CN102533777A - Dairy goat Boule gene and application of dairy goat Boule gene in promoting meiosis of male germ cells - Google Patents
Dairy goat Boule gene and application of dairy goat Boule gene in promoting meiosis of male germ cells Download PDFInfo
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Abstract
The invention discloses a dairy goat Boule gene and application of the dairy goat Boule gene in promoting meiosis of male germ cells. The CDS (coding sequence) of the gene (SEQ.ID.NO.1) is shown as SEQ.ID.NO.2. An expression vector constructed based on the dairy goat Boule gene sequence comprises a Boule gene sequence shown as SEQ.ID.NO.1, a fluorescence labeled gene GFP (green fluorescent protein) is linked downstream of the Boule gene sequence, and an antibiotic screening gene kanr/neor is linked downstream of the GFP. The dairy goat Boule gene sequence is transfected into the male germ cells to promote the meiosis of the male germ cells.
Description
Technical field
The invention belongs to biological technical field, relate to sexual cell reduction division genes involved, particularly a kind of milk goat Boule gene and the maiotic application of short male sex-cell thereof.
Background technology
Mammiferous spermatogeny can be divided into sperma-togonium A propagation and sperma-togonium B is divided into primary spermatocyte, primary spermatocyte forms round spermatid through reduction division and spermatid changes 3 main phase such as The mature sperm into, and wherein reduction division is the committed step in the spermatogeny process.Reduction division is a kind of divisional mode that chromosome number reduces by half in the sexual cell, and in the reduction division process, karyomit(e) only duplicates once, and cell divides twice continuously.Reduction division guaranteeing that the species chromosome number plays a very important role in stable, also is simultaneously a kind of mechanism that species conform and constantly change not only.Boule is the newcomer of DAZ family; Be the maiotic crucial regulatory factor of human sperm's generating process, specific expressed at testis tissue, Boule gene and Boule albumen respectively at 1996 with calendar year 2001 (the Eberhart C G that successively comes to light; Maines J Z; Wasserman S A.Meiotic cell cyclerequirement for a fly homologue of human deleted in Azoospermia [J] .Nature, 1996,381 (6585): 783-785; Xu E Y; Moore F L; Reijo R A.A gene familyrequired for human germ cell development evolved from an ancient meiotic geneconserved in metazoans [J] .Proc Natl Acad Sci USA; 2001,98 (13): 7414-7419).
People's Boule albumen early start is expressed in the spermatocyte of zygotene stage; And up to spherical sperm occurring expression is arranged all in the spermatogeny process; It can pass through to combine 3 ' UTR and then the dephosphorylation Cdc2 of Cdc25A, thereby activates MPF, starts reduction division (Luetjens CM; Xu EY; Et al.Association of meiotic arrest with lack of BOULE protein expression in infertilemen [J] .The Journal Clinical Endocrinology, 2004,89 (4): 1926-1933; Lin YM; Chung CL; And Cheng YS.Posttranscriptional Regulation of CDC25A by BOLLIs a conserved fertility mechanism essential for human spermatogenesis.Endocrine Research; 2009,94 (7): 2650-2657.).For male sex-cell; The shortage of the reduction of BOULE gene expression dose or shortage, BOULE protein expression all can cause reduction division retardance (meiotic arrest; And then cause azoospermia or aspermia or oligospermia and produce sterile (Lin Y M, Kuo P L MA) and the spermatogenesis obstacle; Lin Y H; Et al.Messenger RNA transcripts of the meionticregulator BOULe in the testis of azoospermic men and their application inpredicting the success of sperm retrieval [J] .Hum Reprod, 2005,20 (3): 782-788.).
