Single nucleotide polymorphism (SNP) primer of DSTN gene related to musk ketone content in natural musk and application of single nucleotide polymorphism (SNP) primer

Abstract

The invention relates to a single nucleotide polymorphism (SNP) primer of a DSTN gene related to musk ketone content in natural musk and application of the single nucleotide polymorphism (SNP) primer, and belongs to the technical field of biological detection. The invention provides a single nucleotide polymorphism (SNP) primer of a DSTN gene related to the musk ketone content in natural musk, a molecular marker corresponding to the SNP primer is located at a 682371th basic group of a chromosome 23 of a forest musk deer reference genome, the basic group is mutated into A or C, the genotypes are AA and AC, and deoxyribonucleotide sequences of the SNP primer are shown as SEQ ID NO: 1 and SEQ ID NO: 2. According to a breeding target, by eliminating A / A genotype individuals and retaining A / C genotype individuals, the method has the beneficial effects that the relative content character of musk ketone in musk of the forest musk deer is efficiently and quickly identified, and a scientific basis is provided for early breeding of high-quality forest musk deer. The detection method disclosed by the invention is simple and easy to operate, can be carried out in a laboratory, and can also be applied to a genome breeding technology.

Description

Technical Field

[0001] The present invention relates to a single nucleotide polymorphism (SNP) primer of a DSTN gene related to the content of muscone in natural musk and its application, belonging to the technical field of biological detection. Background Art

[0002] The precious traditional Chinese medicine musk has extremely high economic value and medicinal value. Since ancient times, it has been a precious traditional Chinese medicine and spice, with a large market demand. Moschus berezovskii is the source animal for producing natural musk. The number of wild Moschus berezovskii is scarce and it has become a first-class protected rare animal in the country. Artificial breeding has been carried out in the whole country for more than 60 years, but the overall musk production is low and the quality of musk is uneven. There has always been a serious bottleneck between its production and supply, and the market price of musk remains high. Related research on artificial synthesis of musk has also been carried out, but the role it plays is still not as good as that of natural musk. The components of musk are very complex, mainly including some macrocyclic ketone compounds, steroid compounds, fatty acids, amino acids and inorganic salts, etc. It is widely used in multiple fields and can play a role in cardiovascular diseases, central nervous system diseases, anti-inflammatory and immune, anti-tumor and traditional Chinese medicine in the medical field. Among them, muscone in the components of musk has been proven to be used for dilating coronary arteries and improving microcirculation for angina pectoris, and the drug Shexiang Baoxin Pills have been developed; in addition, muscone also has a neuroprotective effect and is applied to stroke, cerebral ischemia and neurodegenerative diseases. Musk has a high value, and the state also has corresponding standards for the quality of musk. For this volatile organic component of muscone, gas chromatography is mainly used for its quantitative determination. It is described in the Pharmacopoeia that the content of muscone in high-quality natural musk is not less than 2.0% of the dried product. Therefore, carrying out research on improving musk in Moschus berezovskii has become an urgent need for the development of the traditional Chinese medicine industry. It is necessary to find a suitable molecular marker to quickly and accurately identify individuals of Moschus berezovskii with high-yielding muscone, so as to achieve early breeding and improve the quality of natural musk. Summary of the Invention

[0003] The object of the present invention is to propose a single nucleotide polymorphism (SNP) primer of a DSTN gene related to the content of muscone in natural musk and its application, so as to quickly and accurately identify the traits related to the content of muscone in the musk of Moschus berezovskii, aiming at the defects existing in the prior art.

[0004] The DSTN gene, also known as destrin, is a protein belonging to the ADF (actin-depolymerizing factor) family. Its main function is to depolymerize filamentous actin (F-actin) into monomeric form and promote the cleavage of filamentous actin. It is expressed in a variety of cells, including those in the digestive system, urinary system, heart, nervous system, and sensory organs. During musk secretion in forest musk deer, the number of glandular cells in the musk gland increases to support more efficient musk secretion, indirectly promoting the synthesis of more muscone. The DSTN gene has also been shown to play important roles in various biological processes, including cell differentiation, development, and cell migration. Studies on the characteristics of the natural musk components of forest musk deer have shown a significant association between the DSTN gene and the relative content of muscone.

