Sequence of Ovine CYP17A1 Gene and Bioinformatics Characteristics of Its Protein

Abstract

The present invention discloses the sequence of a sheep CYP17A1 gene and the bioinformatics characteristics of its protein, belonging to the technical fields of gene cloning and protein bioinformatics analysis. The present invention first discloses the CDS sequence and protein sequence of a sheep CYP17A1 gene, as well as the physicochemical properties, domains, functional domains, secondary and tertiary structures, phosphorylation and glycosylation modification site information, codon preference, presence and position of signal peptide of the sheep CYP17A1 protein; these disclosed information play an important role in analyzing the synthesis process and mechanism of sex hormones in sheep, and can also evaluate genetic diversity by comparing the gene sequence differences among different sheep breeds, which is crucial for the protection, utilization and evolutionary research of animal genetic resources. In addition, the disclosure of these information helps to optimize the reproductive performance and adaptability of sheep and guide sheep breeding, etc.

Description

Technical Field

[0001] The present invention relates to the technical fields of gene cloning and protein bioinformatics analysis, and particularly relates to the sequence of a sheep CYP17A1 gene and the bioinformatics characteristics of its protein. Background Art

[0002] The protein encoded by the CYP17A1 gene is a multifunctional enzyme called cytochrome P450 17A1, also known as 17α-hydroxylase. Currently, it is known that the main functions of this enzyme in the human body are involved in the biosynthesis of steroid hormones. Specifically, it participates in the following important steps:

[0003] 1) 17α-hydroxylation: CYP17A1 catalyzes the conversion of pregnenolone and progesterone into 17α-hydroxypregnenolone and 17α-hydroxyprogesterone, which are key steps in the synthesis pathways of adrenocortical hormones and sex hormones.

[0004] 2) Having 17,20-lyase activity: Catalyzing the conversion of 17α-hydroxyprogesterone into androstenedione, and the conversion of 17α-hydroxypregnenolone into dehydroepiandrosterone (DHEA). Both androstenedione and dehydroepiandrosterone are precursors of androgens.

[0005] The CYP17A1 enzyme plays a crucial role in the adrenal gland and gonads because it is involved in the synthesis of mineralocorticoids, glucocorticoids, androgens, and estrogens. If CYP17A1 the gene mutates, resulting in a decrease or loss of enzyme activity, it may cause various diseases, including 17α-hydroxylase deficiency congenital adrenal hyperplasia, which can lead to hormone synthesis disorders and affect normal physiological functions such as blood pressure regulation and sexual characteristics development.

[0006] In addition, CYP17A1 it is also the target of some drugs. For example, abiraterone, which is a drug used to treat prostate cancer, reduces androgen production by inhibiting the CYP17A1 enzyme, thereby achieving the therapeutic effect.

[0007] In many countries and regions, sheep farming is an important part of the local rural economy, providing employment opportunities and income sources for farmers. Mutton is one of the traditional foods in many regions of the world and can provide a high-quality source of protein; sheep wool is an important raw material for the textile industry, used to make various clothing and decorations, while sheepskin is used to make leather products; sheep milk can be used to make various dairy products such as cheese, especially very popular in the Mediterranean region. However, currently, in sheep CYP17A1The gene sequence and protein sequence are still unknown, and the characteristic information such as the physical and chemical properties, domains, functional domains, secondary and tertiary structures, modification site information, codon preference, presence and position of signal peptides of the protein has not been made public. Summary of the Invention

[0008] The object of the present invention is to provide the CYP17A1 gene sequence of sheep and the bioinformatics characteristics of its protein, so as to provide accurate gene information for the research on the synthesis process and mechanism of sex hormones in sheep, the optimization of sheep reproductive performance and adaptive breeding.

