Currently, research on the function of the TMPRSS2 gene is mainly focused on human diseases. TMPRSS2 is closely related to the occurrence of prostate cancer, and its expression in prostate cancer is androgen-dependent.

[0005] Goat growth and muscle development are regulated by multiple factors, and the TMPRSS2 gene may play a key role in this. However, a lack of research has yet to uncover its specific mechanisms. This has hindered the application of the TMPRSS2 gene in goat molecular breeding, making it difficult to directly apply it to improve goat growth traits. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention uses the Chr1:141288001-141291000 reference region of the TMPRSS2 gene of Guizhou black goats as the site, detects the copy number variation of this site in the Guizhou black goat population by real-time fluorescence quantitative PCR technology, detects the CNV type of the TMPRSS2 gene and performs type frequency statistics, and conducts association analysis between this site and the growth traits of goats. The results show that the normal type CNV frequency of this detection site is the highest in Guizhou black goats, and the normal type of this site has a significant positive effect on the goat's gait.

[0007] To achieve the above objectives, the present invention discloses the application of TMPRSS2 gene in goat breeding.

[0008] Furthermore, the present invention specifically provides an application of the TMPRSS2 gene in improving the growth traits of goats.

[0009] Furthermore, the invention specifically provides an application of the TMPRSS2 gene copy number variation site in goat breeding.

[0010] Furthermore, the TMPRSS2 gene copy number variation site is used as a molecular genetic marker site for goat growth traits.

[0011] Furthermore, the goat growth trait is goat tube circumference.

[0012] Furthermore, the normal copy number variation site of the TMPRSS2 gene is used as a molecular genetic marker site for early selection of goat gait traits.

[0013] Furthermore, the goats are Guizhou black goats.

[0014] Furthermore, the copy number variation region of the TMPRSS2 gene sequence is Chr1:141288001-141291000.

[0015] Furthermore, real-time fluorescence quantitative PCR analysis was used to amplify the copy number variation region of the TMPRSS2 gene and a partial fragment of the MC1R gene as an internal reference, and then the copy number variation type of the TMPRSS2 gene was identified based on the quantitative results.

[0016] Furthermore, the primer sequences for amplifying the copy number variation region of the TMPRSS2 gene are as follows:

[0017] Upstream primer Fl: 5′-AGCTACTGACAGAGGTCGGA-3′

[0018] Downstream primer R1: 5′-GGTCCCACAACCCTGAATGA-3′

[0019] The primer sequences for amplifying a specific fragment of the MC1R gene are as follows:

[0020] Upstream primer F2: 5′-GGGCAGTCCCTTGACAAAGA-3′

[0021] Downstream primer R2: 5′-ATCTCCCCAGCCTCCTCATT-3′.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects:

[0023] The present invention focuses on the detection of copy number variation (CNV) of the TMPRSS2 gene in Guizhou black goats. One of its biggest highlights is that it has successfully broken through the bottleneck of age restrictions in traditional breeding methods, opening up a new path for the early selection and breeding of Guizhou black goats. In traditional breeding work, the evaluation of goat growth traits often depends on the actual phenotypic characteristics exhibited by individuals after they have developed to a certain stage, such as tube girth size. However, this phenotype-based breeding method has obvious defects. On the one hand, it is necessary to wait for the goats to grow to a certain age to obtain accurate phenotypic data, which greatly prolongs the breeding cycle and increases breeding costs; on the other hand, the phenotype is greatly affected by environmental factors, which may lead to inaccurate breeding results.

[0024] The present invention uses the Guizhou black goat TMPRSS2 gene reference region Chr1:141288001-141291000 as a site, and detects the copy number variation of this site in the group by real-time fluorescence quantitative PCR technology. This technology is not limited by age, and no matter what growth stage the goat is in, it can be genetically tested by collecting a small amount of tissue samples (such as blood, hair, etc.). By detecting the copy number variation type of this site, the genetic potential of the goat can be understood in advance, and the development trend of its growth traits can be accurately predicted. For example, the results of the study show that the normal type CNV frequency of this detection site is the highest, and the normal type has a significant positive effect on the canine circumference of the goat. This means that in the early breeding process, it is only necessary to determine the copy number variation type of the goat at this site by genetic testing, and individuals with normal type CNV can be screened out. These individuals are more likely to show a larger canine circumference in the subsequent growth process, thereby having better growth performance. This early precision breeding method can greatly shorten the breeding cycle, improve breeding efficiency, and make the breeding work of Guizhou black goats more scientific and efficient.