[0005] To eliminate experimental errors caused by environmental factors, all experimental individuals selected in this invention are forest musk deer resources of the same breeding scale. The content of muscone in musk components was measured by gas chromatography-mass spectrometry (GC / MS), and SNP genotyping analysis of forest musk deer was carried out using whole-genome resequencing technology. Through genome-wide association analysis, a molecular marker of the DSTN gene significantly related to the relative content of muscone was screened, providing new gene and molecular marker resources for the breeding of high-yield muscone traits in the musk of forest musk deer. This SNP locus can be used as an efficient primary screening tool to provide a basis for subsequent multi-gene pyramiding breeding or functional research.

[0006] This invention solves the technical problem through the following technical solutions: First, a single nucleotide polymorphism (SNP) primer of the DSTN gene related to the content of muscone in natural musk is provided. The molecular marker corresponding to the SNP primer is located at the 682371st base on chromosome 23 of the forest musk deer reference genome, with a base mutation to A or C, and the genotypes are AA and AC. The deoxyribonucleotide sequences of the SNP primer are shown as SEQ ID NO: 1 and SEQ ID NO: 2. The forest musk deer reference genome is the only high-quality forest musk deer reference genome at the chromosome level successfully assembled in 2022, which is published on the website http: / / muskdb.cn / .

[0007] This invention further provides the application of the SNP primer of the DSTN gene related to the muscone content trait in the musk of forest musk deer, including using it to detect the genotype of the SNP molecular marker for the content of muscone in the musk of forest musk deer.

[0008] The specific detection method includes the following steps: First step: Detect the forest musk deer DNA sample, perform PCR amplification using the SNP primer to obtain an amplification product; Second step: Perform Sanger sequencing on the amplified product; Step 3. Determine the molecular marker genotype of the target site based on the sequencing results obtained in Step 2.

[0009] The SNP primers in the first step of the above method are divided into Forward primer: 5’- ACCCGTTGCTGAATGATGCT-3’ (SEQ ID NO: 1) Reverse primer: 5’- AAACAGCAAGGCGAGGTCTT-3’ (SEQ ID NO: 2). The length of the amplification product is 1027 bp, which contains the base at position 682371 on chromosome 23 of Moschus berezovskii.

[0010] In the first step, the final volume of the reaction system is 50 μl and is DNA of Moschus berezovskii to be detected 4 ng 2 x Accurate Taq Master Mix 25 μl 10 μM / L forward primer 2 μl 10 μM / L reverse primer 2 μl Sterilized water is added to make up to 50 μl. The reaction conditions for PCR amplification are pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 15 sec, for a total of 35 cycles, extension at 72°C for 5 min; store at 4°C.

[0011] The nucleotide sequence of the obtained amplification product is shown in SEQ ID NO: 3 or SEQ ID NO: 4.

[0012] In the third step, the judgment criterion is that the relative content of muskone in individuals with the genotype A / C at the SNP site is higher than that in individuals with the genotype A / A.

[0013] The present invention detects the genotype of the related traits of the muskone content in the musk of Moschus berezovskii through the DSTN gene molecular marker, and finds that the relative content of muskone in individuals with the A / C genotype is higher than that in individuals with the A / A genotype. By using the genomic DNA of the Moschus berezovskii to be detected as a template, specific primer pairs are used for PCR amplification, and then the PCR amplification product is subjected to Sanger sequencing and SNP molecular marker genotyping. Based on the genotype of the SNP molecular marker, the relative content trait of muskone in the musk of Moschus berezovskii can be selected. In breeding, according to the breeding goal, by eliminating individuals with the A / A genotype and retaining individuals with the A / C genotype, the beneficial effect is that it can efficiently and quickly identify the relative content trait of muskone in the musk of Moschus berezovskii, providing a scientific basis for the early selection of high-quality Moschus berezovskii. In addition, the detection method disclosed in the present invention is simple and easy to operate, can be carried out in the laboratory, and can also be applied to genomic breeding technology. Brief Description of the Drawings

[0014] Figure 1 It is the Manhattan plot of the GWAS analysis of the relative content trait of muscone in the musk of Moschus berezovskii.

