尼罗罗非鱼Ikaros基因克

发布时间：2018-03-19 22:23

本文选题：尼罗罗非鱼　切入点：Ikaros基因　出处：《上海海洋大学》2017年硕士论文　论文类型：学位论文

【摘要】：罗非鱼是世界上最重要的水产养殖品种之一,具有生长快、繁殖力高、适应性强和食性广等优点,已在世界各地进行了广泛引种与养殖。但是自2009年以来,罗非鱼链球菌病在罗非鱼养殖中大规模暴发且频繁发生与流行,给罗非鱼养殖产业造成了巨大的经济损失。培育罗非鱼抗病品系是解决罗非鱼链球菌病害的重要途径之一。分子标记辅助育种是一种高效的育种方法,利用分子标记可以定向选育罗非鱼抗病品系,增加选育强度从而加速其遗传改良进程。单核苷酸多态性(single nucleotide polymorphism,SNP)因具有标记数量多且易于检测等特点成为MAS中最为常用的分子标记。Ikaros是一种C2H2型锌指蛋白类转录因子,对淋巴细胞的正确发育及其免疫功能的正常发挥具有重要作用,但其在罗非鱼中的研究还未见相关报道。因此,开展尼罗罗非鱼(Oreochromis niloticus)免疫相关候选基因Ikaros基因的相关功能研究,并从中筛选抗病相关SNP分子标记,这可为实现分子标记辅助罗非鱼抗病选育奠定基础。本研究对尼罗罗非鱼Ikaros基因的cDNA序列和基因组DNA序列进行了克隆,对基因的结构特征、组织分布及其对无乳链球菌感染的响应进行分析,初步了解Ikaros基因的生物学功能;对Ikaros基因5'调控区序列中的SNPs进行检测,并与无乳链球菌(Streptococcus agalactiae)抗性进行相关性分析,以期获得抗病相关的SNPs,为尼罗罗非鱼遗传育种研究提供新的分子标记,研究结果具体如下:1.尼罗罗非鱼Ikaros基因的克隆与组织表达本试验采用RT-PCR和RACE等方法克隆了尼罗罗非鱼Ikaros基因的cDNA序列以及利用PCR和染色体步移技术克隆了Ikaros的基因组DNA序列。对Ikaros基因序列分析发现,Ikaros基因组DNA为20454 bp,包括7个内含子和8个外显子,经可变剪接可形成6种不同的mRNA剪接异构体,其编码的氨基酸序列均具有Ikaros家族典型的锌指结构域且与硬骨鱼类Ikaros氨基酸序列同源性较高(70.6%~93.7%)。采用实时荧光定量PCR分析了Ikaros mRNA在尼罗罗非鱼中的组织分布及其对无乳链球菌感染的响应。结果显示,Ikaros基因在健康尼罗罗非鱼11种被检测的组织或器官中均有表达,其中在血液中的表达量最高,其次为胸腺、脾脏和头肾。人工感染无乳链球菌后,血液、胸腺、脾脏、头肾中Ikaros基因的相对表达量均显著上调,并在48h达到峰值,这表明Ikaros基因参与调控尼罗罗非鱼抵御无乳链球菌的免疫应答反应。2.尼罗罗非鱼Ikaros基因5'调控区序列的克隆与分析通过染色体步移法从尼罗罗非鱼的基因组中克隆获得Ikaros基因5'调控区序列,长度为4178 bp。使用在线生物信息学软件对Ikaros基因5'调控区序列进行预测与分析,预测尼罗罗非鱼Ikaros基因的转录起始位点位于起始密码子(ATG)上游931 bp位置,核心启动子位于转录起始位点-57 bp~48 bp的区间;预测的Ikaros基因启动子区域具有真核生物启动子基本结构元件:TATA框、CCAAT框、八聚体元件等。转录因子结合位点预测显示,在Ikaros基因5'调控区-2200 bp~1200bp的序列中存在丰富的转录因子结合位点,包含GATA-1、Homeobox、CDP CR3+HD、AP-1等。CpG岛分析显示Ikaros基因具有2个CpG岛,分别位于启动子和第一外显子上。3.尼罗罗非鱼Ikaros基因5'调控区序列的SNPs及其与无乳链球菌抗性的关联分析采用直接测序法在亲本(F0)尼罗罗非鱼的Ikaros基因5'调控区序列中筛查到5个SNPs,分别将其命名为SNP1(g.562,GA)、SNP2(g.217,GT)、SNP3(g.-53,CT)、SNP4(g.-220,TC)和SNP5(g.-579,TC)。经分析发现这些SNPs均分布在启动子的各种调控元件中,可能会对Ikaros基因的精确表达产生重要影响。通过Snapshot方法对5个SNPs在子一代(F1)尼罗罗非鱼易感群体和抗病群体中进行基因分型及与无乳链球菌抗性进行关联分析,结果发现SNP2(g.217,GT)、SNP3(g.-53,CT)、SNP4(g.-220,TC)和SNP5(g.-579,TC)的基因型频率和等位基因频率在易感群体和抗病群体中存在显著差异(P0.05),表明这4个SNPs与无乳链球菌抗性显著相关。连锁不平衡分析发现,这5个SNPs可形成一个单倍块和5种单倍型,其中GGCTT单倍型与抗病性显著相关(P0.05),GGTCT和GTCCC单倍型与易感性显著相关(P0.05)。此外,还发现SNP2(g.217,GT)和SNP5(g.-579,TC)处于完全连锁状态(r2=1,LOD=57.25,D'=1),可作为罗非鱼遗传育种的标签SNP。
[Abstract]:Tilapia is one of the most important aquaculture species in the world, with fast growth, high productivity, strong adaptability and wide feeding habits etc., have carried out extensive introduction and Cultivation in the world. But since 2009, tilapia streptococcus disease in tilapia culture in large-scale outbreaks and frequent occurrence and epidemic, caused huge economic losses for tilapia aquaculture industry. Cultivating tilapia varieties is one of the important ways to solve the tilapia streptococcus disease. Molecular marker assisted breeding is an efficient breeding method, can be set to the disease resistant strain of Nile tilapia by molecular marker, increase strength to accelerate the breeding progress of genetic improvement. Single nucleotide polymorphism (single nucleotide polymorphism, SNP) because of the number of markers and easy to detect such characteristics as MAS is the most commonly used molecular markers of.Ikaros is a C2H2 type zinc The protein transcription factor, on lymphocytes development and immune function in normal play an important role, but in Tilapia research has not been reported. Therefore, to carry out the Nile tilapia (Oreochromis niloticus) function of immune related gene Ikaros gene, and screening resistance related molecular markers from SNP. This can lay the foundation for molecular marker assisted breeding of disease resistance. The study of tilapia Oreochromis niloticus Ikaros gene cDNA sequence and genomic DNA sequences were cloned, the structural characteristics of the gene, tissue distribution and the response of group B streptococcus infection were analyzed, a preliminary understanding of the biological function of Ikaros gene; detection of regulatory sequences the Ikaros gene in 5'and SNPs, and Streptococcus agalactiae (Streptococcus agalactiae) resistance correlation analysis, in order