CN103609526B - The application of mitochondrial protein translation factor Guf1 in male sterile research - Google Patents

Abstract

The present invention provides the use of a model organism mouse that knocks out the Guf1 gene in the study of the mechanism of male sterility. More specifically, the invention provides a mouse model for the knockout of the Guf1 gene in the study of human male reproduction, spermatogenesis and male infertility. It may become a diagnostic method for diagnosing male infertility.

Description

Application of Mitochondrial Protein Translation Factor Guf1 in the Study of Male Sterility

technical field

The invention relates to the application of the mitochondrial protein translation factor Guf1 in the research of male reproduction. Specifically, the present invention provides the use of a model organism mouse that knocks out the Guf1 gene in the study of male sterility. More specifically, the present invention provides a mouse model for the knockout of the Guf1 gene in the study of human male reproduction and spermatogenesis. and uses in the mechanisms and treatment of male infertility.

Background technique

At present, about 15% of married couples in the world suffer from infertility, and asthenozoospermia accounts for 19% of male infertility, which has become a common cause of male fertility. The energy required for sperm motility mainly comes from the oxidative phosphorylation of the mitochondrial respiratory chain. If the function of oxidative phosphorylation is abnormal, the change of enzyme activity or expression level and the variation of mitochondrial DNA may lead to mitochondrial energy synthesis disorder and reduce sperm motility. In 1955, Hoffman-Berling was the first to prove that ATP is the energy source for sperm tail movement, so the structural and functional status of mitochondria is one of the important indicators for evaluating sperm quality. The correlation of mitochondrial structural and functional abnormalities with low sperm motility has attracted attention in recent years.

Mitochondria are the main organelles for cells to carry out aerobic oxidation. Almost all life activities of cells are powered by mitochondrial oxidative phosphorylation, and the movement of sperm requires a large amount of energy supply. Therefore, the oxidative phosphorylation function of sperm mitochondrial respiratory chain is important for maintaining The normal motility of sperm plays a vital role. If its function is abnormal, it may directly affect the production and transmission of energy, resulting in sperm motility disorders. At present, research on mitochondrial respiratory function and respiratory chain energy metabolism at home and abroad is mainly focused on some tissues and organs with high oxygen consumption and high energy demand, such as liver tissue, brain tissue, myocardial tissue, etc. Mitochondrial energy metabolism disorders in these high oxygen consumption tissues It will cause a decrease in membrane potential, greatly reduce ATP production, and affect the function of tissues and organs. A large amount of energy supply is required to maintain the normal movement of sperm, so the research on the respiratory function of sperm mitochondria and the energy metabolism of the respiratory chain is of great significance.

As a prokaryotic protein translation elongation factor, LepA can bind to ribosomes to trigger the reverse transport of tRNA, which can increase the fidelity of protein translation in vitro. The eukaryotic homologous protein Guf1 protein of lepA is a very conserved protein located in the mitochondrial matrix. Studies have shown that the mitochondrial protein synthesis rate in yeast cells with Guf1 protein mutations slows down under low temperature conditions, and mitochondrial cytochrome c oxidase is elevated when the temperature rises. activity was reduced, thereby indicating that the Guf1 protein is an important fidelity factor during mitochondrial protein synthesis. Therefore, the functional research of Guf1 protein in higher animals is particularly important.

Contents of the invention

Our study found that Guf1 protein is important for mouse spermatogenesis. Knockout of Guf1 protein led to obstacles in the process of spermatogenesis in mice, a significant decrease in the number of sperm, and decreased sperm motility. Further research found that this was related to the defect of mitochondrial oxidative phosphorylation in the testis. Guf1 protein causes accelerated translation of mitochondrial proteins, but the poor fidelity of translation products leads to accelerated degradation, which may be an important reason for mitochondrial oxidative phosphorylation dysfunction.

We found for the first time that Guf1 protein plays an important role in regulating the function of male reproductive mitochondria in the model organism mouse. Knockout of the gene encoding Guf1 protein can cause spermatogenesis disorder in mice, seriously affect the spermatogenesis and maturation of mice, and provide an important target for the study of the mechanism and treatment of male reproduction.

More specifically, the present invention provides the following:

CLAIMS 1. A method of obtaining a non-human male sterile mammal, said method comprising breeding males of said mammal in which the Guf1 gene has been knocked out.

2. The method according to 1, wherein the mammal is a mouse, and the sequence of the Guf1 gene is as shown in SEQ ID NO:1.

3. A method of rendering sterile a non-human male mammal, said method comprising aberrantly expressing the Guf1 gene in said male mammal.

4. The method according to 3, wherein the mammal is a mouse, and the sequence of the Guf1 gene is as shown in SEQ ID NO:1.

5. Use of the Guf1 gene as a target for rendering sterile a non-human male mammal.

6. purposes according to 5, wherein said mammal is mouse, and the sequence of described Guf1 gene is as shown in SEQ ID NO:1.

7. Use of a reagent for detecting abnormal expression of Guf1 gene in a mammal in the preparation of a diagnostic agent for detecting male sterility in the mammal.