[0015] Figure 2 It is the Sanger sequencing result of the PCR amplification products of two genotypes.

[0016] Figure 3 It is the violin plot of the phenotypic distribution of two genotypes of the molecular marker chr23: 682371. Specific implementation manners

[0017] The following embodiments are applicable to the breeding of Moschus berezovskii.

[0018] Embodiment In this embodiment, the relative content of muscone in the musk of Moschus berezovskii was measured. SNP genotyping was carried out using whole-genome resequencing technology, and molecular markers of the DSTN gene significantly related to the relative content of muscone were screened through genome-wide association analysis. The results are as Figure 1 shown.

[0019] In this embodiment, the following experiments were carried out to identify and apply the molecular markers of the DSTN gene related to the relative content trait of muscone in the musk of Moschus berezovskii 1. Phenotype determination and genotype detection (1) Experimental materials and determination of the relative content of muscone in the musk of Moschus berezovskii 132 adult male Moschus berezovskii were selected as experimental animals and cultivated under the same feeding and management conditions. Free diet and drinking water were adopted throughout the process. Musk was collected from the living Moschus berezovskii harmlessly during the musk maturity season (October every year), and then GC / MS was used to quantitatively analyze the relative content of muscone in the musk components as phenotypic data.

[0020] (2) Extraction of genomic DNA 1. According to the instructions of the TianGen DP602 kit, 20 hairs with relatively round shapes and hair follicles were selected, and the hair follicles (1 cm in length) were aseptically collected and placed in an EP tube. 400 ul of Buffer GHA and 20 ul of Proteinase K reagent were added, and after thorough mixing and centrifugation, digestion was carried out in a water bath at 65 °C overnight. 2. The digested samples were transferred to a TGuide 96 deep-well plate after transient centrifugation. The 96 deep-well plate was placed on the base of the TGuide S16 automatic nucleic acid extraction and purification instrument for 96 deep-well plates.

[0021] Slot Name Mixing Time (min) Magnetic Attraction Segments Magnetic Attraction Time per Segment (sec) Magnetic Attraction Speed (mm / s) 1 Lysis 2 1 0 6 Transfer Magnetic Beads 0.5 5 3 2.5 1 Binding 10 5 4 2.5 2 Rinse 1 5 5 3 2.5 3 Rinse 2 5 5 3 2.5 4 Rinse 3 5 5 3 2.5 5 Elution 10 5 5 2.5 6 Release Magnetic Beads 0.5 1 0 4. After the program runs to completion, the DNA extract is collected aseptically and stored at 4 °C for later use.

[0022] (3) PCR amplification Using the above-extracted genomic DNA as a template, amplify the fragment containing the SNP molecular marker at the 682371st base on chromosome 23 of Moschus berezovskii.

[0023] Forward primer: 5’- ACCCGTTGCTGAATGATGCT-3’ (SEQ ID NO: 1) Reverse primer: 5’- AAACAGCAAGGCGAGGTCTT-3’ (SEQ ID NO: 2) The length of the amplified product is 1027bp, The final concentration of the reaction system is calculated as 50 μl: DNA of Moschus berezovskii to be tested 4 ng 2 x Accurate Taq Master Mix 25 μl Forward primer 2 μl Reverse primer 2 μl Sterilized water is supplemented to 50 μl, The reaction conditions for PCR amplification are pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 15 sec, for a total of 35 cycles, extension at 72°C for 5 min; store at 4°C; take 10 μl for agarose detection, and the amplified product with a single target band length of 1027bp is obtained, and it contains the 682371st base on chromosome 23 of Moschus berezovskii, The sequence of the amplified product is as SEQ ID NO: 3

[0024] (4) Sequencing verification and genotyping The PCR products of each sample were subjected to Sanger sequencing respectively, and the sequencing peak maps are as Figure 2 shown.