Stem cell is one type and has self, the highly propagation and the cell colony of multidirectional differentiation potential.Existing research confirms that stem cell can be an early germ in vitro differentiation; Even further be divided into ovocyte or sperm (H ü bner K; Fuhrmann G, Christenson LK, et al.Derivation ofoocytes from mouse embryonic stem cells [J] .Science; 2003,300:1251-1256; Geijsen N, Horoschak M, Kim K, et al.Derivation of embryonic germ cells andmale gametes from embryonic stem cells [J] .Nature, 2004,427:148-154.).And this process of stem cell experience just must experience reduction division, and it is most important to it how to start reduction division, discovers that overexpression Boule can promote embryonic stem cell to break up to sexual cell with the gene Dazl of family; (Yu Z, Ji P, Cao J; Et al.Dazl promotes germ cell differentiation fromembryonic stem cells [J] .J Mol Cell Biol; 2009,1:93-103.Kee K, Angeles VT; Flores M et al.Human DAZL; DAZ and BOULE genes modulate primordialgerm-cell and haploid gamete formation [J] .Nature, 2009,462:222-225.).Also have research to show certain density vitamin A acid (retinoic acid; RA) Cyp26b1 (oxydase of special metabolism RA) that can degrade; And then the expression of inducing Stra8 (stimulated by retinoic acid gene 8); Thereby start reduction division (Pellegrini M, Filipponi D, Gori M; Et al.ATRA and KLpromote differentiation toward the meiotic program of male germ cell s.CellCycle, 200 (7): 3878-3888.).Stra8 receives the regulation and control of RA, determine maiotic initial, the generation of sperm and ovum (Anderson EL; Baltus AE, Roepers-Gajadien HL, et al.; Stra8and its inducer, retinoic acid, regulate meiotic initiation in both spermatogenesisand oogenesis in mice [J] .Proc Natl Acad Sci USA; 2008,105:14976-14980.).But the research that all at present relevant stem cells break up to sexual cell, but set up the report that effectively can start reduction division and quantitatively evaluating thereof reliably really as yet.
Though set up the multiple research system that is used for sexual cell in-vitro multiplication and differentiation so far; Maiotic key gene of many regulation and control and albumen have also been found in the Knockout experiment; But can't realize setting up external reduction division and the spermatogenetic research model of carrying out fully, external very difficult control sexual cell is maiotic initial.Therefore; Through making up the carrier for expression of eukaryon of some sexual cell specificity key genes; Utilize it to modify the vitro culture sexual cell; Explore the relation between the functional gene, find the maiotic signal pathway of relevant regulation and control, and then interactional molecular mechanism has important scientific meaning between the announcement reduction division process gene.
Summary of the invention
The problem that the present invention solves is to provide a kind of milk goat Boule gene and the maiotic application of short male sex-cell thereof; External source Boule gene transfection male sex-cell can be urged it carry out reduction division, can study the regulating and controlling effect that Boule expresses the sexual cell specific gene.
The present invention realizes through following technical scheme:
A kind of milk goat Boule gene, this gene order is shown in SEQ.ID.NO.1.
A kind of milk goat Boule gene, the CDS sequence of its gene is shown in SEQ.ID.NO.2.
A kind of milk goat Boule gene Fusion gene connects the fluorescence report gene in the downstream of milk goat Boule gene.
Described milk goat Boule gene Fusion gene also connects the antibiotic-screening gene in the downstream of fluorescent mark gene.
A kind of expression vector that makes up based on milk goat Boule gene order comprises the Boule gene order shown in the SEQ.ID.NO.2, connects fluorescent mark gene GFP in the downstream of Boule gene order, connects antibiotic-screening gene kan in the downstream of GFP
r/ neo
r
The described expression vector that makes up based on the Boule gene order is the pIRES2-Boule-EGFP expression vector, through Ecor I and Sal I restriction enzyme site the pIRES2-EGFP expression vector is arrived in the Boule gene clone.
The transfection of milk goat Boule gene order in the male sex-cell to promote the maiotic application of male sex-cell.