8. A kit for diagnosing infertility in a male mammal, said kit comprising a reagent for detecting abnormality of the Guf1 gene in said mammal.

Description of drawings

Figure 1: Guf1 gene knockout strategy and PCR, Western blot detection;

Figure 2: Pathological analysis of male sterility caused by Guf1 knockout;

Figure 3: Guf1 knockout increases sperm cell apoptosis and affects spermatogenesis;

Figure 4: Transmission electron microscope observation of testes and sperm mitochondria of Guf1 knockout mice;

Figure 5: Effect of Guf1 protein on the function of mitochondrial respiratory chain complexes;

Figure 6: The effect of Guf1 knockout on mitochondrial respiratory chain complex subunit proteins;

Figure 7: Guf1 knockout causes decreased cytoplasmic translation and decreased mTOR signaling pathway;

Figure 8: Effects of Guf1 protein knockout on mitochondrial copying, transcription and translation.

Figure 9: Preservation of human spermatozoa and extraction of their genomic DNA.

Detailed ways

Example 1, Guf1 gene knockout strategy and PCR, Western blot detection.

In order to study the function of Guf1 protein in higher organisms and what impact its mutation will have on the body. We chose mice as a model organism, used gene targeting technology to knock out Guf1 protein, and used PCR and Western blot methods to evaluate the knockout efficiency. Figure 1 shows the strategy for knocking out Guf1 protein and the knockout efficiency detected by PCR and Western blot. Figure 1A shows the knockout strategy of Guf1 and Figure 1B represents the genotypes of Guf1 knockout mice identified by PCR. Figure 1B represents the knockout efficiency of Guf1 in mouse tissues detected by western blot. The results showed that the amount of Guf1 protein was significantly reduced in the testis, further evidence that the protein was knocked out in mice.

specific method:

Obtaining Guf1 knockout mice:

Construction of the Guf1 targeting vector: the Guf1 gene was obtained from the 129BAC (bMQ-420K22) clone (purchased from Welcome Trust Sanger Institute) containing the mouse Guf1 gene (the nucleotide sequence of the Guf1 gene is shown in SEQ.ID.NO.1), as shown in the figure 1A. The loxp site (the sequence is GAATTCCTGCAGCCCAATTCCGATC) is added on both sides of the second to the eighth exon of the gene, and the neomycin resistance gene (neo gene, sequence Shown in SEQ ID NO.2) carry out the screening of next step. The sequence of the targeting vector is shown in SEQ ID NO.3.

Embryonic stem cell stage: After the construction of the targeting vector is completed, the midbody targeting plasmid (about 100ug needs to be prepared) is electrotransferred into homologous embryonic stem cell mouse 129 cells (purchased from Nanjing Institute of Biomedicine), so that the Guf1 gene on the targeting vector and the Homologous recombination occurs in the genome of embryonic stem cells, that is, exon 1 and exon 8 of Guf1 on the targeting vector are exchanged with the same sequence on the genome of Guf1 in embryonic stem cells, and then exon 2 to exon 8 of Guf1 are knocked out. Recombinant embryonic stem cell clones were obtained by positive and negative screening with G418 and guanosine, and identified by PCR and southern blot.

Blastocyst injection stage: amplify the correctly targeted ES cell clones, microinject into blastocysts of C57BL/6 mice (Nanjing Institute of Biomedicine), and transplant the blastocysts into surrogate mother mice C57BL/6 ( Nanjing Institute of Biomedicine) in utero, produced 8 male and 2 female chimeric mice. Seven male chimeric mice were crossed with C57BL6 mice to give birth to 20 heterozygous mice (Guf1 flox ^/+ ). Heterozygous mice Guf1 flox ^/+ were housed together with males and females to produce offspring. Guf1 flox ^/fox mice were identified by PCR and mated with EIIa-cre mice (Academy of Military Medical Sciences) to obtain Guf1 knockout and heterozygous mice.

Cutting and digesting the mouse tail: put the Guf1 gene heterozygous mice in the same cage, and when the newborn wild, heterozygous and knockout mice are born to about 18 days old, wean the milk, separate the cages, and prepare sterilized scissors, tweezers, EP tube, alcohol cotton ball. Subtract a section about 0.5 cm from the end of the tail of the newborn mouse and put it into the EP tube. Prepare rat tail digestion solution (20mM Tris-HCl, 5mM EDTA, 0.4M NaCl, 1% SDS, 400μg/ml proteinase K), add 400μl to EP tube, and digest overnight at 55°C.

Extract the mouse genome: centrifuge the mouse digestive solution at 11,000 rpm for 5 minutes, pour the supernatant into a new EP tube, add 200 μl saturated sodium chloride, shake up and down 200 times, place on ice for 10 minutes, and centrifuge at 14,000 rpm for 10 minutes . Pour the supernatant into a new EP tube, add 800 μl of absolute ethanol, centrifuge at 14,000 rpm for 5 minutes, discard the supernatant, and dry the precipitated DNA at room temperature, then add 50 μl of double distilled water.