[0025] Real-time fluorescence quantitative PCR technology can accurately identify the copy number type of an individual. By designing specific primers for the TMPRSS2 gene copy number variation region and an internal reference gene (such as the MC1R gene), the sample is amplified and quantitatively analyzed, and the copy number of the target gene region can be accurately determined based on the difference in fluorescence signals. In the present invention, the primer sequences for amplifying the copy number variation region of the TMPRSS2 gene (upstream primer F1: 5′-AGCTACTGACAGAGGTCGGA-3′, downstream primer R1: 5′-GGTCCCACAACCCTGAATGA-3′) and the primer sequences for amplifying a specific fragment in the MC1R gene (upstream primer F2: 5′-GGGCAGTCCCTTGACAAAGA-3′, downstream primer R2: 5′-ATCTCCCCAGCCTCCTC ATT-3′) have been carefully designed and optimized to ensure the specificity and accuracy of amplification, thereby achieving accurate identification of the gene copy number variation type.

[0026] The present invention detects the copy number variation of the TMPRSS2 gene in Guizhou black goats, providing an important scientific basis for marker-assisted selection (MAS) of Guizhou black goats, and effectively promoting the development of Guizhou black goat breeding. Marker-assisted selection is a breeding technology based on molecular markers, which achieves indirect selection of target traits by detecting molecular markers closely linked to target traits. In the present invention, the TMPRSS2 gene copy number variation site is used as a molecular genetic marker site for goat growth traits, especially the normal copy number variation site is used as a molecular genetic marker site for early selection of goat girth traits, providing a key molecular marker for marker-assisted selection of Guizhou black goats.

[0027] By detecting TMPRSS2 gene copy number variation in a Guizhou Black goat population, calculating the frequencies of different CNV types, and conducting association analysis between this locus and goat growth traits, they confirmed a significant positive effect of the normal CNV on goat gait. This means that this molecular marker can be used for early selection of Guizhou Black goats during breeding, screening for individuals with the normal CNV and thus improving the growth performance of the herd. Compared with traditional breeding methods, marker-assisted selection can more accurately select individuals with superior genetic traits, avoiding the selection bias caused by environmental interference and inaccurate phenotypic identification in traditional breeding methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the amplification curve drawn by qPCR (TMPRSS2 gene) in the examples of the present invention.

[0029] Figure 2 This is a melting curve drawn by qPCR (TMPRSS2 gene) in the examples of the present invention. DETAILED DESCRIPTION

[0030] The invention will be described in detail below with reference to the accompanying drawings and embodiments, which are intended to explain the invention rather than to limit it. All other embodiments obtained by those skilled in the art without creative work are intended to fall within the scope of protection of the present invention.

[0031] The present invention uses real-time fluorescence quantitative PCR (qPCR) technology to detect the copy number variation of the Guizhou black goat TMPRSS2 gene based on the copy number variation interval (Chr1: 141288001-141291000) obtained by resequencing, comprising the following steps:

[0032] (1) Specific primers were designed using the region where the copy number variation of TMPRSS2 gene occurred as a template, and then qPCR amplification was performed using the genomic DNA of Guizhou black goats as a template, and the MC1R gene was used as an internal reference gene. -ΔΔCt Method to determine the copy number type of an individual.

[0033] (2) The present invention uses qPCR technology to detect the copy number variation of the TMPRSS2 gene in Guizhou black goats, and associates different copy number variation types with growth traits to find copy number types with advantageous growth traits.

[0034] The present invention specifically comprises the following steps:

[0035] 1. Sample Collection and Genomic DNA Extraction

[0036] 1.1 Blood sample collection and data collection

[0037] The Guizhou black goats collected in this study were from the Bijie Goat Breeding Farm in Guizhou Province. A total of 310 individuals, aged 24 to 48 months, were collected through the jugular vein. Growth trait data, such as height, length, chest circumference, rump length, ischial width, and crus height, were recorded for subsequent correlation analysis.

[0038] 1.2 Extraction of DNA from blood samples

[0039] 1.2.1. Thaw frozen blood samples (mainly blood cells) at room temperature. Pipette 500 ml of blood into a 1.5 ml centrifuge tube. Add an equal volume of phosphate buffered saline (PBS) and mix thoroughly. Shake gently and centrifuge at 4°C, 12,000 rpm for 5 min. Discard the supernatant and repeat the above steps until the supernatant is transparent and the precipitate is transparent.

[0040] 1.2.2. Add 500 μL of DNA extraction buffer to the centrifuge tube and gently pipette to separate the blood cell pellet from the tube wall. Incubate in a 37°C water bath for 1 hour.

