本文關(guān)鍵詞：MIJ大鼠Klhl10、Insl3、Acr、Zmynd15基因的克隆與序列分析　出處：《河北醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 克隆 測(cè)序 MIJ大鼠 雄性不育

【摘要】：目的:有接近15%新組成的家庭會(huì)承受不孕癥的困擾,大部分是由于分子和遺傳因素導(dǎo)致的不孕。雖然有許多基因涉及不育,但是目前只有一小部分基因被確定。遺傳因素導(dǎo)致男性不育受到越來越多的關(guān)注。盡管做了許多努力,但是臨床上只有少數(shù)相關(guān)基因多態(tài)性被確定。這主要是由于男性不育癥的多因素的性質(zhì),研究設(shè)計(jì)難度大以及沒有好的實(shí)驗(yàn)?zāi)Ｐ偷脑?因此迫切需要建立能用于男性不育研究的動(dòng)物模型。MIJ大鼠的雄性后代中穩(wěn)定出現(xiàn)不育個(gè)體,經(jīng)遺傳測(cè)交實(shí)驗(yàn)證實(shí)為常染色體上的單一隱性基因突變所致。MIJ近交系大鼠有望成為生殖領(lǐng)域研究的良好動(dòng)物模型。因此突變基因的定位將極大地幫助MIJ大鼠的保種、繁殖及其推廣應(yīng)用。通過前期的表達(dá)譜基因芯片篩選,篩選到10個(gè)和雄性不育相關(guān)的差異表達(dá)基因,然而并不清楚是否是由于表達(dá)差異基因的突變導(dǎo)致了MIJ大鼠的不育。為了進(jìn)一步研究MIJ雄性不育大鼠的基因突變情況,本實(shí)驗(yàn)對(duì)MIJ雄性不育大鼠,MIJ雄性正常大鼠和MIJN雄性大鼠Klhl10(kelch-like family member 10)、Insl3(insulin-like 3)、Acr(acrosin)和Zmynd15(zinc finger,MYND-type containing 15)四個(gè)基因進(jìn)行了克隆測(cè)序和序列比對(duì)研究。方法:根據(jù)搜索RGD(Rat Genome Database)得到大鼠的DNA基因序列,用Primer 5.0軟件對(duì)Klhl10、Insl3、Acr、Zmynd15四個(gè)基因的序列進(jìn)行引物設(shè)計(jì),并送公司進(jìn)行合成,對(duì)MIJ雄性不育大鼠、MIJ雄性正常大鼠和MIJN雄性大鼠基因組DNA分別進(jìn)行PCR擴(kuò)增,將擴(kuò)增出來的特異性片段連接T克隆載體,對(duì)連接成功的質(zhì)粒進(jìn)行酶切鑒定,將酶切鑒定克隆成功的陽性質(zhì)粒送公司測(cè)序,將三種大鼠的基因片段測(cè)序結(jié)果進(jìn)行比對(duì),對(duì)各個(gè)片段進(jìn)行拼接分析,以期得到MIJ雄性不育大鼠相對(duì)于MIJ雄性正常大鼠、MIJN雄性大鼠的突變所在。結(jié)果:1完成了對(duì)MIJ雄性不育大鼠,MIJ雄性正常大鼠和MIJN雄性大鼠的Klhl10、Insl3、Acr、Zmynd15四個(gè)基因的克隆將PCR產(chǎn)物連接T載體,篩選酶切鑒定連接成功克隆的質(zhì)粒,完成了對(duì)MIJ雄性不育大鼠,MIJ雄性可育大鼠和MIJN大鼠的Klhl10、Insl3、Acr、Zmynd15四個(gè)基因克隆。Klhl10、Insl3、Acr和Zmynd15基因分別制備36,2,8,8個(gè)克隆,3種大鼠共制備54個(gè)克隆。2完成了對(duì)MIJ雄性不育大鼠,MIJ雄性可育大鼠和MIJN大鼠Klhl10、Insl3、Acr、Zmynd15四個(gè)基因的54個(gè)克隆片段的測(cè)序?qū)⒖寺〕晒Φ腗IJ雄性不育大鼠,MIJ雄性可育大鼠和MIJN大鼠的Klhl10、Insl3、Acr、Zmynd15四個(gè)基因的54個(gè)克隆片段進(jìn)行了克隆測(cè)序。3對(duì)MIJ雄性不育大鼠,MIJ雄性正常大鼠和MIJN雄性大鼠的Klhl10、Insl3、Acr、Zmynd15四個(gè)基因的測(cè)序結(jié)果進(jìn)行拼接及比對(duì)分析Klhl10基因的堿基有16367pb,共制備36個(gè)克隆,重疊堿基為3251bp;Insl3基因的堿基有1940pb,共制備2個(gè)克隆,重疊堿基為75bp;Acr基因的堿基有6187pb,共制備8個(gè)克隆,重疊堿基為646pb;Zmynd15基因的堿基有6614pb,共制備8個(gè)克隆,重疊堿基為623pb,通過DNAstar軟件進(jìn)行拼接,并去除多余部分堿基。將MIJ雄性不育大鼠,MIJ雄性可育大鼠和MIJN大鼠Klhl10、Insl3、Acr、Zmynd15四個(gè)基因的序列拼接結(jié)果用DNAMEN軟件進(jìn)行序列比對(duì),未發(fā)現(xiàn)突變堿基。結(jié)論:1首次報(bào)道了MIJ雄性不育大鼠,MIJ雄性正常大鼠和MIJN雄性大鼠Klhl10、Insl3、Acr、Zmynd15四個(gè)基因的DNA序列。2排除了MIJ雄性不育大鼠的突變基因位于Klhl10、Insl3、Acr、Zmynd15四個(gè)基因序列的可能性。本實(shí)驗(yàn)為進(jìn)一步對(duì)MIJ大鼠突變基因的鑒定研究,確定人類與動(dòng)物同源的類似基因,為人類男性不育研究提供了科學(xué)實(shí)驗(yàn)依據(jù)。
[Abstract]:Objective: nearly 15% new families will suffer from infertility, most of which are caused by molecular and genetic infertility. Although many genes are involved in infertility, only a small portion of the genes are identified at present. Genetic factors have caused more and more attention to male infertility. Although many efforts have been made, only a few related gene polymorphisms have been identified clinically. This is mainly due to the multi factorial nature of male infertility. Because of the difficulty of research and design and the lack of good experimental models, it is urgent to establish an animal model that can be used for male infertility research. The male sterile in the male offspring of MIJ rats was stable, and the genetic test proved to be the result of a single recessive gene mutation on the autosomes. The MIJ inbred rat is expected to be a good animal model in the field of reproductive research. Therefore, the location of the mutant gene will greatly help the MIJ rats to