本文選題：總DNA提取 + 個(gè)體識(shí)別　； 參考：《南昌大學(xué)》2017年碩士論文

【摘要】：本研究采用基于COI基因的DNA條形碼技術(shù)和微衛(wèi)星分子標(biāo)記技術(shù)對(duì)草魚個(gè)體識(shí)別進(jìn)行研究,為后續(xù)草魚育種養(yǎng)殖提供理論基礎(chǔ)。傳統(tǒng)酚-氯仿法是提取DNA的一種常用方法。該法的優(yōu)勢(shì)是DNA提取量大,但質(zhì)量偏低,且傳統(tǒng)法提取DNA通常選取樣本肌肉或血液,對(duì)樣本傷害極大。本研究在傳統(tǒng)酚-氯仿法基礎(chǔ)上進(jìn)行改良,結(jié)合Tris飽和酚不含醌等氧化物、Chelex 100法提取DNA時(shí)間短的優(yōu)勢(shì),優(yōu)化獲得一種以魚鱗為原材料的,改良DNA提取法。此法所提取DNA效果明顯優(yōu)于傳統(tǒng)法,并通過(guò)PCR實(shí)驗(yàn)顯示,所提取的核基因和線粒體基因都可用于后續(xù)各類研究。COI又稱細(xì)胞色素C氧化酶亞基I(cytochrome c oxidase subunit I),COI基因(cytochrome oxidase I)即為線粒體DNA上編碼該亞基的基因,長(zhǎng)度大約600bp,主要用于物種鑒定或生物系統(tǒng)發(fā)育研究。通過(guò)使用Dnaman等生物軟件,對(duì)草魚COI基因序列比對(duì)分析,了解草魚COI基因之間相互關(guān)系,進(jìn)一步探討基于COI基因的DNA條形碼技術(shù)在草魚個(gè)體識(shí)別方面的可行性。結(jié)果顯示,草魚COI基因片段大小約為1440bp,堿基T、A、C、G的平均含量分別為30.2%、27.1%、25.0%,17.7%呈依次遞減趨勢(shì),其中G的含量明顯低于其它的含量,表現(xiàn)出很強(qiáng)的堿基組成偏向性。9尾草魚COI基因序列共有24處堿基突變,在序列第280-440bp之間和第1300-1410bp之間分別有7個(gè)和6個(gè)突變位點(diǎn),屬于突變頻率較高區(qū)段,可考慮作為DNA微型條形碼。各草魚之間的遺傳距離為0.1-0.6%,平均遺傳距離0.39%,小于同一物種內(nèi)遺傳距離的臨界值2%,因此線粒體COI基因作為DNA條形碼技術(shù)目的基因,不適用于草魚的個(gè)體識(shí)別。微衛(wèi)星又稱簡(jiǎn)單重復(fù)序列(simple sequence repeats,SSR),存在于絕大多數(shù)真核生物的基因組中。利用微衛(wèi)星突變率高、中性DNA、存在廣泛且序列兩端的保守性較好等特點(diǎn),微衛(wèi)星分子標(biāo)記技術(shù)被廣泛運(yùn)用于各種生物領(lǐng)域。本研究利用該技術(shù)將少量多態(tài)性豐富的微衛(wèi)星位點(diǎn)排列組合,從85對(duì)微衛(wèi)星引物進(jìn)行篩選出8對(duì)多態(tài)性較好的引物(HLJC115、HLJC104、HLJC107、13118、HLJC2、HLJC111、HLJC9和HLJC126),分別用于11尾草魚樣本和63尾草魚樣本的個(gè)體識(shí)別,11尾草魚樣本共擴(kuò)增出19對(duì)等位基因,平均每個(gè)位點(diǎn)含4.75對(duì)等位基因,等位基因數(shù)目范圍2-7之間。63尾草魚樣本共擴(kuò)增出38對(duì)等位基因,平均每個(gè)位點(diǎn)含6.33對(duì)等位基因,等位基因數(shù)目范圍4-9之間。研究結(jié)果顯示微衛(wèi)星分子標(biāo)記技術(shù)對(duì)小范圍樣本(11尾)或大范圍樣本(63尾)的草魚,個(gè)體識(shí)別率均可達(dá)100%。
[Abstract]:In this study, DNA barcode based on COI gene and microsatellite molecular marker were used to study the individual identification of grass carp, which provided a theoretical basis for the subsequent breeding and breeding of grass carp. Traditional phenol-chloroform method is a common method for extracting DNA. The advantage of this method is that the quantity of DNA is large, but the quality is low, and the traditional method of DNA extraction usually selects the sample muscle or blood, which is very harmful to the sample. Based on the traditional phenol-chloroform method and combined with the advantage of short extraction time of Tris saturated phenol without quinone, an improved DNA extraction method with fish scales as raw material was obtained. The DNA extracted by this method is obviously superior to that of the traditional method, and the results of PCR experiments show that, The extracted nuclear genes and mitochondrial genes can be used in all kinds of further studies. The cytochrome oxidase I gene of cytochrome C oxidase subunit I(cytochrome c oxidase subunit is the gene encoding this subunit on mitochondrial DNA. About 600 BP in length, mainly for species identification or phylogenetic research. By using Dnaman and other biological software to analyze the sequence alignment of COI gene of grass carp, to understand the relationship between COI gene of grass carp, and to explore the feasibility of DNA barcode based on COI gene in individual identification of grass carp. The results showed that the COI gene fragment size of grass carp was about 1440 BP, and the average content of the base TGG was 30.2% 27.1% and 25.0%, respectively, and the content of G was significantly lower than that of the other ones. The results showed that there were 24 base mutations in the COI gene sequence of grass carp with strong base composition bias. There were 7 and 6 mutation sites between 280-440bp and 1300-1410bp, respectively, which belonged to the higher mutation frequency region. Can be considered as a DNA minibar code. The genetic distance between grass carp is 0.1-0.6, and the average genetic distance is 0.39, which is smaller than the critical value of genetic distance within the same species. Therefore, mitochondrial COI gene is not suitable for individual identification of grass carp as the target gene of DNA barcode technology. Microsatellites, also known as simple sequence repeats, are present in the genomes of most eukaryotes. Microsatellite molecular marker technology has been widely used in various biological fields because of its high mutation rate, neutral DNA, wide range and good conservation at both ends of the sequence. In this study, a small number of polymorphic microsatellite loci were arranged and combined using this technique. Eight pairs of polymorphic primers HLJC115, HLJC104, HLJC107, HLJC2HJC111HLJC9 and HLJC126N were screened from 85 pairs of microsatellite primers. The alleles were amplified by 19 pairs of alleles in 11 samples of grass carp and 63 samples of grass carp, respectively. There were 4.75 alleles per locus, 38 alleles were amplified from 2-7 to 63.The average alleles were 6.33 alleles and 4-9 alleles per locus. The results show that the individual recognition rate of grass carp with microsatellite molecular marker technique can reach 100% for small samples (11 tails) or large scale samples (63 tails).
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S917.4

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