PCR identification: Synthetic PCR identification primers (Shanghai Sangong Synthetic), the primer sequence is as follows:

Loxpt F2: 5'TTTGTCCTAAATGCGTGGTG 3'

Loxpt R2: 5'CCCGCTCCCTAATAAAGATG 3'

Frtt R2: 5'CGATCCCTGTACTCAAGACC 3'

The reaction system is as follows:

2x Taq mix: 10μl

Loxpt F2: 0.5 μl

Loxpt R2 (Frtt R2): 0.5μl

Genomic DNA: 1.5 μl

RT-PCR amplification conditions: denaturation -95 degrees, 5 minutes; annealing -60 degrees, 45 seconds; extension -72 degrees, 1 minute; number of cycles: 30 times; final extension conditions -72 degrees, 10 minutes. PCR samples were subjected to agarose gel electrophoresis (gel concentration: 1%).

Western blotting: 6-8 week-old wild-type and Guf1 knockout mice obtained by PCR identification of mating cages were killed by neck dislocation, heart and testis tissues were taken out, and rinsed with PBS. Add 1ml of RIPA lysate, grind in a tissue grinder, place the homogenate on ice for 30 minutes, centrifuge at 12000rpm for 30 minutes, and use the BCA kit to measure the protein concentration of the supernatant.

Prepare 15% SDS-PAGE gel, add a certain volume of sample buffer to the sample whose protein concentration has been measured, heat at 95 degrees for 15 minutes, take 50 μg of protein and add it to 15% SDS-PAGE gel, 100v constant pressure, Run for about 3 hours.

The protein samples on the gel were transferred to PVDF membrane, blocked with 5% skimmed milk powder for 1 hour, then added 1:1000 diluted anti-Guf1 antibody (sigma) and incubated overnight. Wash the membrane 5 times with TBST, 5 minutes each time. Horseradish peroxidase-labeled rabbit secondary antibody (JacksonImmunoResearch) diluted 1:3000 was added and incubated for 1.5 hours. After washing the membrane three times with TBST (150mM NaCl, 20mM Tris-HCl, pH7.4, Tween-20 0.05%), add ultra-sensitive ECL chemiluminescence reagent (Beiyuntian Company), and put it into a cassette for exposure.

Example 2. Pathological analysis of male sterility caused by Guf1 gene knockout.

In order to further study the dysfunction caused by the loss of Guf1, we focused on the pathological study of the reproduction of Guf1 knockout mice, including the number of offspring, sperm motility, and pathological sections and transmission electron microscope analysis of testes. Figure 2A represents the analysis of the number of offspring, which shows that after male knockout mice and normal female mice were caged, there was mating behavior but no offspring were produced. Figure 2B counts the sperm counts of wild-type and Guf1-knockout mice, showing that the number of sperm in Guf1-knockout mice is significantly reduced. Figure 2C is the observation of testis morphology, and it was found that the testis morphology of Guf1 knockout mice became smaller. Figure 2D shows the morphology of sperm observed under a confocal fluorescence microscope. It was found that the mitochondrial sheath of the knockout sperm was missing, bent, and abnormal mitochondria.

specific method:

Analysis of male reproductive ability: 20 wild-type and Guf1 knockout mice aged 6-8 weeks were taken respectively, and each mouse was co-caged with two CD1 female mice aged 6-7 weeks. The mice with vaginal plugs were taken out and raised separately, and the number of offspring was counted. It was found that 40 female mice mated with wild-type mice all had plugs, only two had no offspring, and the rest gave birth to 4-12 pups. In contrast, 40 CD1 female mice mated with knockout mice had no offspring.

Sperm count statistics: 6-8 weeks old wild and knockout mice (n>3) were killed by neck dislocation, the epididymis was taken out with scissors, put into a plate filled with phosphate buffered saline (PBS), and injected with a syringe. The epididymis was shattered and counted on a blood cell count.

Observation of sperm morphology by confocal fluorescence microscope: 6-8 weeks old wild and knockout mice (n>3) were killed by neck dislocation, the epididymis was taken out with scissors, the epididymis was crushed with a syringe, Hoechest and Mitotracker (invitrogen) were washed with PBS 1:10000 dilution, spermatozoa were observed by confocal fluorescence microscope.

Example 3. Guf1 knockout increases sperm cell apoptosis and affects spermatogenesis.

In order to study the effect of Guf1 on the process of mouse spermatogenesis, we stained the reproductive organs of wild-type and Guf1-knockout male mice, including testes and epididymis, and analyzed sperm motility. Figure 3A, C are the H&E staining of the knockout epididymis and testis, the results show that there are a large number of immature sperm cells in the knockout mouse epididymis. Figure 3B is TUNEL detection of apoptosis in testis and epididymis, indicating that Guf1 knockout will produce obvious apoptosis. Figure 3D and E are the analysis of sperm motility by computer assisted sperm analyzer (CASA), and the results show that the sperm motility of knockout mice is significantly reduced. Through the above pathological analysis of Guf1 knockout, it was found that Guf1 knockout would cause spermatogenesis disorder and lead to male sterility.

specific method:

H&E staining of epididymis and testis:

Paraffin section: Remove the testis and epididymis tissues of wild-type and Guf1 knockout mice, and fix them in 4% perfluoroalkoxy (Polyfluoroalkoxy, PFA) to prevent autolysis or bacterial decomposition after cell death. In order to maintain the original shape and structure of the cells.