[0041] 1.2.3. Add 5 μL of proteinase K (20 mg / mL) and mix well. Digest in a 55°C water bath overnight (about 16 hours) until the flocculent precipitate disappears and the solution becomes clear. If it is still not clear, add 10 μL of proteinase K, mix well, and continue digesting until it becomes clear.

[0042] 1.2.4. Cool the reaction solution to room temperature, add 500 μL Tris-saturated phenol, shake gently for 15 minutes to mix thoroughly, centrifuge at 12,000 rpm at 4°C for 10 minutes, transfer the upper aqueous phase to another sterile centrifuge tube, and repeat the above steps once.

[0043] 1.2.5. Add 500 ml of chloroform and shake gently for 20 minutes to mix thoroughly. Centrifuge at 12,000 rpm at 4°C for 15 minutes. Transfer the upper aqueous phase into another sterilized 1.5 ml centrifuge tube.

[0044] 1.2.6. Add 500 mL of a mixture of chloroform and isoamyl alcohol (24:1), mix thoroughly for 20 min, and centrifuge at 12,000 rpm for 10 min at 4°C. Transfer the supernatant to another 1.5 mL centrifuge tube.

[0045] 1.2.7. Add 0.1 volumes of NaAc buffer and 2 volumes of ice-cold anhydrous ethanol, mix and rotate the centrifuge tube until a white flocculent precipitate is separated.

[0046] 1.2.8. Centrifuge at 12,000 rpm for 10 min at 4°C, discard the supernatant, and rinse the DNA pellet twice with 70% ice-cold ethanol.

[0047] 1.2.9. Centrifuge at 12000 rpm for 10 min at 4°C, discard the supernatant, and allow the ethanol to evaporate completely at room temperature.

[0048] 1.2.10. Dissolve the dried DNA solution in 80-100 μL of TE. Store at 4°C until the DNA is completely dissolved. Check the quality of the DNA using a UV spectrophotometer and store at -80°C.

[0049] 2. Design of specific primers for amplification of target genes and internal reference genes

[0050] Using the NCBI-published goat TMPRSS2 gene (NC_030808.1) as the reference sequence, we located the copy number variation region identified during resequencing, from positions 141288001 to 141291000 of the TMPRSS2 gene (target gene) genomic sequence. Primers encompassing this region were designed using Prime 5.0 software and compared using NCBI-BLAST. The primer sequences are as follows (primer pair P1):

[0051] Sequence 1-upstream primer Fl: 5′-AGCTACTGACAGAGGTCGGA-3′

[0052] Sequence 2-downstream primer R1: 5′-GGTCCCACAACCCTGAATGA-3′

[0053] At the same time, using the goat MC1R gene sequence published by NCBI as a reference sequence, the same method was used to design primers for amplifying a specific fragment of the internal reference gene (MC1R gene sequence). The primer sequences are as follows (primer pair P2):

[0054] Sequence 3 - Upstream primer F2: 5′-GGGCAGTCCCTTGACAAAGA-3′

[0055] Sequence 4 - downstream primer R2: 5′-ATCTCCCCAGCCTCCTCATT-3′.

[0056] 3. Real-time Quantitative PCR

[0057] The qPCR reaction system is shown in Table 1 below.

[0058] Table 1 qPCR reaction system

[0059]

[0060] The qPCR reaction procedure is:

[0061] (1) Pre-denaturation at 95°C for 3 min;

[0062] (2) Amplification reaction: denaturation at 95°C for 15 s, annealing at 60°C for 30 s, for a total of 40 cycles.

[0063] The primers were determined to be suitable for real-time fluorescence quantitative PCR analysis by drawing amplification curves and melting curves. A smooth amplification curve indicated that the qPCR reagents were of good quality and the amplification system and conditions were appropriate ( Figure 1 ); The melting curves drawn are consistent with each other, and the curves are smooth, with high and sharp peaks, and no miscellaneous peaks caused by primer dimers or non-specific amplification, indicating that the primers are of good quality ( Figure 2 ).

[0064] 4. Determination of individual copy number types

[0065] The experimental results are based on 2*2 -ΔΔCt The specific calculation method is: ΔΔCt = ΔCt (experimental group) - ΔCt (reference group). Where ΔCt (experimental group) = Ct (target gene of experimental group) - Ct (reference gene of experimental group), ΔCt (reference group) = Ct (target gene of reference group) - Ct (reference gene of reference group). In the formula, the experimental group is the individual sample to be tested for copy number variation. The reference group is the individual sample known to have no copy number variation. The reference group Guizhou black goat individuals selected in the resequencing experiment can be used. 2* 2-ΔΔCt It represents the multiple of the target sequence copy number in the experimental group relative to the control group.

