MIJ大鼠Klhl10、Insl3、Acr、Zmynd15基因的克隆与序列分析

发布时间：2017-12-27 10:34

本文关键词：MIJ大鼠Klhl10、Insl3、Acr、Zmynd15基因的克隆与序列分析　出处：《河北医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:有接近15%新组成的家庭会承受不孕症的困扰,大部分是由于分子和遗传因素导致的不孕。虽然有许多基因涉及不育,但是目前只有一小部分基因被确定。遗传因素导致男性不育受到越来越多的关注。尽管做了许多努力,但是临床上只有少数相关基因多态性被确定。这主要是由于男性不育症的多因素的性质,研究设计难度大以及没有好的实验模型的原因,因此迫切需要建立能用于男性不育研究的动物模型。MIJ大鼠的雄性后代中稳定出现不育个体,经遗传测交实验证实为常染色体上的单一隐性基因突变所致。MIJ近交系大鼠有望成为生殖领域研究的良好动物模型。因此突变基因的定位将极大地帮助MIJ大鼠的保种、繁殖及其推广应用。通过前期的表达谱基因芯片筛选,筛选到10个和雄性不育相关的差异表达基因,然而并不清楚是否是由于表达差异基因的突变导致了MIJ大鼠的不育。为了进一步研究MIJ雄性不育大鼠的基因突变情况,本实验对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)四个基因进行了克隆测序和序列比对研究。方法:根据搜索RGD(Rat Genome Database)得到大鼠的DNA基因序列,用Primer 5.0软件对Klhl10、Insl3、Acr、Zmynd15四个基因的序列进行引物设计,并送公司进行合成,对MIJ雄性不育大鼠、MIJ雄性正常大鼠和MIJN雄性大鼠基因组DNA分别进行PCR扩增,将扩增出来的特异性片段连接T克隆载体,对连接成功的质粒进行酶切鉴定,将酶切鉴定克隆成功的阳性质粒送公司测序,将三种大鼠的基因片段测序结果进行比对,对各个片段进行拼接分析,以期得到MIJ雄性不育大鼠相对于MIJ雄性正常大鼠、MIJN雄性大鼠的突变所在。结果:1完成了对MIJ雄性不育大鼠,MIJ雄性正常大鼠和MIJN雄性大鼠的Klhl10、Insl3、Acr、Zmynd15四个基因的克隆将PCR产物连接T载体,筛选酶切鉴定连接成功克隆的质粒,完成了对MIJ雄性不育大鼠,MIJ雄性可育大鼠和MIJN大鼠的Klhl10、Insl3、Acr、Zmynd15四个基因克隆。Klhl10、Insl3、Acr和Zmynd15基因分别制备36,2,8,8个克隆,3种大鼠共制备54个克隆。2完成了对MIJ雄性不育大鼠,MIJ雄性可育大鼠和MIJN大鼠Klhl10、Insl3、Acr、Zmynd15四个基因的54个克隆片段的测序将克隆成功的MIJ雄性不育大鼠,MIJ雄性可育大鼠和MIJN大鼠的Klhl10、Insl3、Acr、Zmynd15四个基因的54个克隆片段进行了克隆测序。3对MIJ雄性不育大鼠,MIJ雄性正常大鼠和MIJN雄性大鼠的Klhl10、Insl3、Acr、Zmynd15四个基因的测序结果进行拼接及比对分析Klhl10基因的碱基有16367pb,共制备36个克隆,重叠碱基为3251bp;Insl3基因的碱基有1940pb,共制备2个克隆,重叠碱基为75bp;Acr基因的碱基有6187pb,共制备8个克隆,重叠碱基为646pb;Zmynd15基因的碱基有6614pb,共制备8个克隆,重叠碱基为623pb,通过DNAstar软件进行拼接,并去除多余部分碱基。将MIJ雄性不育大鼠,MIJ雄性可育大鼠和MIJN大鼠Klhl10、Insl3、Acr、Zmynd15四个基因的序列拼接结果用DNAMEN软件进行序列比对,未发现突变碱基。结论:1首次报道了MIJ雄性不育大鼠,MIJ雄性正常大鼠和MIJN雄性大鼠Klhl10、Insl3、Acr、Zmynd15四个基因的DNA序列。2排除了MIJ雄性不育大鼠的突变基因位于Klhl10、Insl3、Acr、Zmynd15四个基因序列的可能性。本实验为进一步对MIJ大鼠突变基因的鉴定研究,确定人类与动物同源的类似基因,为人类男性不育研究提供了科学实验依据。
[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.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R711.6

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