Dehydration and transparency: Use ethanol from low concentration to high concentration as dehydrating agent to gradually remove the water in the tissue. Then put it in transparent agent xylene to make it transparent.

Wax-immersion embedding: Pour melted paraffin into the small box, put the tissue in it, and let it cool and solidify.

Slicing and patching: put the embedded wax block in a microtome, cut into thin slices, 5-8μm, put the thin slices in hot water at 42 degrees to spread the slices, paste them on glass slides, and put them in a constant temperature box for drying Dry.

Dewaxing staining: Before staining, put the dried paraffin sections in xylene to deparaffinize, then pass through high-concentration to low-concentration ethanol, and finally add distilled water. The slices were stained in hematoxylin aqueous solution for several minutes, rinsed with running water for 1 hour, added distilled water for a while, and dehydrated in 70% and 90% alcohol for 10 minutes each. Add eosin staining solution and stain for 2-3 minutes.

Dehydration and transparency: After staining, the sections were dehydrated with different concentrations of ethanol, and then xylene was used to make the sections transparent.

Mounting: Drop the transparent slices with gum and cover with a cover slip for mounting.

TUNEL cell apoptosis detection:

Paraffin sections of the testis and epididymis were made, and the sections were dipped in xylene twice for 5 minutes each time. The transparent slices were dipped in graded ethanol (100%, 95%, 90%, 80%, 70%) for 3 minutes each time. The slides were washed twice with PBS. Treat tissue with proteinase K working solution for 20 minutes. Then washed twice with PBS. Prepare TUNEL reaction mixture, mix 50μl TdT+450μl fluorescein-labeled dUTP solution. Add 50 μl TUNEL reaction mixture after the slides are dry, and react in a dark and humid box at 37°C for 1 hour. Rinse with PBS 3 times. Photograph.

Computer-assisted sperm analysis (CASA) to evaluate various sperm movement parameters: take 4 wild-type mice and 4 knock-out mice aged 6-8 weeks, take out the epididymis, put the sperm in a 37-degree water bath, and put the counting pool in the analyzer to maintain a constant temperature Pre-warm on the plate, after the sperm sample is liquefied, take 1 drop of the mixed sample semen and drop it in the middle of the sample pool, and then analyze it. Using computer-aided semen analysis software of Chinese Academy of Sciences (Institute of Zoology, Chinese Academy of Sciences), the main parameters of semen, namely sperm motility, and sperm average curve velocity (VCL), average linear velocity (VSL), average path velocity (VAP), average Whiplash frequency (BCF) etc. were analyzed.

Example 4. Transmission electron microscope observation of testes and sperm mitochondria of Guf1 knockout mice.

Previous studies have shown that Guf1 is a mitochondria-localized protein. In order to study the effect of Guf1 protein on mitochondrial function, we studied the mitochondrial structure and function of the reproductive organs of Guf1 knockout mice. Figure 4 shows the effect of Guf1 knockout on the mitochondrial morphology of mouse testis and sperm. Figure 4A and C represent the transmission electron microscope of Guf1 protein-knockout spermatogonia and spermatocytes, respectively, and the results show that the mitochondrial morphology of spermatocytes in Guf1 knockout mice changes during development. Figure 4B and D represent the sperm mitochondrial sheath of Guf1-knockout mice. The results show that the absence of Guf1 can cause significant changes in the morphology of sperm mitochondria, which is reflected in the irregular arrangement of mitochondria in the sperm mitochondrial sheath and defects in the inner ridge.

specific method:

Transmission electron microscopy of testis and epididymis: testes and epididymis were removed from wild and knockout mice, and fixed in osmium tetroxide for 1-2 hours. After fixation, rinse with buffer for 20 minutes and then dehydrate. The dehydration gradient is as follows: 30% acetone, 50% acetone, 70% acetone, 80% acetone, 90% acetone, each step acts for 30 minutes. Pure acetone was applied 3 times, 30 minutes each time. The samples were infiltrated and made ultra-thin sections, and finally stained, first stained with lead citrate for 10 minutes, washed 3 times with double distilled water to remove carbon dioxide, then stained with uranyl acetate for 30 minutes, washed 3 times with double distilled water. After the ultra-thin sections are dry, they can be observed with a transmission electron microscope Tecnai Spirit (120KV).

Example 5. Effect of Guf1 protein on the function of mitochondrial respiratory chain complex.