[0009] To achieve the above object, the present invention provides the CYP17A1 gene of sheep, CYP17A1 The CDS sequence of the

[0010] gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. CYP17A1 Nested PCR primers for cloning the

[0011] gene of sheep include a total of 6 sets of primers, namely P1 - P6. The P1 set of primers includes RT1793 - P1 - F, RT1793 - P1 - R', RT1793 - P1 - R; the P2 set of primers includes RT1793 - P2 - F, RT1793 - P2 - F', RT1793 - P2 - R, RT1793 - P2 - R'; the P3 set of primers includes RT1793 - P3 - F, RT1793 - P3 - F', RT1793 - P3 - R, RT1793 - P3 - R'; the P4 set of primers includes RT1793 - P4 - F, RT1793 - P4 - F', RT1793 - P4 - R', RT1793 - P4 - R; the P5 set of primers includes RT1793 - P5 - F, RT1793 - P5 - F', RT1793 - P5 - R', RT1793 - P5 - R; the P6 set of primers includes RT1793 - P6 - F, RT1793 - P6 - F', RT1793 - P6 - R.Preferably, the RT1793-P1-F sequence is as shown in SEQ ID NO.3, the RT1793-P1-R' sequence is as shown in SEQ ID NO.4, the RT1793-P1-R sequence is as shown in SEQ ID NO.5, the RT1793-P2-F sequence is as shown in SEQ ID NO.6, the RT1793-P2-F' sequence is as shown in SEQ ID NO.7, the RT1793-P2-R sequence is as shown in SEQ ID NO.9, the RT1793-P2-R' sequence is as shown in SEQ ID NO.8, the RT1793-P3-F sequence is as shown in SEQ ID NO.10, the RT1793-P3-F' sequence is as shown in SEQ ID NO.11, the RT1793-P3-R sequence is as shown in SEQ ID NO.13, the RT1793-P3-R' sequence is as shown in SEQ ID NO.12, the RT1793-P4-F sequence is as shown in SEQ ID NO.14, the RT1793-P4-F' sequence is as shown in SEQ ID NO.15, the RT1793-P4-R' sequence is as shown in SEQ ID NO.16, the RT1793-P4-R sequence is as shown in SEQ ID NO.17, the RT1793-P5-F sequence is as shown in SEQ ID NO.18, the RT1793-P5-F' sequence is as shown in SEQ ID NO.19, the RT1793-P5-R' sequence is as shown in SEQ ID NO.20, the RT1793-P5-R sequence is as shown in SEQ ID NO.21, the RT1793-P6-F sequence is as shown in SEQ ID NO.22, the RT1793-P6-F' sequence is as shown in SEQ ID NO.23, and the RT1793-P6-R sequence is as shown in SEQ ID NO.24.

[0012] Clone the above-mentioned sheep CYP17A1 The nested PCR system for cloning the above-mentioned sheep gene is a 50 µL system, including 2 µL One Step Enzyme Mix, 2 µL upstream primer with a concentration of 10 µM, 2 µL downstream primer with a concentration of 10 µM, 1 µL total RNA, 25 µL 2×One-Step Reaction Solution A or B, and made up to 50 µL with Rnase free water.

[0013] Clone the above-mentioned sheep CYP17A1 The amplification conditions for the nested PCR of cloning the above-mentioned sheep gene are: reverse transcription at 50 °C for 30 min; pre-denaturation at 94 °C for 2 min; denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min, for 33 cycles; final extension at 72 °C for 5 min.

[0014] A sheep as described aboveCYP17A1 Application of gene in optimizing sheep reproductive performance and adaptive breeding

[0015] Therefore, the sheep provided by the present invention CYP17A1 sequence of the gene and bioinformatics characteristics of its protein, and the specific technical effects are as follows:

[0016] The present invention discloses for the first time the CDS sequence and protein sequence of the sheep CYP17A1 gene, as well as the physicochemical properties, domains, functional domains, secondary and tertiary structures, phosphorylation and glycosylation modification site information, codon preference, presence and position of signal peptide of the sheep CYP17A1 protein; these disclosed information play an important role in analyzing the synthesis process and mechanism of sex hormones in sheep, and can also evaluate genetic diversity by comparing the gene sequence differences among different sheep breeds, which is crucial for the protection, utilization and evolutionary research of animal genetic resources. In addition, the disclosure of these information helps to optimize the reproductive performance and adaptability of sheep and guide sheep breeding, etc.