The maiotic method of a kind of promotion male sex-cell may further comprise the steps:
1) the milk goat Boule gene order of clone shown in SEQ.ID.NO.2, and the Boule gene order is cloned in the pIRES2-EGFP expression vector through Ecor I and Sal I restriction enzyme site, the pIRES2-Boule-EGFP expression vector obtained;
2) will treat that the male sex-cell of transfection and pIRES2-Boule-EGFP expression vector hatch jointly, with the transfection of pIRES2-Boule-EGFP expression vector among male sex-cell;
3～4h after the transfection male sex-cell is added the foetal calf serum of its volume 10% and 1% non-essential amino acid on minimum medium, and the L-glutaminate of the beta-mercaptoethanol of 0.1mmol/L and 2mmol/L is cultivated; Described basic culture solution is DM/F12 nutrient solution or H-DMEM nutrient solution;
And under fluorescent microscope, the fluorescent mark gene is observed, screening reduction division starts the cell that the related gene expression level increases.
Describedly treat that the male sex-cell of transfection is that mouse propagation cell is GC1 or people's a 293T cell, the basic culture solution that is adopted is the H-DMEM nutrient solution;
Describedly treat that the male sex-cell of transfection is the milk goat male sex-cell, the basic culture solution that is adopted is the DM/F12 nutrient solution.
Compared with prior art, the present invention has following beneficial technical effects:
1, the present invention has cloned the Boule gene of milk goat, and its CD district size is 816bp (SEQ.ID.NO.2), reaches more than 90% in the conservative property of its CDS sequence; This gene can promote male sex-cell reduction division.
2, the present invention has made up the pIRES2-Boule-EGFP recombinant vectors on the basis of the Boule gene of clone milk goat, utilizes pIRES2-Boule-EGFP recombinant vectors transfectional cell, after the transfection, prolongs in time, and the cell count of expressing GFP increases.
Behind the pIRES2-Boule-EGFP transfection GC1, noticeable change does not take place in cellular form, but the GFP positive cell rate is significantly higher than control group.Behind the pIRES2-Boule-EGFP transfection 293T, noticeable change does not take place in cellular form yet, but the expression level of reproducibility marker gene slightly raises, and shows that cell has the maiotic trend of entering; Behind the transfection milk goat male sex-cell, noticeable change does not take place in cellular form, starts reduction division Expression of Related Genes level and significantly increases.Through with pIRES2-Boule-EGFP with have the luciferase reporting carrier cotransfection 293T cell of Stra8 gene 3 ' UTR; Find that fluorescence intensity has the growth about 40% than control group; Explain that the Boule gene may directly act on reduction division specific gene Stra8, and then promote that mammalian cell is maiotic initial.
Description of drawings
Fig. 1 is the agarose gel electrophoresis figure of the Boule gene of pcr amplification;
Fig. 2 is that Ecor I and Sal I double digestion are identified positive recombinant plasmid pIRES2-Boule-EGFP;
Fig. 3 is the more constructed systematic evolution trees of species sequence such as milk goat Boule gene C DS sequence and people, ox, Henghe macaque, dog, pig, goat;
Fig. 4 is the expression of fluorescence microscope recombinant plasmid pIRES2-Boule-EGFP at the GC1 cell;
Fig. 5 is the expression of fluorescence microscope recombinant plasmid pIRES2-Boule-EGFP at the 293T cell;
Fig. 6 is the expression of fluorescence microscope recombinant plasmid pIRES2-Boule-EGFP at the milk goat male sex-cell;
Fig. 7 is sexual cell specific marker expression of gene result behind the milk goat male sex-cell transfection pIRES2-Boule-EGFP 48h;
Fig. 8 be 293T cell cotransfection pIRES2-Boule-EGFP with the luciferase reporting carrier 48h of band Stra8 gene 3 ' UTR after fluorescence intensity figure as a result.
Embodiment
Below in conjunction with concrete embodiment the present invention is done further detailed description, said is to explanation of the present invention rather than qualification.