In order to further study the effect of Guf1 on mitochondrial function, we studied the mitochondrial oxidative phosphorylation system, while using normal tissue heart as a control. Figure 3A represents the ATP levels of wild-type and knockout testis and epididymis, and the results show that testis and epididymis ATP levels were decreased in the case of Guf1 knockout. Figure 3B represents the activity of mitochondrial cytochrome c oxidase, and the results show that the activity of cytochrome c oxidase is reduced in Guf1 knockout testis and epididymis. Figure 3C represents the Blue native PAGE analysis of the complexes of the mitochondrial respiratory chain, and the results showed that the amount of cytochrome c oxidase was significantly reduced in the knockout testis and epididymis. Figure 3D represents the BNG staining of cytochrome c oxidase, which shows that the activity of cytochrome c oxidase is significantly reduced. Figure 3E represents the activity staining of complex I, complex II and complex V in mitochondria, and the results showed that the activity of complex I, V, II did not change significantly.

specific method:

Determination of ATP level: the testes and epididymis of 6-8 week old wild-type and knockout mice were washed three times with PBS. Remove the tunica from the testis, chop the testis and epididymis with a sterilized blade, and digest with 0.25% trypsin, 5 minutes each time, 15 minutes in total. Single cells were obtained. After the cells were lysed, they were centrifuged at 13,000 rpm for 10 minutes, and the protein concentration of the supernatant was determined with a BCA kit. Take 5 μl of sample and add 50 μl of ATP detection buffer, and detect with a chemiluminescent microplate reader (Perkin Elmer, USA) detector.

Blue Native PAGE: According to the Mitochondria Isolation Kit (Abcam), mouse heart and testicular mitochondria were separated, and the BCA kit was used to determine the protein concentration. Centrifuge 400 μg mitochondria at 12000 rpm for 5 minutes, add 40 μl buffer A (50 mM sodium chloride, 50 mM imidazole/hydrochloric acid, 1 mM EDTA, pH 7.0) to resuspend. Add 12μl Digitonin to each tube, mix well and act on ice for 10 minutes. Centrifuge at 100,000g at 4°C for 30 minutes, take the supernatant, add 5 μl glycerol and 6 μl Coomassie brilliant blue. Take 20 μl samples for Blue-native PAGE to separate mitochondrial complexes, see the reference (Ilka Wittig et al., nature 2006) for the method.

Complex I activity: Incubate the Blue-native PAGE separation gel in 50mM Tris-Hcl, pH 7.4 buffer (containing 0.5mM NBT (Nitroblue tetrazolium chloride, nitrotetrazolium chloride), 5mM NADH (Nicotinamide adenine dinucleotide, Nicotinamide adenine dinucleotide)) at room temperature for 1 hour.

Complex II activity: Incubate the Blue-native PAGE separation gel in 50mM Tris-Hcl, pH 7.4 buffer (0.2mM PMS (Methylphenaziniummethylsulfate, phenazine methylsulfate), 84mM succinic acid, 50mM NBT), room temperature for 1 Hour.

Complex IV activity: incubate Blue-native PAGE gel in 50mM Tris-Hcl, pH 7.4 buffer (containing 0.1% diaminobenzidine, 24 units/ml catalase, 0.1% cytochrome c), 37 degrees for 3-6 hours.

Complex V activity: Rinse the Blue-native PAGE separation gel with water for 10 minutes, and put it into 0.1M glycine buffer (pH 8.6) for 1 hour. Then put the separating gel into the buffer containing the following components: 35mM Tris base, 270mM glycine, 14mM magnesium sulfate, 5mMATP, 0.2% silver nitrate, and act for 3-6 hours at 37 degrees.

Example 6. Effect of Guf1 knockout on mitochondrial respiratory chain complex subunit proteins.

The above studies have shown that Guf1 knockout can cause a significant decrease in the activity of cytochrome c oxidase in the electron transport chain of the mouse testis and epididymis, so we studied the amount of each complex subunit of the mitochondrial electron transport chain, and at the same time, the normal tissue heart was used as a control . Panel A shows the western blot results of each subprotein of complex I-V in the heart, testis and epididymis, and the results show that Cox IV (encoded by nuclear genes), Cox1 and Cox2 (encoded by mitochondrial genome) in complex IV in testis and epididymis of knockout mice coding) is significantly reduced. The amount of ND2 was reduced in complex I without any change in the amount of NDUFA9. The amount of cytochrome b (CYTB) in complex III was reduced in knockout testes, whereas the amount of nuclear-encoded Core2 was unchanged. The amount of 70kd Fp in complex II was unchanged. There was also no change in the amount of ATP5A in complex V. Panel B is the result of Blue native western blot, which further shows that the amount of mitochondrial complex IV is reduced. Figure C is the result of Blue-native 2D, which shows that the amount of each subprotein of complex IV is more significantly reduced than that of other complexes.

specific method:

Western blot: 6-8 week old wild-type and Guf1 knockout mice were killed by neck dislocation, and the mouse heart, testis and epididymis tissues were taken out and washed with PBS. Add 1ml of RIPA lysate and protease inhibitor cocktail (Roche) at the same time, grind in a tissue grinder, place the homogenate on ice for 30 minutes, centrifuge at 12000rpm for 30 minutes, and use the BCA kit to measure the protein concentration of the supernatant.