[0066] The -ΔΔCt of each individual to be tested was calculated according to the formula, and the copy number type of the tested Guizhou black goat individual was determined according to the CNV type judgment standard: -ΔΔCt>0.5 is Gain type (insertion type); -0.5≤-ΔΔCt≤0.5 is Median type (normal type); -ΔΔCt<0.5 is Loss type (deletion type).

[0067] 5. Association analysis between CNV loci of TMPRSS2 gene and growth traits in Guizhou black goats

[0068] Count the number of individuals with each type (Gain, Median, and Loss) in the test population, and calculate the frequency of each type. The calculation formula is: Pc = Nc / N. Where Pc represents the frequency of a certain copy number type; Nc represents the number of individuals with CNV type C in the population; and N represents the total number of individuals in the test population.

[0069] Correlation analysis was performed using SPSS (18.0). In data processing, a fixed model was used for analysis based on the different factors affecting body size traits, taking into account environmental effects, age, sex, genetic effects, and their interaction effects, while also being simplified based on actual conditions. The complete model is as follows:

[0070] Yijk=μ+Mi+Nj+CNVj+eijk

[0071] Where: Yijk represents the observed trait value, μ represents the population mean, Ai represents the age of the i-th individual, CNVj represents the fixed effect of the j-th copy number variation type, and eijk represents the random error. Differences between groups were tested using LSD multiple comparisons, and the results are presented as mean ± SE. The results of the data processing are shown in Table 2.

[0072] Table 2 Association analysis between TMPRSS2 gene copy number variation and growth traits in Guizhou black goats

[0073]

[0074]

[0075] Note: Mean values ​​with different letters in the shoulder indicate significant differences (P<0.05). *P<0.05. The values ​​in brackets indicate the frequencies of copy number types.

[0076] Association analysis results showed that the copy number variation site of the TMPRSS2 gene in Guizhou Black goats is significantly associated with the growth trait of canine girth. The normal-type individuals exhibited significantly better growth traits than the insertion-type individuals. Therefore, the normal-type copy number variation of the TMPRSS2 gene could serve as a molecular marker for early selection of canine girth in Guizhou Black goats, potentially enabling rapid breeding of these species.

[0077] 6. Application of CNV markers in Guizhou Black Goat Breeding

[0078] The above results indicate that the TMPRSS2 gene can be used as a candidate gene for improving the growth traits of Guizhou black goats, and its copy number variation site can be used as a candidate molecular genetic marker site for improving the growth traits of Guizhou black goats, which will help accelerate the breeding process.

Claims

1. Application of TMPRSS2 gene in goat breeding.

2. The use according to claim 1, characterized in that Specifically, the TMPRSS2 gene is used to improve the growth traits of goats.

3. The use according to claim 1, characterized in that Specifically, the application of TMPRSS2 gene copy number variation sites in goat breeding.

4. The use according to claim 3, characterized in that The TMPRSS2 gene copy number variation site is used as a molecular genetic marker site for goat growth traits.

5. The use according to claim 4, characterized in that The goat growth trait is goat tube girth.

6. The use according to claim 4, characterized in that The normal copy number variation site of the TMPRSS2 gene is used as a molecular genetic marker site for early selection of goat gait traits.

7. The use according to any one of claims 1 to 6, characterized in that The goats are Guizhou black goats.

8. The use according to any one of claims 3 to 6, characterized in that The TMPRSS2 gene sequence copy number variation region is Chr1:141288001-141291000.

9. The use according to claim 6, characterized in that Real-time fluorescence quantitative PCR was used to amplify the copy number variation region of the TMPRSS2 gene and a partial fragment of the MC1R gene as an internal reference, and then the copy number variation type of the TMPRSS2 gene was identified based on the quantitative results.

10. The use according to claim 9, characterized in that The primer sequences for amplifying the copy number variation region of the TMPRSS2 gene are as follows: Upstream primer Fl: 5′-AGCTACTGACAGAGGTCGGA-3′ Downstream primer R1: 5′-GGTCCCACAACCCTGAATGA-3′ The primer sequences for amplifying a specific fragment of the MC1R gene are as follows: Upstream primer F2: 5′-GGGCAGTCCCTTGACAAAGA-3′ Downstream primer R2: 5′-ATCTCCCCAGCCTCCTCATT-3′.

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