[0025] 2. Result analysis 132 forest musk deer were selected to measure the relative content of muscone in their mature musk by GC / MS for correlation analysis. The t.test function of R4.0 software was used for statistical test, and the average comparison mode between each pair was selected to statistically test the genotypes and the relative content of muscone of the experimental forest musk deer. P < 0.001 indicates a highly significant difference. The results are shown in Table 1 and Figure 3 as follows. Among the tested individuals, there were 18 individuals with AC genotype and 114 individuals with AA genotype. The difference in the relative content of muscone in the musk of the two genotypes of forest musk deer was highly significant ( P < 0.001). The average relative content of muscone in the AC genotype was 0.559, which was significantly higher than that in the AA genotype, which was 0.204. The results show that the molecular marker of the DSTN gene is significantly correlated with the relative content trait of muscone in the musk of forest musk deer. Individuals with A / C genotype can be selected and bred according to the actual breeding goal to increase the content of muscone and improve the uniformity of muscone in the total musk.

[0026] Genotype Number per Head Relative Content of Muscone / g Standard Deviation CV A / A 114 <![CDATA[0.204 a > 0.1016 48.82% A / C 18 <![CDATA[0.559 b > 0.1400 25.04% Note: Data in the same column with the same superscript letter indicate no significant difference, and data with different superscript letters indicate significant difference ( P <0.001).

[0027] Except for the above embodiments, the present invention may have other embodiments. All technical solutions formed by equivalent replacement or equivalent transformation fall within the protection scope required by the present invention.

Claims

1. A single nucleotide polymorphism SNP primer of the DSTN gene related to the content of muscone in natural musk, characterized in that: The molecular marker corresponding to the SNP primer is located at the 682,371st base on chromosome 23 of the Moschus berezovskii reference genome, with the base mutation being A or C, and the genotypes being AA and AC. The deoxyribonucleotide sequences of the SNP primers are as shown in SEQ ID NO: 1 and SEQ ID NO:

2.

2. Use of the SNP primer of the DSTN gene related to the content of muscone in natural musk according to claim 1, characterized in that It includes the SNP genotype for detecting the content of muscone in the musk of Moschus berezovskii.

3. Use of the SNP primer of the DSTN gene related to the content of muscone in natural musk according to claim 2, characterized in that, The detection method includes the following steps: First step: Detect the Moschus berezovskii DNA sample, and perform PCR amplification with the SNP primer to obtain an amplification product. Second step: Perform Sanger sequencing on the amplification product obtained. Third step: Determine the SNP genotype of the target site according to the sequencing result obtained in the second step.

4. Use of the SNP primer of the DSTN gene related to the content of muscone in natural musk according to claim 3, characterized in that: In the first step, the length of the amplification product is 1027 bp, including the 682,371st base on chromosome 23 of the Moschus berezovskii reference genome.

5. Use of the SNP primer of the DSTN gene related to the content of muscone in natural musk according to claim 4, characterized in that In the first step, the final volume of the reaction system is 50 μl and it is 4 ng of the Moschus berezovskii DNA to be detected 25 μl of 2 x Accurate Taq Master Mix 2 μl of 10 μM / L upstream primer 2 μl of 10 μM / L downstream primer Make up to 50 μl with sterilized water. The reaction conditions for PCR amplification are pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 58°C for 15 sec, extension at 72°C for 15 sec, for a total of 35 cycles, extension at 72°C for 5 min; store at 4°C.

6. Use of the SNP primer of the DSTN gene related to the content of muscone in natural musk according to claim 5, characterized in that: The nucleotide sequence of the amplification product is as shown in SEQ ID NO: 3 or SEQ ID NO:

4.

7. Use of the SNP primer of the DSTN gene related to the content of muscone in natural musk according to claim 3, characterized in that: In the third step, the judgment criterion is that the relative content of muscone in individuals with the genotype A / C at the SNP site is higher than that in individuals with the genotype A / A.

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