猪MLH1、KLK7基因的分子鉴定、多态性及产仔数关联分析

发布时间：2018-03-10 23:55

本文选题：猪　切入点：MLH1　出处：《云南农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：云南地方猪种丰富且独特,是构成云南养猪业重要的种质基础,云南地方猪种产仔数普遍较低,严重制约了云南养猪业的发展。国内外相关研究表明,决定猪产仔数的高低的分子机制异常复杂,涉及到众多基因包括非编码RNA的表达与调控。目前关于云南地方猪种产仔数高低的分子遗传学机制方面的研究取得了一定的进展,主要从候选基因水平即DNA的水平探讨云南地方猪种产仔数高低的遗传机理,本研究以低产仔数的6个云南地方猪种为研究对象,以较高高产仔数的大白猪和长白猪为参照,研究与繁殖相关的两个基因(MutL同源体1,直肠癌,非息肉病性型2(MLH1)基因和激肽释放酶7(KLK7)基因),以探讨这两个基因是否可以作为与猪产仔数相关的候选基因进行后续研究,具体为:1、Mut L同源体1,直肠癌,非息肉病性型2(MLH1)是与繁殖相关的重要基因。在本研究中,猪MLH1基因的全长cDNA通过RACE技术被克隆。该基因编码蛋白质具有757个氨基酸残基,和藏羚羊、羊、牛、南方白犀牛、山羊分别具有:96%、95%、93%、92%、92%的高度同源性。这个基因归属于GeneID:100337665。基因进化分析表明猪MLH1基因和南方白犀牛的MLH1基因具有较近的遗传关系。通过PCR-Hha I-RFLP分析发现在该基因mRNA 395-bp处具有一个G/A突变(GU373696:c395 GA),8个猪种在该突变位点等位基因频率和基因型频率具有很大的差异。该突变多态性和大白猪(n=200)、长白猪(n=200)的产子数进行关联分析表明该突变与总产子数关联极显著(P0.01)。因此,猪MLH1基因可以作为猪产子数候选基因以来提高猪产子数。本实验研究结果为猪MLH1基因的进一步研究奠定了基础。2、激肽释放酶7(Kallikrein-related peptidase 7,KLK7)是另外一个与繁殖相关的重要基因。在本研究中,通过RT-PCR技术,克隆猪KLK7基因的全长编码序列。该基因的序列分析表明,猪KLK7基因编码含有257个氨基酸的蛋白质,该蛋白质与6种生物KLK7蛋白具有高度同源性:北极熊(95%)、威德尔海豹(94%)、狗(92%)、太平洋海象(95%)、家猫(92%)、东北虎(91%)具有高度同源性。通过计算机辅助分析表明该基因结构有5个外显子和4个内含子。基因进化分析表明,猪KLK7基因和狗的KLK7基因具有较近的亲源关系。通过PCR-Alu I-RFLP分析发现在该基因mRNA 390-bp处具有一个T/C突变(GU373714:c390 TC),8个猪种在该突变位点等位基因频率和基因型频率具有很大的差异。该突变位点的多态性与大白猪(n=200)、长白猪(n=200)的产仔数进行关联分析,结果表明该突变位点与猪总产仔数关联显著差异(P0.05)。因此,猪KLK7基因可以作为猪产仔数候选基因用来提高猪产仔数。本实验研究结果为猪KLK7基因的进一步研究奠定了基础。
[Abstract]:Yunnan local pig breeds are rich and unique, which constitute the important germplasm basis of Yunnan pig industry. Yunnan local pig breeds are generally low in litter size, which seriously restricts the development of Yunnan pig industry. The molecular mechanism of determining piglets' litter size is very complicated and involves the expression and regulation of many genes, including non-coding RNA. At present, some progress has been made in the study of molecular genetic mechanism of piglets' litter size in Yunnan. The genetic mechanism of litter size of Yunnan local pigs was discussed from the candidate gene level (DNA level). Six Yunnan local breeds with low litter size were used as the research objects, and the large white pigs and Landrace pigs with higher litter size were used as reference. To study two genes related to reproduction, namely MutL homologue 1, rectal cancer, non-polyposis type 2MLH1) gene and KLK7 gene, to explore whether these two genes can be used as candidate genes for porcine litter size. In this study, the full-length cDNA of porcine MLH1 gene was cloned by RACE technique. And Tibetan antelope, sheep, cattle, southern white rhinoceros, The gene belongs to gene ID: 1003376655.The gene evolution analysis shows that the gene of pig MLH1 has a close genetic relationship with the MLH1 gene of southern white rhinoceros. By PCR-Hha I-RFLP analysis, it is found that the gene is located at mRNA 395-bp. There is a G / A mutation GU373696: c395GAA. The allele frequency and genotypic frequency of 8 pig breeds at this mutation site are very different. This mutation polymorphism is associated with the number of births of the large white pig and Landrace pig. The results show that the mutation is associated with the total yield of GU373696: c395GA.At the same time, there is a significant difference between the allele frequency and the genotypic frequency of the mutant. The subnumber correlation is extremely significant (P0.01). Porcine MLH1 gene can be used as a candidate gene to increase the number of pigs giving birth. The results of this study laid a foundation for further study of porcine MLH1 gene. KLK7 is another important factor related to reproduction. Genes. In this study, The full-length encoding sequence of porcine KLK7 gene was cloned by RT-PCR technique. The sequence analysis of the gene showed that porcine KLK7 gene encodes 257 amino acid proteins. This protein has high homology with six biological KLK7 proteins: polar bear 95, Weddell seal 94, dog 92, Pacific walrus 95, domestic cat 922, Amur tiger 91. the structure of the gene is confirmed by computer aided analysis. 5 exons and 4 introns. The relationship between porcine KLK7 gene and dog KLK7 gene is close. PCR-Alu I-RFLP analysis shows that there is a T / C mutation GU373714: C390 TCN at mRNA 390-bp, and the allele frequency and genotype frequency of 8 pig breeds at this mutation site are very large. The polymorphism of the mutation locus was associated with the litter size of Landrace and Landrace. The results showed that there was a significant difference between the mutation site and the total litter size of pigs. Therefore, the porcine KLK7 gene could be used as a candidate gene to increase the litter size of pigs. The results of this study laid a foundation for the further study of porcine KLK7 gene.
【学位授予单位】：云南农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

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