1, the clone of milk goat Boule gene and vector construction
At first extract the total RNA of milk goat testis tissue, the reverse transcription test kit obtains cDNA, as the template of amplification;
According to ox b-Boule gene cDNA complete sequence (NM_001102115) in the GenBank DB, utilize Primer Premier 5.0 primer-design software design of amplification primers, and select suitable restriction enzyme, the following primer of concrete design:
Upstream primer: tc
GaattcGa aagtctccgt cgggaagcgt
Downstream primer: ct
GtcgacGg cagcttctag ccggttcatt g
The line part is an Ecor I restriction enzyme site in the upstream primer, and underscore partly is the SalI restriction enzyme site in the downstream primer;
Boule gene PCR amplification reaction system is 15 μ L, comprising: 10 * buffer, 1.5 μ L, MgCl
2(25mmol/L) 1.6 μ L, dNTP (2.5mmol/L) 1.2 μ L, Taq archaeal dna polymerase 0.1 μ L, each 0.3 μ L of upstream and downstream primer (10 μ mol/L), cDNA template 0.5 μ L, ddH
2O 9.5 μ L.
Response procedures: 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 90s, and totally 35 circulations are mended extension 10min, 4 ℃ of preservations for last 72 ℃.
Reclaim and purified pcr product, product locates to occur the specific amplification band through 1% agarose gel electrophoresis at about 1091bp (detected result is as shown in Figure 1), obtains the Boule gene of milk goat;
Then PCR product, pIRES2-EGFP carrier for expression of eukaryon (Addgene) are connected with behind EcorI and the Sal I double digestion respectively, the pIRES2-EGFP carrier for expression of eukaryon are gone in the Boule gene clone, construction recombination plasmid pIRES2-Boule-EGFP.
Recombinant vectors pIRES2-Boule-EGFP is carried out Ecor I and the evaluation of Sal I double digestion; Product is through 1% agarose gel electrophoresis; The double digestion qualification result is as shown in Figure 2; At 1091bp and 5300bp place the purpose band appears, remaining pIRES2-Boule-EGFP carrier framework after Boule gene target fragment of also promptly being cloned into and enzyme are cut.
Cut the order-checking of checking back through a plurality of mono-clonal enzymes of picking, size is consistent with sequence, obtains the Boule gene order after the order-checking, and nucleotide sequence is shown in SEQ.ID.NO.1.
With the CDS sequence in the milk goat Boule gene; (the CDS sequence is the sequence of one section protein product of coding shown in SEQ.ID.NO.2; The height in CDS district is similar between each species; Just mean that also the amino acid that albumen constituted that they translate is more similar, the function of performance is also more similar, and promptly proteic function is more conservative).Compare with the Boule gene C DS sequence of species such as the last people who announces of GenBank, ox, Henghe macaque, dog, pig, goat, compare with DNAMAN software, the constructing system evolutionary tree, the result is as shown in Figure 3.Bioinformatic analysis shows that the CDS sequence of Boule gene at a plurality of species high conservatives, that is to say that the function of Boule albumen in each species is comparatively conservative.
2, the observation of cell after the transfection of recombinant plasmid and the transfection
GC1 cell, 293T cell and the milk goat male sex-cell that will be in logarithmic phase are blown and beaten into single cell suspension with 0.25% tryptic digestion, are inoculated in 24 well culture plates that are covered with gelatin.Culturing cell is to about 70% fusion, according to the TurboFect of Fermentas company
TMThe transfection specification sheets carries out the transfection of pIRES2-Boule-EGFP recombinant plasmid.Be specially:
2 μ g plasmids are added in the 300 μ l opti-MEM nutrient solutions, add 4 μ l TurboFect transfection reagents again, gentle mixing.Behind the incubated at room 20min, the TurboFect/DNA mixture is evenly added in the petridish.3-4h after the transfection, the substratum of GC1 and 293T are replaced by the foetal calf serum cultivation of H-DMEM+0.1mmol/L beta-mercaptoethanol+2mmol/L L-glutaminate+1% non-essential amino acid+10% volumetric concentration; The substratum of milk goat male sex-cell is replaced by the foetal calf serum of DM/F 12+0.1mmol/L beta-mercaptoethanol+2mmol/L L-glutaminate+1% non-essential amino acid+10% volumetric concentration.