[0017] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1 is the electrophoresis pattern of RNA extracted from sheep liver tissue in Example 1 of the present invention;

[0020] Figure 2 is the electrophoresis pattern of nested PCR in Example 1 of the present invention; where M is the marker, 1-6 in part A are the electrophoresis results of sample 1; 7-12 in part B are the electrophoresis results of sample 2; 13-18 in part C are the electrophoresis results of sample 3, 19-24 are the electrophoresis results of sample 4, and 25-30 are the electrophoresis results of sample 5;

[0021] Figure 3 is the analysis result of the signal peptide of the CYP17A1 protein in Example 2 of the present invention;

[0022] Figure 4 is the prediction result of the transmembrane structure of the CYP17A1 protein in Example 2 of the present invention;

[0023] Figure 5 is the analysis result of the hydrophilicity and hydrophobicity of the CYP17A1 protein in Example 2 of the present invention;

[0024] Figure 6 is the prediction result of the secondary structure of CYP17A1 protein in Example 2 of the present invention;

[0025] Figure 7 is the prediction result of the tertiary structure of CYP17A1 protein in Example 2 of the present invention;

[0026] Figure 8 is the analysis result of the conserved functional domain of CYP17A1 protein in Example 2 of the present invention;

[0027] Figure 9 is the prediction result of the phosphorylation site of CYP17A1 protein in Example 2 of the present invention;

[0028] Figure 10 is the prediction result of the glycosylation site of CYP17A1 protein in Example 2 of the present invention;

[0029] Figure 11 is in Example 3 of the present invention CYP17A1 gene function analysis result diagram;

[0030] Figure 12 is in Example 3 of the present invention CYP17A1 relative expression levels of mRNA in different organs;

[0031] Figure 13 is the relative expression level of CYP17A1 protein in the HPG axis in Example 3 of the present invention;

[0032] Figure 14 is the expression of CYP17A1 protein in the HPG axis in Example 3 of the present invention; where NC is nerve cells, GC is granulosa cells, AC is basophilic cells, BC is eosinophilic cells, CC is chromophobe cells, S is spermatogonia, Sc is Sertoli cells, and Ic is interstitial cells. Detailed implementation manners

[0033] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and examples.

[0034] In order to make the objectives, technical solutions and advantages of the present application clearer, more thorough and complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and examples. The following detailed descriptions are all descriptions of examples, aiming to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meanings as those generally understood by those of ordinary skill in the technical field to which this application belongs.

[0035] The instruments, equipment, reagents and materials used in the examples are all obtained through commercial channels.

[0036] Example 1

[0037] Obtain CYP17A1 the sequence of the gene, the specific steps are as follows:

[0038] S11. Take 1 cm of liver tissue from freshly slaughtered Qianhua meat Merino sheep (obtained from Qian'an Zhihua Sheep Breeding Co., Ltd., address: Wudaoling, Fuzicun, Zanzi Township, Qian'an City, Jilin Province). A total of 5 samples are taken and stored in liquid nitrogen. After bringing them back to the laboratory, use the Trizol extraction kit to extract the RNA of the liver tissue. Aspirate 5 μL of the RNA solution, perform 1.5% agarose gel electrophoresis with 1×TAE as the electrophoresis buffer. After electrophoresis, take a photo under ultraviolet transillumination. The results are shown in 3 . Figure 1 .

[0039] S12. Design primers according to the goat homologous gene sequence of the CYP17A1 gene published in NCBI, as shown in Table 1. Use the primers shown in Table 1, the system shown in Table 2, and the amplification program shown in Table 3 to perform nested PCR.

[0040] Table 1

[0041] ;

[0042] Table 2

[0043] ;

[0044] Table 3

[0045] ;

[0046] Perform 1% agarose gel electrophoresis on the PCR product with 1×TAE as the electrophoresis buffer. After electrophoresis, take a photo under ultraviolet transillumination. The results are shown in Figure 2 .

