Sequence of sheep CYP17A1 gene and bioinformatics characteristics of protein thereof

Abstract

The invention discloses a sequence of a sheep CYP17A1 gene and bioinformatics characteristics of protein thereof, and belongs to the technical field of gene cloning and protein bioinformatics analysis. The invention discloses a CDS sequence and a protein sequence of the sheep CYP17A1 gene as well as physicochemical properties, a structural domain, a functional domain, secondary and tertiary structures, phosphorylation and glycosylation modification site information, codon preference, and existence and position of signal peptide of the sheep CYP17A1 protein for the first time; the public information plays an important role in analyzing the synthesis process and mechanism of sex hormones in sheep bodies, genetic diversity can be evaluated by comparing gene sequence differences among different sheep varieties, and the public information is crucial to protection, utilization and evolution research of animal genetic resources. The disclosure of the information is also helpful for optimizing the reproductive performance and adaptability of the sheep, guiding the breeding of the sheep and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of gene cloning and protein bioinformatics analysis, in particular to sheep CYP17A1 The sequence of the 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. It is currently known that the main function of this enzyme in the human body involves the biosynthesis of steroid hormones. Specifically, it is involved in the following important steps: 1) 17α-Hydroxylation: CYP17A1 catalyzes the conversion of pregnenolone and progesterone into 17α-hydroxypregnenolone and 17α-hydroxyprogesterone, which is a key step in the synthesis pathway of adrenal cortical hormones and sex hormones.

[0003] 2) It has 17,20-lyase activity: it catalyzes the conversion of 17α-hydroxyprogesterone to androstenedione, and 17α-hydroxypregnenolone to dehydroepiandrosterone (DHEA). Both androstenedione and dehydroepiandrosterone are precursors of androgens.

[0004] The CYP17A1 enzyme plays a vital role in the adrenal and gonadal glands as it is involved in the synthesis of mineralocorticoids, glucocorticoids, androgens, and estrogens. CYP17A1 Gene mutations lead to reduced or lost enzyme activity, which may cause a variety of diseases, including 17α-hydroxylase-deficient congenital adrenal hyperplasia, which can lead to hormone synthesis disorders and affect normal physiological functions such as blood pressure regulation and sexual development.

[0005] also, CYP17A1 It is also the target of certain drugs, such as abiraterone, which is a drug used to treat prostate cancer. It achieves its therapeutic effect by inhibiting the CYP17A1 enzyme to reduce the production of androgens.

[0006] In many countries and regions, sheep farming is an important part of the local rural economy, providing employment opportunities and income sources for farmers. Sheep meat is one of the traditional foods in many parts of the world and can provide a high-quality protein source; sheep wool is an important raw material for the textile industry, used to make various clothes and decorations, while sheepskin is used to make leather products; sheep milk can be used to make a variety of dairy products, such as cheese, which is very popular in the Mediterranean region. However, at present, sheep CYP17A1 The gene sequence and protein sequence are still unknown, and the protein's physical and chemical properties, structural domains, functional domains, secondary and tertiary structures, modification site information, codon preference, presence and location of signal peptides and other characteristic information have never been made public. Summary of the invention

[0007] The present invention aims to provide sheep CYP17A1 The gene sequence and bioinformatics characteristics of its protein provide accurate genetic information for studying the synthesis process and mechanism of sex hormones in sheep, optimizing sheep reproductive performance and adaptive breeding.

[0008] To achieve the above object, the present invention provides sheep CYP17A1 Gene, CYP17A1 The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0009] Cloning of the sheep CYP17A1 The nested PCR primers for the gene include 6 groups of primers from P1 to P6. The primers of group P1 include RT1793-P1-F, RT1793-P1-R', and RT1793-P1-R. The primers of group P2 include RT1793-P2-F, RT1793-P2-F', RT1793-P2-R, and RT1793-P2-R'. The primers of group P3 include RT1793-P3-F, RT1793-P3-F', RT1793-P3-R, and RT1793-P3-R'. T1793-P3-R', P4 group primers include RT1793-P4-F, RT1793-P4-F', RT1793-P4-R', RT1793-P4-R, P5 group primers include RT1793-P5-F, RT1793-P5-F', RT1793-P5-R', RT1793-P5-R, P6 group primers include RT1793-P6-F, RT1793-P6-F', RT1793-P6-R.