After the transfection, under fluorescent microscope, all observe the expression of green fluorescent protein (GFP) in GC1,293T and the milk goat sexual cell.Prolong in time, the cell count of expressing GFP increases.Behind pIRES2-Boule-EGFP transfection GC1 (Fig. 4) and the 293T (Fig. 5); Noticeable change does not take place in cellular form; But the GFP positive cell number is significantly higher than control group, and sexual cell specific marker gene expression dose raises, and shows that cell has the maiotic trend of entering.
Contrast among Fig. 4,5 is respectively GC1 and the 293T of transfection pIRES2-EGFP, is respectively the observations under light field and fluorescent microscope; The figure of no phosphor dot is GC1 and the 293T that does not change among two width of cloth figure of Fig. 4,5 transfer Boule, and the figure that phosphor dot occurs is GC1 and the 293T that changes Boule over to.
Behind the pIRES2-Boule-EGFP recombinant plasmid transfection milk goat male sex-cell; The Fluirescence observation result is shown in 6; Noticeable change does not take place in cellular form yet; Contrast in the middle of Fig. 6 is the milk goat male sex-cell of transfection pIRES2-EGFP, is respectively the observations under light field and fluorescent microscope; The figure of no phosphor dot is the milk goat male sex-cell that does not change, and the figure that phosphor dot occurs is the milk goat male sex-cell that changes Boule over to.And the expression level that starts reduction division genes involved Stra8, Scp3, Vasa, Cdc25a, Cdc2, Cyp26b1 significantly increases (concrete detected result is as shown in Figure 7).
Described reduction division genes involved adopts QRT-PCR to detect, and amplification reaction system is 15 μ L: comprising: ddH
2O 6.3 μ L, 2 * SYBR, 7.5 μ L, Taq archaeal dna polymerase 0.1 μ L, each 0.3 μ L of upstream and downstream primer (10 μ mol/L), cDNA template 0.5 μ L.
The QRT-PCR response procedures is: 94 ℃ of annealing 5min, and then 40 circulations of repetition comprise 94 ℃ of sex change 20s in each circulation, and 58 ℃ are extended 30s; Then 70 ℃ of 10s begin solubility curve, and since 70 ℃, to 95 ℃ of end, each gradient is gathered first order fluorescence, time spent 10s at a distance from 0.5 ℃.
Further,, carry out two luciferase reporter genes and detect, be specially through with pIRES2-Boule-EGFP and the luciferase reporting carrier cotransfection 293T cell that has Stra8 gene 3 ' UTR:
The two luciferase reporting carriers of pIRES2-Boule-EGFP eukaryotic vector and Stra8 by behind 1: 1 transfectional cell 48h, behind the exhaustion cell culture fluid, are added the cell pyrolysis liquid mixing, fully lysing cell.Subsequently, dissolving luciferase detection reagent and luciferase detect damping fluid, need the amount of 100 μ L according to each sample, get an amount of luciferase and detect damping fluid and added luciferase detection substrate according to 1: 100 and be mixed with luciferase testing liquid.Opening fluor tester measures the fluorescent value of each sample.
Detected result is as shown in Figure 8; The cell of transfection pIRES2-Boule-EGFP has the growth about 40% than the fluorescence intensity of the control cells of untransfected; Explain that the Boule gene may directly act on male sex-cell reduction division specific gene Stra8, and then promote that mammalian cell is maiotic initial.
Claims (10)
1. a milk goat Boule gene is characterized in that its gene order is shown in SEQ.ID.NO.1.
2. a milk goat Boule gene is characterized in that, the CDS sequence of this gene is shown in SEQ.ID.NO.2.
3. a milk goat Boule gene Fusion gene is characterized in that, connects the fluorescence report gene in the downstream of milk goat Boule gene.