Prepare 15% SDS-PAGE gel, add a certain volume of protein loading buffer to the sample whose protein concentration has been measured, heat at 95 degrees for 15 minutes, take a certain amount of protein and add it to 15% SDS-PAGE gel, 100v constant Pressure, electrophoresis about 3 hours.

The protein samples on the gel were transferred to PVDF membrane, blocked with 5% skimmed milk powder for 1 hour, and then diluted with different primary antibodies (Abcam) according to the instructions, and incubated overnight. Wash the membrane 5 times with TBST (150mM NaCl, 20mM Tris-HCl, pH7.4, Tween-20 0.05%), 5 minutes each time. The secondary antibody was diluted 1:3000 and incubated for 1.5 hours. After washing the membrane three times with TBST, an ultra-sensitive ECL chemiluminescent reagent (Biyuntian Company) was added and placed in a cassette for exposure.

Blue-native 2D: First, perform Blue native PAGE electrophoresis according to the above experimental method, then fix the gel in fixative solution for 1 hour, and stain with Coomassie brilliant blue for more than 2 hours. The gel was excised and placed in 1% mercaptoethanol (trace bromophenol blue) for 1 hour. Prepare 10% Tricine-SDS-PAGE, put the gel in the stacking gel of Tricine-SDS-PAGE, constant current 20mA, after electrophoresis, Coomassie brilliant blue staining and decolorization.

Example 7. Guf1 knockout causes decreased cytoplasmic translation and mTOR signaling pathway.

In order to study the function of Guf1 gene in eukaryotic cells, we studied the changes of signaling pathway and cytoplasmic translation level caused by Guf1 to reveal the significance of Guf1 in vivo. Figure 4A is the result of the phosphorylation microarray of the testis, the results show that Guf1 is mainly related to cancer, and the expression of proteins related to the mTOR signaling pathway is reduced. Figure 4B shows the expression of mTOR signaling pathway-related proteins verified by western blot. The results show that the phosphorylation level of Thr37/46 of the substrate 4E-BP downstream of mTOR decreases, which is consistent with the chip results. Figure 4C shows the observation of cytoplasmic translation level by cytoplasmic ribosome display technology, and it was found that the cytoplasmic translation of knockout mice was reduced.

specific method:

Western blot: 6-8 week-old wild-type and Guf1 knockout mice were killed by neck dislocation, and the heart and testis tissues of the mice were taken out and rinsed with PBS. Add 1ml of RIPA lysate, add protease inhibitor cocktail (Roche) and phosphatase inhibitor (Roche), put it into a tissue grinder for grinding, place the homogenate on ice for 30 minutes, centrifuge at 12000rpm for 30 minutes, and measure the supernatant with a BCA kit protein concentration.

Prepare 15% SDS-PAGE gel, add a certain volume of protein loading buffer to the sample whose protein concentration has been measured, heat at 95 degrees for 15 minutes, take a certain amount of protein and add it to 15% SDS-PAGE gel, 100v constant Pressure, electrophoresis about 3 hours.

The protein samples on the gel were transferred to PVDF membrane, blocked with 5% skimmed milk powder for 1 hour, and then different primary antibodies (Cell signal technology) were diluted according to the instructions and incubated overnight. After washing the membrane 5 times with TBST (150mM NaCl, 20mM Tris-HCl, pH7.4, Tween-20 0.05%), the secondary antibody (labeled with horseradish peroxidase) was diluted 1:3000 and incubated for 1.5 hours. After washing the membrane 5 times with TBST, the supersensitive ECL chemiluminescent reagent (Biyuntian Company) was added, and the membrane was placed in a cassette for exposure.

Cytoplasmic ribosome display technology:

To prepare TMK Lysis Buffer:

10mM Tris-Cl, pH 7.4: 1ml of 1M Tris-Cl

10mM MgCl: 0.5ml of 1M MgCl

100mM KCl: 10ml of 1M KCl

1% (v/v) Triton X-100: 1ml

0.5% (w/v) sodium deoxycholate: 0.5g

1U/ml RNase inhibitor: 2.5μl 40U/μl

2mM DTT: 200 μl of 1M DTT

Cycloheximide: 0.1mg/ml

Add DEPC- _H2O to 100ml.

Prepare 10%-50% sucrose: prepare with DEPC water at 4 degrees.

Preparation of 10% (w/v) sucrose solution (200ml):

Sucrose: 20g

100mM KCl: 20ml of 1M KCl

5mM MgCl: 1ml MgCl

2mM DTT: 400 μl of 1M DTT

20mM HEPES, pH 7.4: 4ml of 1M HEPES

Preparation of 50% (w/v) sucrose solution (200ml):

Sucrose: 100g

100mM KCl: 20ml of 1M KCl

5mM magnesium chloride: 1ml of magnesium chloride

2mM DTT: 400 μl of 1M DTT

20mM HEPES, pH 7.4: 4ml of 1M HEPES

Add 2 μl (40 U/μl) RNase inhibitor and 50 μl cycloheximide (100 μg/μl) in 50 ml sucrose just before use. Put it in the gradient mixer to prepare the gradient.