to obtain resistance The SNPs provides a new molecular marker for genetic breeding of Tilapia nilotica, the results of the study are as follows: the expression of the test using RT-PCR and RACE methods to clone the Ikaros gene of Nile tilapia cDNA sequence and the use of PCR and chromosome walking technique to clone the genomic DNA sequence Ikaros gene and Ikaros gene in 1. tissues of Nile tilapia of the Ikaros gene. Sequence analysis showed that the genome of Ikaros DNA was 20454 BP, including 7 introns and 8 exons, by alternative splicing can form 6 kinds of mRNA splice variants, encoding the amino acid sequence of the Ikaros family have typical zinc finger domain and teleost Ikaros amino acid sequence high (70.6%~93.7%). By using real-time quantitative PCR analysis of tissue distribution of Ikaros mRNA in Nile tilapia and the response of Streptococcus agalactiae infection. The results showed that I The Karos gene in 11 healthy tilapia were detected in the tissues or organs were expressed, the expression in blood is the highest, followed by the thymus, head kidney and spleen. The artificial infection of Streptococcus agalactiae, blood, thymus, spleen, head kidney Ikaros gene expression were significantly up-regulated, and achieve the peak at 48h, which indicates that the cloning and analysis of Ikaros gene involved in the regulation of immune responses against the Nile tilapia Oreochromis niloticus.2. Ikaros 5'gene regulatory sequence of Streptococcus agalactiae was cloned from the Nile tilapia genome sequences in promoter region of Ikaros gene 5' by chromosome walking method, the length of 4178 bp. using bioinformatics software prediction and analysis of the promoter region of Ikaros gene 5'sequence, the predicted transcription start site of tilapia Ikaros gene in initiation codon (ATG) 931 bp upstream of the core position, Rev. The dynamic range of sub transcription initiation site is located -57 bp~48 BP; Ikaros gene promoter prediction with eukaryotic promoter basic structural element sub region: TATA box, CCAAT box, eight poly element. Transcription factor binding site prediction shows that in the sequence of Ikaros gene 5'regulatory region of -2200 bp~ 1200bp in the presence of abundant transcription factor the binding sites, including GATA-1, Homeobox, CDP, CR3+HD, AP-1 and.CpG analysis showed that Ikaros gene has 2 Island CpG Island, located in the promoter and the first exon sequences in promoter region of.3. gene 5' SNPs Ikaros Nile tilapia and the resistance of Streptococcus agalactiae and correlation analysis by direct sequencing in the parent (F0) Ikaros 5'gene control region sequence of Nile tilapia screening to 5 SNPs, named SNP1 (g.562, GA), SNP2 (g.217, GT), SNP3 (g.-53, CT), SNP4 (g.-220, TC) and SNP5 (g.-579, TC). The analysis shows that this Some SNPs are distributed in a variety of regulatory elements in the promoter, may have an important effect on the expression of Ikaros gene will be accurate. Through the method of Snapshot 5 SNPs in the first generation of Nile tilapia (F1) in susceptible and resistant populations were genotyped and association analysis with Streptococcus agalactiae resistance results SNP2 (g.217, GT), SNP3 (g.-53, CT), SNP4 (g.-220, TC) and SNP5 (g.-579, TC) genotype and allele frequencies in susceptible and resistant populations showed significant difference in (P0.05), the 4 SNPs showed significant correlation with the resistance of Streptococcus agalactiae. Analysis of linkage disequilibrium, the 5 SNPs can form a single block and 5 haplotypes, the GGCTT haplotype was significantly associated with disease resistance (P0.05), GGTCT and GTCCC haplotypes were significantly correlated with susceptibility (P0.05). In addition, also found that SNP2 (g.217, GT) and SNP5 (g.-579, TC) is completely chain shape State (r2=1, LOD=57.25, D'=1), which can be used as a label SNP. for the genetic breeding of Tilapia

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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