[0010] Preferably, the RT1793-P1-F sequence is shown in SEQ ID NO.3, the RT1793-P1-R' sequence is shown in SEQ ID NO.4, the RT1793-P1-R sequence is shown in SEQ ID NO.5, the RT1793-P2-F sequence is shown in SEQ ID NO.6, the RT1793-P2-F' sequence is shown in SEQ ID NO.7, the RT1793-P2-R sequence is shown in SEQ ID NO.9, the RT1793-P2-R' sequence is shown in SEQ ID NO.8, the RT1793-P3-F sequence is shown in SEQ ID NO.10, the RT1793-P3-F' sequence is shown in SEQ ID NO.11, the RT1793-P3-R sequence is shown in SEQ ID NO.13, the RT1793-P3-R' sequence is shown in SEQ ID NO.12, and the RT1793-P4-F sequence is shown in SEQ ID NO. NO.14, the RT1793-P4-F' sequence is shown in SEQ ID NO.15, the RT1793-P4-R' sequence is shown in SEQ ID NO.16, the RT1793-P4-R sequence is shown in SEQ ID NO.17, the RT1793-P5-F sequence is shown in SEQ ID NO.18, the RT1793-P5-F' sequence is shown in SEQ ID NO.19, the RT1793-P5-R' sequence is shown in SEQ ID NO.20, the RT1793-P5-R sequence is shown in SEQ ID NO.21, the RT1793-P6-F sequence is shown in SEQ ID NO.22, the RT1793-P6-F' sequence is shown in SEQ ID NO.23, and the RT1793-P6-R sequence is shown in SEQ ID NO.24.

[0011] Cloning of the sheep CYP17A1 The nested PCR system for genes is a 50µL 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 RNase free water to make up to 50 µL.

[0012] Cloning of the sheep CYP17A1 The amplification conditions of the nested PCR of the gene were as follows: 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, 33 cycles; and final extension at 72°C for 5 min.

[0013] A sheep as above CYP17A1 The use of genetics in optimizing sheep reproductive performance and adaptive breeding.

[0014] Therefore, the sheep provided by the present invention CYP17A1 The bioinformatics characteristics of the gene sequence and its protein, and its specific technical effects are as follows: The present invention discloses sheep for the first time CYP17A1 The CDS sequence and protein sequence of the gene, as well as the physicochemical properties, structural domains, functional domains, secondary and tertiary structures, phosphorylation and glycosylation modification site information, codon preference, presence and position of signal peptides of sheep CYP17A1 protein; these public information are important for analyzing the synthesis process and mechanism of sex hormones in sheep, and can also be used to evaluate genetic diversity by comparing the gene sequence differences between different sheep breeds. It is crucial for the protection, utilization and evolution research of animal genetic resources. In addition, the disclosure of this information will also help optimize sheep reproductive performance and adaptability, and guide sheep breeding.

[0015] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in 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 ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative labor.

[0017] Figure 1 is an electrophoresis diagram of RNA extracted from sheep liver tissue in Example 1 of the present invention; Figure 2 It is the electrophoresis diagram of the nested PCR in Example 1 of the present invention; wherein M is a 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; Figure 3 is the result of the CYP17A1 protein signal peptide analysis in Example 2 of the present invention; Figure 4 is the prediction result of the transmembrane structure of CYP17A1 protein in Example 2 of the present invention; Figure 5 is the result of the hydrophilicity analysis of the CYP17A1 protein in Example 2 of the present invention; Figure 6 is the prediction result of the secondary structure of CYP17A1 protein in Example 2 of the present invention; Figure 7 is the prediction result of the tertiary structure of CYP17A1 protein in Example 2 of the present invention; Figure 8 It is the result of the analysis of the conserved functional domain of CYP17A1 protein in Example 2 of the present invention; Fig. 9 is the prediction result of the phosphorylation site of CYP17A1 protein in Example 2 of the present invention; Fig.10 is the prediction result of glycosylation site of CYP17A1 protein in Example 2 of the present invention; Fig.11 In the third embodiment of the present invention CYP17A1 Gene function analysis results diagram; Fig.12 In the third embodiment of the present invention CYP17A1 Relative expression of mRNA in different organs; Fig.13 is the relative expression of CYP17A1 protein in the HPG axis in Example 3 of the present invention; Fig.14 It is the expression of CYP17A1 protein in the HPG axis in Example 3 of the present invention; wherein NC is a nerve cell, GC is a granulosa cell, AC is a basophil, BC is an eosinophil, CC is a chromophobe cell, S is a spermatogonia, Sc is a supporting cell, and Ic is an interstitial cell. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0019] In order to make the purpose, technical scheme and advantages of the present application clearer, more thorough and more complete, the technical scheme of the present invention is clearly and completely described below through the accompanying drawings and examples. The following detailed descriptions are all descriptions of the embodiments, and are intended to provide further detailed descriptions of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present application belongs.