Wild-type and Guf1-knockout mice were killed by neck dislocation, and the heart and testis were immediately taken out and frozen in liquid nitrogen, ground into powder in a mortar without RNase, poured into EP tubes, added TMK lysate, and lysed on ice 5 minutes. Centrifuge at 13000g for 20 minutes. The supernatant was plated in a 10%-50% sucrose gradient. Centrifuge at 26000 rpm for 4 hours. Nucleic acid detector A260 detection.

Example 8. Effect of Guf1 protein knockout on mitochondrial copy, transcription and translation.

Studies in yeast have shown that Guf1 is an important mitochondrial protein translation factor. To further study the effect of Guf1 on mitochondrial protein synthesis in eukaryotic cells. We did the following research. Figure A is to measure the copy number of mitochondrial DNA in the heart, testis and epididymis of wild-type and Guf1 knockout mice, and the results show that the copy number of mitochondrial DNA has increased. Figures C and E represent the mRNA levels of mitochondrial complex proteins, where C is the result of testis, and Figure E is the result of heart. The results show that the mRNA levels of mitochondrial complex proteins have no difference in wild-type and knockout heart and testis Clearly, Guf1 knockdown did not affect mitochondrial DNA transcription. Panel B shows the changes in the amount of the large mitochondrial ribosomal subunit L11 and the small ribosomal subunit S18, and the results show that the amounts of L11 and S18 in the knockout testis were slightly increased. Panel D is the in vitro translation of mitochondrial proteins, and the results show that in Guf1 knockout testes, the amount of mitochondrial protein translation in vitro increases.

specific method:

Determination of mitochondrial DNA copy number:

The heart and testis DNA of wild-type and Guf1 knockout mice were extracted, and the primers of cox1 gene encoded by mitochondrial DNA (mtDNA) and ndufv1 gene encoded by nuclear DNA (nDNA) were synthesized. The cox1 and ndufv genes were amplified by qPCR using genomic DNA from the heart and testis as templates. The ratio of cox1 and ndufv represents mtDNA/nDNA.

Mitochondrial complex protein qPCR: Trizol reagent was used to extract the total RNA of heart and testis of wild-type and Guf1 knockout mice, using 2 μg of total RNA as template, using Molony murine leukemia virus reverse transcriptase (Molony murine leukemia virus reverse transcriptase, MoMuLVRT) as a template and polythymine (oligo(dT)) as a primer to synthesize cDNA. Refer to the literature to synthesize 16S rRNA, 12S rRNA, ND1, ND6, NDUFS3, NDUFS7, NDUFB6, NDUFA9 (NADH dehydrogenase subunit ), Cox1, Cox2 (cytochrome oxidase subunit), CytB (cytochrome reductase subunit, cytochrome b), ATP6 (ATP synthase subunit), EF-Tu (elongation factor TU) primers. RT-PCR reaction was carried out in ABIStepOne plus quantitative PCR instrument with SYBR Green Master Mix (Takara, Japan) reagent.

Mitochondrial protein translation in vitro: mitochondrial isolation kit (Abcam) was used to extract heart and testicular mitochondria of wild-type and Guf1 knockout mice, and BCA kit was used to measure mitochondrial protein concentration. 100μg mitochondria were centrifuged at 12000g, and the pellet was reacted with reaction buffer at 37°C for 1 hour. The reaction buffer composition is as follows: 25mM sucrose, 75mM sorbitol, 1mM magnesium chloride, 0.05mM EDTA, 10mM Tris-HCl, 10mM dipotassium hydrogen phosphate, 10mM glutamate, 2.5mM malic acid, 1mM ADP, 1mg/ml defatted bovine serum Albumin, 100 μg/ml ipeacin, 100 μg/ml cycloheximide, 0.2 mM amino acid mixture, 1 unit of RNase inhibitor.

After the reaction was completed, the sample was centrifuged at 12000g for 5 minutes, washed 3 times with cold buffer (320mM sucrose; 1mM EDTA; 10mM Tris-HCl, pH7.4), and 25μl of 1x protein loading buffer was added to the precipitate, and heated at 95°C for 10 minutes. Perform 15%-20% SDS-PAGE electrophoresis. Make dry glue, press phosphor screen.

Example 9, Preservation of Human Sperm and Extraction of Genomic DNA

Take 400 microliters of sperm from infertile patients (Tianjin General Hospital) to extract the genome, and then perform sequencing. Figure 9A shows the extracted patient genomes numbered 1, 2, and 3, and the results show that the extraction was successful. The above DNA was sequenced, and FIG. 9B shows the sequencing results, and the results showed that there was a deletion of two bases (TC) at the 5' untranslated end of the human guf1 gene with reproductive abnormalities. The experimental program we adopted can successfully obtain the DNA sequence of the sperm exon, and analyze the mutation site by comparison, so it can become a basis for making a kit.

specific method:

Sampling: Aqueous solution at room temperature (or liquefaction in a water bath at 37°C)

Freeze:

Mix the liquefied sperm and cryopreservation solution at a ratio of 1:1 and add slowly drop by drop, mixing evenly while adding; freeze at 4°C for 30 minutes, and store in liquid nitrogen for 15 minutes.