preserve, reproduce and promote their application. We screened 10 differentially expressed genes related to male sterility by screening gene chips, but it was not clear whether the mutation of MIJ gene caused the sterility of the rats. In order to further study the MIJ male sterile rat gene mutation, the experiments of MIJ male sterile rats, normal male MIJ rats and MIJN rats (Klhl10 Kelch-like family member 10), Insl3 (insulin-like 3), Acr (acrosin) and Zmynd15 (zinc finger, MYND-type containing 15) four gene the study of cloning sequencing and sequence alignment. Methods: according to the search of RGD (Rat Genome Database) DNA gene sequence of rat, using Primer 5 software, Insl3 and Acr sequences of Klhl10 and Zmynd15 four genes were amplified and sent to the company for synthesis of MIJ male sterile rats, normal male MIJ rats and MIJN male rat genomic DNA were amplified by PCR specific fragment of the PCR product of cloned into vector T plasmid, for successful identification of enzyme digestion, the positive plasmid was identified by restriction enzyme cloning sequencing will be sent to the company, gene sequencing results of three rats were compared by splicing analysis of each fragment, in order to get MIJ male sterile rats relative to the mutation site of MIJ MIJN male rats in normal male rats. Results: 1 completed the MIJ male sterile rats, cloning of MIJ in normal male rats and MIJN male rats of Klhl10, Insl3, Acr, Zmynd15 four gene PCR was ligated to T vector plasmid screening, enzyme digestion connection successfully cloned, completed the MIJ male sterile rats, Klhl10, cloning Insl3, Acr, Zmynd15 four male fertile gene MIJ and MIJN rats. 36,2,8,8 clones were prepared by Klhl10, Insl3, Acr and Zmynd15 genes, and 54 clones were prepared in 3 rats. 2 completed the MIJ male sterile rats, male MIJ sequencing 54 clones with fragment and MIJN rats Klhl10, Insl3, Acr, Zmynd15 of the four genes cloned MIJ male sterile rats, 54 male fertile clones MIJ and MIJN rats, Klhl10 Insl3, Acr and Zmynd15 four genes were cloned and sequenced. 3 pairs of MIJ male sterile rats, sequencing of MIJ in normal male rats and MIJN male rats of Klhl10, Insl3, Acr, Zmynd15 four gene results 16367pb splicing and alignment analysis of Klhl10 gene nucleotide, 36 clones, overlapping bases 3251bp; base of Insl3 gene 1940pb. A total of 2 clones were prepared, overlapping base for 75bp; Acr gene sequence of 6187pb, 8 clones, overlapping bases 646pb; Zmynd15 gene sequence of 6614pb, 8 clones, overlapping bases 623pb were spliced by DNAstar software, and removing the excess part of the base. Sequence alignment of four genes of MIJ male sterility rat, MIJ male fertile rat and MIJN rat Klhl10, Insl3, Acr and Zmynd15 were compared with DNAMEN software, and no mutation base was found. Conclusion: 1 the DNA sequence of four genes of MIJ male sterile rats, MIJ male normal rats and MIJN male rats, Klhl10, Insl3, Acr and Zmynd15 genes were reported for the first time. 2 the possibility of the mutation of MIJ Male Sterile Rats in Klhl10, Insl3, Acr and Zmynd15 gene sequences was excluded. In order to further identify the mutant genes in MIJ rats and identify homologous genes in humans and animals, we provide a scientific basis for the study of human male infertility.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R711.6

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