[0020] The instruments, equipment, reagents and materials used in the examples were all obtained through commercial channels.

[0021] Embodiment 1

[0022] get CYP17A1 The specific steps are as follows: S11, take 1cm from a Qianhua meat Merino sheep just slaughtered (from Qian'an Zhihua Sheep Breeding Co., Ltd., address: Wudaoling, Fuzi Village, Zanzi Township, Qian'an County, Changchun City, Jilin Province) 3Liver tissue, a total of 5 samples were taken and stored in liquid nitrogen. After being brought back to the laboratory, the RNA of the liver tissue was extracted using the Trizol extraction kit. 5 μL of RNA solution was taken and subjected to 1.5% agarose electrophoresis with 1×TAE as the electrophoresis buffer. After electrophoresis, the samples were photographed under ultraviolet transmission light. The results are shown in Figure 1 .

[0023] S12. According to CYP17A1 Primers were designed based on the goat homologous gene sequence published in NCBI, as shown in Table 1. Nested PCR was performed using the primers shown in Table 1, the system shown in Table 2, and the amplification program shown in Table 3.

[0024] Table 1 ;

[0025] Table 2 ;

[0026] Table 3 ;

[0027] The PCR products were subjected to 1% agarose gel electrophoresis using 1×TAE as electrophoresis buffer and photographed under ultraviolet light. Figure 2 .

[0028] S13. Use a column DNA gel recovery kit to cut the target band and perform 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 connected and genetically transformed. The specific steps are as follows: ① After the product obtained using the system shown in Table 2-3 is evenly mixed with an equal amount of PMD 18-T vector connection liquid, the mixture is reacted at 16°C for 30 minutes.

[0029] ②Put 100μL SK2301 competent cells on ice, thaw completely, gently suspend the cells evenly, then add 10μL of the ligation product obtained in step ①, gently mix, and place on ice for 30 min. Heat shock in a 42℃ water bath for 60s, place on ice for 15min, add 400μL LB medium, and culture at 37℃, 200~250rpm for 1h. Centrifuge at 4000rpm for 5min at room temperature, aspirate 400μL supernatant with a pipette, and suspend the cells with the remaining medium. Spread the cell suspension on an ampicillin plate pre-coated with 20μL 100mM IPTG and 100μL 20mg / ml X-gal, and culture overnight at 37℃.

[0030] PCR was performed 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 the PCR product was sent to the company for sequencing.

[0031] Table 4 ;

[0032] Table 5 ;

[0033] Sequencing results: Sheep CYP17A1 The CDS sequence is shown in SEQ ID NO.1.

[0034] SEQ ID NO.1

[0035] Embodiment 2

[0036] Will obtain CYP17A1 The CDS sequence was translated into protein using SnapGene software to obtain the protein sequence, which is shown in SEQ ID NO.2.