4. milk goat Boule gene Fusion gene as claimed in claim 3 is characterized in that, also connects the antibiotic-screening gene in the downstream of fluorescent mark gene.
5. expression vector that makes up based on milk goat Boule gene order; It is characterized in that; Comprise the Boule gene order shown in the SEQ.ID.NO.1, connect fluorescent mark gene GFP, connect antibiotic-screening gene kan in the downstream of GFP in the downstream of Boule gene order
r/ neo
r
6. the expression vector that makes up based on milk goat Boule gene order as claimed in claim 5; It is characterized in that; The described expression vector that makes up based on the Boule gene order is the pIRES2-Boule-EGFP expression vector, through Ecor I and Sal I restriction enzyme site the pIRES2-EGFP expression vector is arrived in the Boule gene clone.
The transfection of milk goat Boule gene order in the male sex-cell to promote the maiotic application of male sex-cell.
8. one kind promotes the maiotic method of male sex-cell, it is characterized in that, may further comprise the steps:
1) the milk goat Boule gene order of clone shown in SEQ.ID.NO.1, and the Boule gene order is cloned in the pIRES2-EGFP expression vector through Ecor I and Sal I restriction enzyme site, the pIRES2-Boule-EGFP expression vector obtained;
2) will treat that the male sex-cell of transfection and pIRES2-Boule-EGFP expression vector hatch jointly, with the transfection of pIRES2-Boule-EGFP expression vector among male sex-cell;
3～4h after the transfection male sex-cell is added the foetal calf serum of its volume 10% and 1% non-essential amino acid on minimum medium, and the L-glutaminate of the beta-mercaptoethanol of 0.1mmol/L and 2mmol/L is cultivated; Described basic culture solution is DM/F12 nutrient solution or H-DMEM nutrient solution;
And under fluorescent microscope, the fluorescent mark gene is observed, screening reduction division starts the cell that the related gene expression level increases.
9. the maiotic method of promotion male sex-cell as claimed in claim 8 is characterized in that, describedly treats that the male sex-cell of transfection is the milk goat male sex-cell, and the basic culture solution that is adopted is the DM/F12 nutrient solution.
10. the maiotic method of promotion male sex-cell as claimed in claim 8 is characterized in that, describedly treats that the male sex-cell of transfection is that mouse propagation cell is GC1 or people's a 293T cell, and the basic culture solution that is adopted is the H-DMEM nutrient solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102888407A (en) * | 2012-10-08 | 2013-01-23 | 西北农林科技大学 | Milk goat PLZF gene and application thereof to promoting self-renewal of androgenous stem cells |
| CN111100927A (en) * | 2019-12-18 | 2020-05-05 | 华中科技大学 | Application of seminal plasma BOLL and kit for diagnosing azoospermia meiosis block |
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| CN102181462A (en) * | 2011-02-24 | 2011-09-14 | 中国农业科学院北京畜牧兽医研究所 | New method for improving transgenic efficiency of animals |
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| CN102181462A (en) * | 2011-02-24 | 2011-09-14 | 中国农业科学院北京畜牧兽医研究所 | New method for improving transgenic efficiency of animals |
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| LI X ET AL.: "Gene Bank：FJ623266.1", 《GENE BANK》, 31 March 2009 (2009-03-31) * |
| 李新福 等: "黄犏牛与黄牛b-Boule基因编码区序列、结构、表达模式和睾丸组织表达水平的差异分析", 《自然科学进展》, vol. 19, no. 10, 30 October 2009 (2009-10-30), pages 1042 - 1048 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102888407A (en) * | 2012-10-08 | 2013-01-23 | 西北农林科技大学 | Milk goat PLZF gene and application thereof to promoting self-renewal of androgenous stem cells |
| CN111100927A (en) * | 2019-12-18 | 2020-05-05 | 华中科技大学 | Application of seminal plasma BOLL and kit for diagnosing azoospermia meiosis block |
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