發(fā)布時(shí)間：2018-03-10 23:55

  本文選題：豬　切入點(diǎn)：MLH1　出處：《云南農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：云南地方豬種豐富且獨(dú)特,是構(gòu)成云南養(yǎng)豬業(yè)重要的種質(zhì)基礎(chǔ),云南地方豬種產(chǎn)仔數(shù)普遍較低,嚴(yán)重制約了云南養(yǎng)豬業(yè)的發(fā)展。國(guó)內(nèi)外相關(guān)研究表明,決定豬產(chǎn)仔數(shù)的高低的分子機(jī)制異常復(fù)雜,涉及到眾多基因包括非編碼RNA的表達(dá)與調(diào)控。目前關(guān)于云南地方豬種產(chǎn)仔數(shù)高低的分子遺傳學(xué)機(jī)制方面的研究取得了一定的進(jìn)展,主要從候選基因水平即DNA的水平探討云南地方豬種產(chǎn)仔數(shù)高低的遺傳機(jī)理,本研究以低產(chǎn)仔數(shù)的6個(gè)云南地方豬種為研究對(duì)象,以較高高產(chǎn)仔數(shù)的大白豬和長(zhǎng)白豬為參照,研究與繁殖相關(guān)的兩個(gè)基因(MutL同源體1,直腸癌,非息肉病性型2(MLH1)基因和激肽釋放酶7(KLK7)基因),以探討這兩個(gè)基因是否可以作為與豬產(chǎn)仔數(shù)相關(guān)的候選基因進(jìn)行后續(xù)研究,具體為:1、Mut L同源體1,直腸癌,非息肉病性型2(MLH1)是與繁殖相關(guān)的重要基因。在本研究中,豬MLH1基因的全長(zhǎng)cDNA通過RACE技術(shù)被克隆。該基因編碼蛋白質(zhì)具有757個(gè)氨基酸殘基,和藏羚羊、羊、牛、南方白犀牛、山羊分別具有:96%、95%、93%、92%、92%的高度同源性。這個(gè)基因歸屬于GeneID:100337665�；�因進(jìn)化分析表明豬MLH1基因和南方白犀牛的MLH1基因具有較近的遺傳關(guān)系。通過PCR-Hha I-RFLP分析發(fā)現(xiàn)在該基因mRNA 395-bp處具有一個(gè)G/A突變(GU373696:c395 GA),8個(gè)豬種在該突變位點(diǎn)等位基因頻率和基因型頻率具有很大的差異。該突變多態(tài)性和大白豬(n=200)、長(zhǎng)白豬(n=200)的產(chǎn)子數(shù)進(jìn)行關(guān)聯(lián)分析表明該突變與總產(chǎn)子數(shù)關(guān)聯(lián)極顯著(P0.01)。因此,豬MLH1基因可以作為豬產(chǎn)子數(shù)候選基因以來提高豬產(chǎn)子數(shù)。本實(shí)驗(yàn)研究結(jié)果為豬MLH1基因的進(jìn)一步研究奠定了基礎(chǔ)。2、激肽釋放酶7(Kallikrein-related peptidase 7,KLK7)是另外一個(gè)與繁殖相關(guān)的重要基因。在本研究中,通過RT-PCR技術(shù),克隆豬KLK7基因的全長(zhǎng)編碼序列。該基因的序列分析表明,豬KLK7基因編碼含有257個(gè)氨基酸的蛋白質(zhì),該蛋白質(zhì)與6種生物KLK7蛋白具有高度同源性:北極熊(95%)、威德爾海豹(94%)、狗(92%)、太平洋海象(95%)、家貓(92%)、東北虎(91%)具有高度同源性。通過計(jì)算機(jī)輔助分析表明該基因結(jié)構(gòu)有5個(gè)外顯子和4個(gè)內(nèi)含子�；�因進(jìn)化分析表明,豬KLK7基因和狗的KLK7基因具有較近的親源關(guān)系。通過PCR-Alu I-RFLP分析發(fā)現(xiàn)在該基因mRNA 390-bp處具有一個(gè)T/C突變(GU373714:c390 TC),8個(gè)豬種在該突變位點(diǎn)等位基因頻率和基因型頻率具有很大的差異。該突變位點(diǎn)的多態(tài)性與大白豬(n=200)、長(zhǎng)白豬(n=200)的產(chǎn)仔數(shù)進(jìn)行關(guān)聯(lián)分析,結(jié)果表明該突變位點(diǎn)與豬總產(chǎn)仔數(shù)關(guān)聯(lián)顯著差異(P0.05)。因此,豬KLK7基因可以作為豬產(chǎn)仔數(shù)候選基因用來提高豬產(chǎn)仔數(shù)。本實(shí)驗(yàn)研究結(jié)果為豬KLK7基因的進(jìn)一步研究奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：云南農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S828

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