[0047] S13. Use the column DNA gel recovery kit to cut out the target band for gel recovery of the target DNA. Divide the recovered product into two parts. One part is sent to the company for sequencing, and the other part is used for ligation and genetic transformation. The specific steps are as follows:

[0048] ① Mix the product obtained using the system shown in Table 2-3 evenly with an equal amount of PMD 18-T vector ligation solution and react at 16°C for 30 minutes.

[0049] ② Place 100 μL of SK2301 competent cells on ice. After complete thawing, gently and evenly suspend the cells, then add 10 μL of the ligation product obtained in step ①, gently mix well, and place on ice for 30 min. Heat shock in a 42 °C water bath for 60 s, place on ice for 15 min, then add 400 μL of LB medium, and culture with shaking at 37 °C and 200 - 250 rpm for 1 h. Centrifuge at 4000 rpm for 5 min at room temperature, pipette off 400 μL of the supernatant, and suspend the cells with the remaining medium. Spread the cell suspension on an ampicillin plate pre-coated with 20 μL of 100 mM IPTG and 100 μL of 20 mg / ml X-gal, and incubate inverted at 37 °C overnight.

[0050] Perform PCR using the universal primers of the pMD 18-T vector, the system shown in Table 4, and the amplification program shown in Table 5, and send the PCR product to the company for sequencing.

[0051] Table 4

[0052] ;

[0053] Table 5

[0054] ;

[0055] The sequencing result is: The CYP17A1 CDS sequence of sheep is shown in SEQ ID NO.1.

[0056] SEQ ID NO.1

[0057]

[0058] Example 2

[0059] The obtained CYP17A1 CDS sequence was subjected to protein translation using SnapGene software to obtain the protein sequence, and the sequence is shown in SEQ ID NO.2.

[0060] SEQ ID NO.2

[0061] MWVLLAVFLLTLAYLFWPKTKHSGAKYPRSLPSLPLVGSLPFLPRRGQQHENFFKLQEKYGPIYSFRLGSKTTVMIGHHQLAREVLLKKGKEFSGRPKVATLDILSDNQKGIAFADHGAHWQLHRKLVLNAFALFKDGNLKLEKIINQEANVLCDFLATQHGQSIDLSEPLSLAVTNIISFICFNFSFKNEDPALKAIQNVNDGILEVLSKEVLLDIFPALKIFPSKAMEKMKGCVETRNELLSEILEKCQENFTSDSITNLLHILMQAKVNADNNNTGPEQDSKLLSNRHMLATIADIFGAGVETTTSVIKWIVAYLLHHPSLKKRIQDSIDQNIGFNRTPTISDRNRLVLLEATIREVLRIRPVAPMLIPHKAIIDSSIGDLTIDKGTDVVVNLWALHHNEKEWQQPDLFMPERFLDPTGTQLISPSLSYLPFGAGPRSCVGEMLARQELFLFMSRLLQRFNLEIPDDGKLPSLEGNPSLVLQIKPFKVKIEVRQAWKEAQAEGSTS*

[0062] The physical and chemical properties of the CYP17A1 protein were analyzed using the ProtParam tool, and the results were as follows: number of amino acids: 509; molecular weight: 57337.58; isoelectric point: 8.60.

[0063] The amino acid composition is shown in Table 6. The total number of negatively charged residues: 54, and the total number of positively charged residues: 58. Structural formula: C 2600 H 4137 N 697 O 731 S 15 , total number of atoms: 8180. The atomic composition is shown in Table 7.

[0064] Table 6

[0065] ;

[0066] Table 7

[0067] ;

[0068] Protein signal peptide analysis was performed using SignalP, and the results are as Figure 3 shown. CYP17A1 has a signal peptide, and the signal peptide position is: amino acids 1 - 17.

[0069] Protein transmembrane structure prediction and hydrophilicity / hydrophobicity analysis were performed using Detaibio. The results of protein transmembrane structure prediction are as Figure 4 shown. The transmembrane domain position is: amino acids 5 - 22. The results of hydrophilicity / hydrophobicity analysis are as Figure 5 shown.

[0070] Protein secondary structure prediction was performed using SOPMA, and the results are as Figure 6 shown. Alpha helix (Hh): 216 is 42.44%; Extended strand (Ee): 56 is 11.00%; Beta turn (Tt): 25 is 4.91%; Random coil (Cc): 212 is 41.65%.