[0037] SEQ ID NO.2 MWVLLAVFLLTLAYLFWPKTKHSGAKYPRSLPSLPLVGSLPFLPRRGQQHENFFKLQEKYGPIYSFRLGSKTTVMIGHHQLAREVLLKKGKEFSGRPKVATLDILSDNQKGIAFADHGAHWQLHRKL VLNAFALFKDGNLKLEKIINQEANVLCDFLATQHGQSIDLSEPLSLAVTNIISFICFNFSFKNEDPALKAIQNVNDGILEVLSKEVLLDIFPALKIFPSKAMEKMKGCVETRNELLSEILEKCQENFT SDSITNLLHILMQAKVNADNNNTGPEQDSKLLSNRHMLATIADIFGAGVETTTSVIKWIVAYLLHHHPSLKKRIQDSIDQNIGFNRTPTISDRNRLVLLEATIREVLRIRPVAPMLIPHKAIIDSSIG DLTIDKGTDVVVNLWALHHNEKEWQQPDLFMPERFLDPTGTQLISPSLSYLPFGAGPRSCVGEMLARQELFLFMSRLLQRFNLEIPDDGKLPSLEGNPSLVLQIKPFKVKIEVRQAWKEAQAEGSTS* The ProtParam tool was used to analyze the physicochemical properties of CYP17A1 protein. The results showed: number of amino acids: 509; molecular weight: 57337.58; isoelectric point: 8.60.

[0038] The amino acid composition is shown in Table 6. The total number of negatively charged residues is 54, and the total number of positively charged residues is 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.

[0039] Table 6 ;

[0040] Table 7 ;

[0041] SignalP was used to analyze protein signal peptides. The results are as follows: Figure 3 As shown, CYP17A1 has a signal peptide, and the signal peptide position is: 1st to 17th amino acids.

[0042] Detaibio was used to predict the protein transmembrane structure and analyze its hydrophilicity. Figure 4 As shown in Figure 1, the transmembrane domain is located at amino acids 5-22. The hydrophilicity analysis results are shown in Figure 1. Figure 5 shown.

[0043] SOPMA was used to predict the secondary structure of proteins. The results are as follows: Figure 6 As shown, Alpha helix (Hh): 216is 42.44%; Extended strand (Ee): 56 is 11.00%; Beta turn (Tt): 25 is 4.91%; Random coil (Cc): 212 is 41.65%.

[0044] SWISS-MODEL was used to predict the protein tertiary structure. The results are as follows: Figure 7 shown.

[0045] The protein conserved functional domain analysis was performed using the SMART online website. The results are as follows: Figure 8 and as shown in Table 8.

[0046] Table 8 ;

[0047] Cell-PLoc was used for subcellular localization analysis, and the results showed that CYP17A1 was localized in Endoplasmicreticulum, Microsome, and Mitochondrion.

[0048] SignalP was used to predict phosphorylation sites (see Fig. 9 ) and glycosylation site prediction (prediction results see Fig.10 ), a total of 37 phosphorylation sites and 5 glycosylation sites were obtained.

[0049] EMBOSS was used to perform codon preference analysis, and the results are shown in Table 9.

[0050] Table 9 ;

[0051] Embodiment 3 CYP17A1 is a key enzyme in the androgen biosynthesis pathway and plays a role in the testes 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 the interstitial cells of the testis, and initiates the synthesis 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 produces progesterone under the catalysis of 3B-hydroxysteroid dehydrogenase (3B-HSD). Subsequently, progesterone is gradually converted into 17a-androstenedione and androstenedione under the action of 17a-hydroxylase (CYP17A1), and finally testosterone is produced (see Fig.11 ).

[0052] Five healthy male Qianhua merino sheep before sexual maturity (4 months old) under the same feeding and management conditions were selected as the test subjects. The liver, testis, pituitary, hypothalamus, epididymal caput, epididymal body, epididymal tail, and quadriceps femoris tissues were collected and analyzed by fluorescence quantitative PCR (primer sequences were based on the sheep published by NCBI CYP17A1 and β-actin mRNA sequence was designed, see Table 10), immunohistochemistry and western blotting were used to detect CYP17A1 mRNA and protein localization and expression levels. CYP17A1 It may affect spermatogenesis by affecting hypothalamic nerve cells and granulosa cells, and then acting on the eosinophilic cells and basophilic cells of the pituitary gland, and finally regulating the supporting cells, interstitial cells and germ cells of the testis, thereby regulating male reproduction. CYP17A1 The mRNA expression of the gene was high in the testis, liver and hypothalamus, and the CYP17A1 protein was expressed in the hypothalamus, pituitary gland and testis, with the expression trend showing pituitary gland > hypothalamus > testis. Figure 12-14 .