Sperm DNA Extraction:

1. Take 200μl sperm + 200μl Buffer X2 in a 1.5ml centrifuge tube, put it in a 55°C water bath for at least 1 hour (1.5 hours), and mix it upside down every 10 minutes;

2. Add 400 μl of Buffer AL (a component of the QIAGEN DNA extraction kit) (precipitation may occur, it can be dissolved at 70°C) and vortex for 15 seconds, incubate at 70°C for 10 minutes, centrifuge briefly, and remove the water droplets from the lid;

3. Add 400 μl of ethanol (95%-100%), vortex and mix for 15 seconds, and simply dry the water droplets on the lid (precipitation may occur, which has no effect on the results);

4. Add the mixture in step 3 to the reaction column, centrifuge at 8000rpm for 1 minute, and replace the column in a new 2ml collection tube (if it is not clean, centrifuge again);

5. Add 500μl AW1 (the component in the QIAGEN DNA extraction kit), centrifuge at 8000rpm for 1 minute, and replace the column in a new collection tube;

6. Add 500μl AW2 (component in QIAGEN DNA extraction kit), centrifuge at 14000rpm for 3 minutes;

7. Replace the collection tube with a new one, and centrifuge the empty column at 14000rpm for 1 minute;

8. Change to a new 1.5ml centrifuge tube, add 100μl Buffer AE (a component in the QIAGEN DNA extraction kit) or distilled water, place at room temperature for 1 minute, 8000rpm, and centrifuge for 1 minute (here, AE should be added in several times to ensure that the column sufficient DNA elution).

Table 1: Primer list for exon sequencing of human guf1 gene

Primer ID Primer sequence Sequencing exons 1F ACTGACAGTACACACACCACAG exon 1 1R AAGTTAGTGACTTGCCCAAGG exon 1 2F CTGATTGGCCCTAAAAATTG 2nd to 4th exons 2R ACTAGTCAATTCCAGAGAATGC 2nd to 4th exons 3F TGACCCCAAAAGACATAAGTTG Exon 5 3R GTGAGTTTCAGTTGCACTTCTG Exon 5 4F TATTGTAGAGGGGTCAGTTAGG exon 6 4R AGTGATAAAACCTTTGAGCCTC exon 6 5F TACCCAGATTTCGAACTATTCC 7th to 8th exons 5R TCAACTGACGTTTCTATTGTCC 7th to 8th exons 6F TAAGACCTGGTGTGTTCATTTG exon 9 6R AACACATCCTAAATGATCTCCC exon 9 7F TTGGAGTGCAAATCTAGTTG Exons 10 and 11 7R_1 AATCCGCAACGTGAAACAG Exon 10 7R_2 GTTCTTCATCATTAGCCATAGG exon 11 8F CATGTAAGTGGCAATCTGTTC exon 12 8R CTTTCCTTTGGGCTAACATATC exon 12

9F TTTGGTAGCAAGCCTAATGC exon 13 9R TGTTCCAAGTCATTCTAACC exon 13 10F GATGGAAACATATCTCTTTGGG Exon 14 10R GGTGAAGGGAAACATTTCTATG Exon 14 11F TCCATTTGAACCTCCTTAAGAG Exon 15 11R AAACTGGAGAGCACCATCATAC Exon 15 12F CAAGCCTTGACCATTTTTTC exon 16 12R CATGGTCAAGCTGAAATTTACC exon 16 13F CTCCATCCAGATTAAATCCTG Exon 17 13R CATGGCTTCAAAATCTAAGCTC Exon 17

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Claims (8)

1. obtain an inhuman male sterile mammiferous method, described method comprises cultivate that Guf1 gene is knocked described mammiferous male.

2. method according to claim 1, wherein said Mammals is mouse, and the sequence of described Guf1 gene is as shown in SEQ ID NO:1.

3. cause the method that inhuman boar is sterile, described method comprises the Guf1 abnormal gene expression making described boar.

4. method according to claim 3, wherein said Mammals is mouse, and the sequence of described Guf1 gene is as shown in SEQ ID NO:1.

5.Guf1 gene is as the purposes of the target spot causing inhuman boar sterile.

6. purposes according to claim 5, wherein said Mammals is mouse, and the sequence of described Guf1 gene is as shown in SEQ ID NO:1.

7. for the reagent that detects mammiferous Guf1 abnormal gene expression for the preparation of the purposes detected in described mammiferous male sterile diagnostic reagent caused by Guf1 abnormal gene expression.

8., for diagnosing the sterile test kit of boar caused by Guf1 abnormal gene expression, described test kit comprises the reagent for detecting described mammiferous Guf1 gene unconventionality.