[0071] Protein tertiary structure prediction was performed using SWISS - MODEL, and the results are as Figure 7 shown.

[0072] Protein conserved functional domain analysis was performed using the SMART online website, and the results are as Figure 8 and Table 8 shown.

[0073] Table 8

[0074] ;

[0075] Subcellular localization analysis was performed using Cell - PLoc, and the result is that CYP17A1 is localized in Endoplasmic reticulum. Microsome. Mitochondrion.

[0076] Phosphorylation site prediction (the prediction results are shown in Figure 9 ) and glycosylation site prediction (the prediction results are shown in Figure 10 ) were performed using SignalP. A total of 37 phosphorylation sites and 5 glycosylation sites were obtained.

[0077] Codon preference analysis was performed using EMBOSS, and the results are shown in Table 9.

[0078] Table 9

[0079] ;

[0080]

[0081] Example 3

[0082] CYP17A1 is a key enzyme in the androgen biosynthesis pathway and plays a role in the testis and adrenal glands. Gonadotropin-releasing hormone released by the hypothalamus acts on luteinizing hormone (LH) released by the pituitary gland, binds to the luteinizing hormone receptor on Leydig cells in the testis, and initiates the synthesis process of testosterone. Under the regulation of LH, cholesterol is rapidly transported into the mitochondria through the mediation of steroidogenic acute regulatory protein (StAR). In the matrix of cell mitochondria, cholesterol is converted into pregnenolone through the action of cholesterol side-chain cleavage enzyme (CYP11A1 or P450scc). Next, pregnenolone is catalyzed by 3β-hydroxysteroid dehydrogenase (3β-HSD) to generate progesterone. Subsequently, progesterone is gradually converted into 17α-androstenedione and androstenedione under the action of 17α-hydroxylase (CYP17A1), and finally testosterone is generated (see Figure 11 ).

[0083] Five healthy pre-pubertal (4-month-old) male Qianhua meat Merino sheep under the same feeding and management conditions were selected as experimental subjects. Liver, testis, pituitary gland, hypothalamus, head of epididymis, body of epididymis, tail of epididymis, and quadriceps femoris tissues were collected and detected for the localization and expression levels of CYP17A1 mRNA and protein by fluorescence quantitative PCR (primer sequences were designed according to the sheep CYP17A1 and β-actin mRNA sequences published by NCBI, see Table 10), immunohistochemistry, and Western blotting. The results showed that CYP17A1 It may affect spermatogenesis and then regulate male reproduction by acting on hypothalamic nerve cells and granulosa cells, and then acting on eosinophilic and basophilic cells of the pituitary gland, and finally regulating in Sertoli cells, Leydig cells and germ cells of the testis. CYP17A1 The gene has relatively high mRNA expression in the testis, liver and hypothalamus. CYP17A1 protein is expressed in the hypothalamus, pituitary gland and testis, and the expression level trend is pituitary gland > hypothalamus > testis. See Figures 12 - 14 .

[0084] Table 10

[0085] ;

[0086] Therefore, the present invention discloses for the first time sheep CYP17A1The CDS sequence and protein sequence of the gene, as well as the physicochemical properties, domains, functional domains, secondary and tertiary structures, phosphorylation and glycosylation modification site information, codon preference, presence and location of signal peptides of the sheep CYP17A1 protein; these publicly available information play an important role in analyzing the synthesis process and mechanism of sex hormones in sheep, and can also evaluate genetic diversity by comparing the gene sequence differences among different sheep breeds, which is crucial for the protection, utilization and evolutionary research of animal genetic resources. In addition, the disclosure of this information also helps to optimize the reproductive performance and adaptability of sheep and guide sheep breeding, etc.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. Use of ovine CYP17A1 protein in the preparation of a reagent for promoting spermatogenesis in male sheep for non-therapeutic purposes, characterized in that: The reagent uses sheep CYP17A1 protein as the active ingredient; the amino acid sequence of the CYP17A1 protein is as shown in SEQ ID NO.2.

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