[0053] Table 10 ;

[0054] Therefore, the present invention discloses sheep for the first time CYP17A1The CDS sequence and protein sequence of the gene, as well as the physicochemical properties, structural domains, functional domains, secondary and tertiary structures, phosphorylation and glycosylation modification site information, codon preference, presence and position of signal peptides of sheep CYP17A1 protein; these public information are important for analyzing the synthesis process and mechanism of sex hormones in sheep, and can also be used to evaluate genetic diversity by comparing the gene sequence differences between different sheep breeds. It is crucial for the protection, utilization and evolution research of animal genetic resources. In addition, the disclosure of this information will also help optimize sheep reproductive performance and adaptability, and guide sheep breeding.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. Sheep CYP17A1 A gene characterized by: CYP17A1 The CDS sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.

2.

2. Cloning the sheep described in claim 1 CYP17A1 A nested PCR primer for a gene, characterized in that: There are 6 primers in total, including P1-P6. The primers in the P1 group include RT1793-P1-F, RT1793-P1-R', and RT1793-P1-R. The primers in the P2 group include RT1793-P2-F, RT1793-P2-F', RT1793-P2-R, and RT1793-P2-R'. The primers in the P3 group include RT1793-P3-F, RT1793-P3-F', RT1793-P3-R, and RT1793 -P3-R', P4 group primers include RT1793-P4-F, RT1793-P4-F', RT1793-P4-R', RT1793-P4-R, P5 group primers include RT1793-P5-F, RT1793-P5-F', RT1793-P5-R', RT1793-P5-R, P6 group primers include RT1793-P6-F, RT1793-P6-F', RT1793-P6-R.

3. The cloned sheep according to claim 2 according to claim 1 CYP17A1 A nested PCR primer for a gene, characterized in that: The RT1793-P1-F sequence is shown in SEQ ID NO.3, the RT1793-P1-R' sequence is shown in SEQ ID NO.4, the RT1793-P1-R sequence is shown in SEQ ID NO.5, the RT1793-P2-F sequence is shown in SEQ ID NO.6, the RT1793-P2-F' sequence is shown in SEQ ID NO.7, the RT1793-P2-R sequence is shown in SEQ ID NO.9, the RT1793-P2-R' sequence is shown in SEQ ID NO.8, the RT1793-P3-F sequence is shown in SEQ ID NO.10, the RT1793-P3-F' sequence is shown in SEQ ID NO.11, the RT1793-P3-R sequence is shown in SEQ ID NO.13, the RT1793-P3-R' sequence is shown in SEQ ID NO.12, and the RT1793-P4-F sequence is shown in SEQ ID NO. NO.14, the RT1793-P4-F' sequence is shown in SEQ ID NO.15, the RT1793-P4-R' sequence is shown in SEQ ID NO.16, the RT1793-P4-R sequence is shown in SEQ ID NO.17, the RT1793-P5-F sequence is shown in SEQ ID NO.18, the RT1793-P5-F' sequence is shown in SEQ ID NO.19, the RT1793-P5-R' sequence is shown in SEQ ID NO.20, the RT1793-P5-R sequence is shown in SEQ ID NO.21, the RT1793-P6-F sequence is shown in SEQ ID NO.22, the RT1793-P6-F' sequence is shown in SEQ ID NO.23, and the RT1793-P6-R sequence is shown in SEQ ID NO.

24.

4. Cloning the sheep according to claim 1 CYP17A1 A system for nested PCR of genes, characterized in that: The system is 50µL, including 2µL One-Step Enzyme Mix, 2µL 10µM upstream primer, 2µL 10µM downstream primer, 1µL total RNA, 25µL 2× One-Step Reaction Solution A or B, and RNase free water to make up to 50µL.

5. Cloning the sheep according to claim 1 CYP17A1 The amplification conditions of the nested PCR of the gene are characterized by: 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, 33 cycles; final extension at 72°C for 5 min.

6. A sheep as claimed in claim 1 CYP17A1 The use of genetics in optimizing sheep reproductive performance and adaptive breeding.

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