兰州鲇微卫星多态性及生长相关基因分析

发布时间：2018-04-29 22:34

本文选题：兰州鲇 + EST-SSR　；参考：《宁夏大学》2017年硕士论文

【摘要】：兰州鲇(Silurus lanzhouensis),又名黄河鲶,是黄河中上游特有的优质经济鱼类,因其肉质细嫩、刺多肉少、营养价值高等优点,深受养殖户和消费者的喜爱,市场需求量大,消费市场前景广阔。但是目前兰州鲇野生种群数量日益减少,养殖群体选育程度较低。鉴于此,本研究采用EST-SSR标记技术开展兰州鲇遗传多样性研究;运用基因克隆技术开展兰州鲇核糖体蛋白质L15(RPL15)基因完整CDS区、生肌决定因子Myf5(Myf5)基因部分CDS区生物信息学分析,同时采用实行荧光定量法分析上述两个基因在兰州鲇8个不用组织、不同性别个体间的表达;采用TA克隆和单链构象多态性的方法,分析兰州鲇生肌决定因子MyoD(MyoD)基因、肌细胞生成素MyoG(MyoG)基因的SNPs和多态性,筛选出与生长有关的标记位点。通过以上研究为今后兰州鲇分子标记辅助选育和基因工程育种技术奠定了一定的理论基础和技术支撑,并取得以下研究结果:试验一:获得 218 个 EST-SSR 序列,GenBank 登录号:JZ84579-JZ845687;JZ845227-JZ845367;81个不含SSR的EST序列,GenBank登录号:JZ845478-JZ84559。根据侧翼序列设计出299对引物,选取53对进行扩增检验,有22对引物可扩增出目的条带,用PAGE-银染法在100尾兰州鲇个体间进行多态性检测,发现11个EST-SSR位点具有多态性。试验二:首次克隆测序获得兰州鲇RPL15基因的完整CDS区序列长度为615bp(GenBank登录号:KT1969343)和Myf5基因的部分cDNA序列长度为477 bp(GenBank登录号:KT580948)。RPL15基因编码由355个氨基酸残基组成的可溶碱性蛋白质。通过荧光定量PCR分析兰州鲇RPL15基因主要在脑组织和肝组织中特异性表达,且脑组织和肝组织极显著的高于其它组织(P0.01)。脑组织RPL15的表达雌鱼显著高于雄鱼(P0.05)。兰州鲇Myf5基因编码由122个氨基酸残基组成的可溶酸性蛋白质。兰州鲇Myf5基因主要在肌肉组织中特异性表达;雌雄异体间的表达,肌肉组织Myf5的表达雄鱼显著高于雌鱼(P0.05)。试验三:MyoD基因仅在第1外显子中发现1处突变G250A。MyoD有3种基因型(AA、BB和AB)。体重、体高表型值以AB基因型的为最高,以BB基因型的为最低,且3种基因型所对应的表型值组间差异显著(P0.05)。体长表型值在3种基因型中表现出AAABBB的关系,且不同基因型对应的表型值组间差异显著(P0.05)。MyoG基因仅在第1外显子中发现1处突变C213T突变。MyoG基因有3种带型命名为:CC、EE和EC。3种不同基因型对体重、体长和体高表型值的影响表现出EE基因型EC基因型CC基因型的变化关系,且EE基因型显著高于基因型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).
【学位授予单位】：宁夏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

【参考文献】