本文選題：蘭州鲇 + EST-SSR�。� 參考：《寧夏大學》2017年碩士論文

【摘要】：蘭州鲇(Silurus lanzhouensis),又名黃河鯰,是黃河中上游特有的優(yōu)質(zhì)經(jīng)濟魚類,因其肉質(zhì)細嫩、刺多肉少、營養(yǎng)價值高等優(yōu)點,深受養(yǎng)殖戶和消費者的喜愛,市場需求量大,消費市場前景廣闊。但是目前蘭州鲇野生種群數(shù)量日益減少,養(yǎng)殖群體選育程度較低。鑒于此,本研究采用EST-SSR標記技術(shù)開展蘭州鲇遺傳多樣性研究;運用基因克隆技術(shù)開展蘭州鲇核糖體蛋白質(zhì)L15(RPL15)基因完整CDS區(qū)、生肌決定因子Myf5(Myf5)基因部分CDS區(qū)生物信息學分析,同時采用實行熒光定量法分析上述兩個基因在蘭州鲇8個不用組織、不同性別個體間的表達;采用TA克隆和單鏈構(gòu)象多態(tài)性的方法,分析蘭州鲇生肌決定因子MyoD(MyoD)基因、肌細胞生成素MyoG(MyoG)基因的SNPs和多態(tài)性,篩選出與生長有關(guān)的標記位點。通過以上研究為今后蘭州鲇分子標記輔助選育和基因工程育種技術(shù)奠定了一定的理論基礎(chǔ)和技術(shù)支撐,并取得以下研究結(jié)果:試驗一:獲得 218 個 EST-SSR 序列,GenBank 登錄號:JZ84579-JZ845687;JZ845227-JZ845367;81個不含SSR的EST序列,GenBank登錄號:JZ845478-JZ84559。根據(jù)側(cè)翼序列設(shè)計出299對引物,選取53對進行擴增檢驗,有22對引物可擴增出目的條帶,用PAGE-銀染法在100尾蘭州鲇個體間進行多態(tài)性檢測,發(fā)現(xiàn)11個EST-SSR位點具有多態(tài)性。試驗二:首次克隆測序獲得蘭州鲇RPL15基因的完整CDS區(qū)序列長度為615bp(GenBank登錄號:KT1969343)和Myf5基因的部分cDNA序列長度為477 bp(GenBank登錄號:KT580948)。RPL15基因編碼由355個氨基酸殘基組成的可溶堿性蛋白質(zhì)。通過熒光定量PCR分析蘭州鲇RPL15基因主要在腦組織和肝組織中特異性表達,且腦組織和肝組織極顯著的高于其它組織(P0.01)。腦組織RPL15的表達雌魚顯著高于雄魚(P0.05)。蘭州鲇Myf5基因編碼由122個氨基酸殘基組成的可溶酸性蛋白質(zhì)。蘭州鲇Myf5基因主要在肌肉組織中特異性表達;雌雄異體間的表達,肌肉組織Myf5的表達雄魚顯著高于雌魚(P0.05)。試驗三:MyoD基因僅在第1外顯子中發(fā)現(xiàn)1處突變G250A。MyoD有3種基因型(AA、BB和AB)。體重、體高表型值以AB基因型的為最高,以BB基因型的為最低,且3種基因型所對應的表型值組間差異顯著(P0.05)。體長表型值在3種基因型中表現(xiàn)出AAABBB的關(guān)系,且不同基因型對應的表型值組間差異顯著(P0.05)。MyoG基因僅在第1外顯子中發(fā)現(xiàn)1處突變C213T突變。MyoG基因有3種帶型命名為:CC、EE和EC。3種不同基因型對體重、體長和體高表型值的影響表現(xiàn)出EE基因型EC基因型CC基因型的變化關(guān)系,且EE基因型顯著高于基因型EC和CC基因型(P0.05)。
[Abstract]:Silurus lanzhouensis, also known as the Yellow River catfish, is a special high quality economic fish in the middle and upper reaches of the Yellow River. Because of its advantages of delicate meat, less prickly meat and high nutritional value, it is loved by breeders and consumers. The market demand is large and the consumption market prospect is broad. But at present, the wild population of Silurus meridionalis in Lanzhou is decreasing day by day, and the breeding degree of breeding population is low. In this study, the genetic diversity of Silurus meridionalis was studied by EST-SSR marker technique, and the complete CDS region of the ribosomal protein L15RPL15 and the bioinformatics analysis of partial CDS region of myogenic determinant Myf5) gene were carried out by gene cloning technique. At the same time, the expression of the two genes in 8 non-tissue and different sex individuals of Silurus meridionalis was analyzed by using fluorescence quantitative method, TA cloning and single strand conformation polymorphism were used to analyze the MyoDU MyoD) gene of the muscle determinant of Silurus meridionalis. The SNPs and polymorphism of myopoietin myopoietin (MyoG) gene were used to screen the marker sites related to the growth of myopoietin. These studies have laid a theoretical foundation and technical support for molecular marker-assisted breeding and genetic engineering breeding of Silurus meridionalis in the future. The main results are as follows: experiment 1: the accession number of 218 EST-SSR sequences was obtained: JZ84579-JZ845687, JZ845227-JZ8453677, and 81 EST sequences without SSR were identified as GenBank accession No.: JZ845478-JZ84559. According to the flanking sequence, 299 pairs of primers were designed and 53 pairs were selected for amplification test. 22 pairs of primers were used to amplify the target bands. The polymorphism of 11 EST-SSR loci was detected by PAGE-silver staining method. Experiment 2: the length of complete CDS region of RPL15 gene of Silurus meridionalis was 615bp(GenBank accession number: KT1969343 for the first time and part of cDNA sequence of Myf5 gene was 477 bp(GenBank accession number: KT580948. RPL15 gene encoded soluble basic protein composed of 355 amino acid residues. Fluorescence quantitative PCR was used to analyze the specific expression of RPL15 gene in the brain and liver tissues of Silurus meridionalis, and it was significantly higher in the brain and liver tissues than in other tissues (P 0.01). The expression of RPL15 in brain of female was significantly higher than that of male. The Myf5 gene encodes a soluble acidic protein composed of 122 amino acid residues. The expression of Myf5 gene in the muscle tissues of Silurus meridionalis was mainly specific, and the expression of Myf5 in the muscle tissue was significantly higher in the male than in the female. Experiment 3: MyoD gene only found that there were three genotypes of G250A.MyoD in exon 1. The phenotypic values of body weight and body height were the highest in AB genotype and the lowest in BB genotype. The phenotypic values of body length showed the relationship of AAABBB among the three genotypes. Moreover, there were significant differences in phenotypic values of different genotypes among groups. Only one mutation C213T mutation. MyoG gene was found in exon 1. There were three banding types named as the weight of different genotypes of EC.3 and 10% CCEE. The effect of body length and body height on CC genotype of EE genotype was significantly higher than that of genotype EC and CC genotype (P 0.05).
【學位授予單位】：寧夏大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S917.4

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