本文選題：尼羅羅非魚　切入點：Ikaros基因　出處：《上海海洋大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：羅非魚是世界上最重要的水產(chǎn)養(yǎng)殖品種之一,具有生長快、繁殖力高、適應(yīng)性強和食性廣等優(yōu)點,已在世界各地進(jìn)行了廣泛引種與養(yǎng)殖。但是自2009年以來,羅非魚鏈球菌病在羅非魚養(yǎng)殖中大規(guī)模暴發(fā)且頻繁發(fā)生與流行,給羅非魚養(yǎng)殖產(chǎn)業(yè)造成了巨大的經(jīng)濟(jì)損失。培育羅非魚抗病品系是解決羅非魚鏈球菌病害的重要途徑之一。分子標(biāo)記輔助育種是一種高效的育種方法,利用分子標(biāo)記可以定向選育羅非魚抗病品系,增加選育強度從而加速其遺傳改良進(jìn)程。單核苷酸多態(tài)性(single nucleotide polymorphism,SNP)因具有標(biāo)記數(shù)量多且易于檢測等特點成為MAS中最為常用的分子標(biāo)記。Ikaros是一種C2H2型鋅指蛋白類轉(zhuǎn)錄因子,對淋巴細(xì)胞的正確發(fā)育及其免疫功能的正常發(fā)揮具有重要作用,但其在羅非魚中的研究還未見相關(guān)報道。因此,開展尼羅羅非魚(Oreochromis niloticus)免疫相關(guān)候選基因Ikaros基因的相關(guān)功能研究,并從中篩選抗病相關(guān)SNP分子標(biāo)記,這可為實現(xiàn)分子標(biāo)記輔助羅非魚抗病選育奠定基礎(chǔ)。本研究對尼羅羅非魚Ikaros基因的cDNA序列和基因組DNA序列進(jìn)行了克隆,對基因的結(jié)構(gòu)特征、組織分布及其對無乳鏈球菌感染的響應(yīng)進(jìn)行分析,初步了解Ikaros基因的生物學(xué)功能;對Ikaros基因5'調(diào)控區(qū)序列中的SNPs進(jìn)行檢測,并與無乳鏈球菌(Streptococcus agalactiae)抗性進(jìn)行相關(guān)性分析,以期獲得抗病相關(guān)的SNPs,為尼羅羅非魚遺傳育種研究提供新的分子標(biāo)記,研究結(jié)果具體如下:1.尼羅羅非魚Ikaros基因的克隆與組織表達(dá)本試驗采用RT-PCR和RACE等方法克隆了尼羅羅非魚Ikaros基因的cDNA序列以及利用PCR和染色體步移技術(shù)克隆了Ikaros的基因組DNA序列。對Ikaros基因序列分析發(fā)現(xiàn),Ikaros基因組DNA為20454 bp,包括7個內(nèi)含子和8個外顯子,經(jīng)可變剪接可形成6種不同的mRNA剪接異構(gòu)體,其編碼的氨基酸序列均具有Ikaros家族典型的鋅指結(jié)構(gòu)域且與硬骨魚類Ikaros氨基酸序列同源性較高(70.6%~93.7%)。采用實時熒光定量PCR分析了Ikaros mRNA在尼羅羅非魚中的組織分布及其對無乳鏈球菌感染的響應(yīng)。結(jié)果顯示,Ikaros基因在健康尼羅羅非魚11種被檢測的組織或器官中均有表達(dá),其中在血液中的表達(dá)量最高,其次為胸腺、脾臟和頭腎。人工感染無乳鏈球菌后,血液、胸腺、脾臟、頭腎中Ikaros基因的相對表達(dá)量均顯著上調(diào),并在48h達(dá)到峰值,這表明Ikaros基因參與調(diào)控尼羅羅非魚抵御無乳鏈球菌的免疫應(yīng)答反應(yīng)。2.尼羅羅非魚Ikaros基因5'調(diào)控區(qū)序列的克隆與分析通過染色體步移法從尼羅羅非魚的基因組中克隆獲得Ikaros基因5'調(diào)控區(qū)序列,長度為4178 bp。使用在線生物信息學(xué)軟件對Ikaros基因5'調(diào)控區(qū)序列進(jìn)行預(yù)測與分析,預(yù)測尼羅羅非魚Ikaros基因的轉(zhuǎn)錄起始位點位于起始密碼子(ATG)上游931 bp位置,核心啟動子位于轉(zhuǎn)錄起始位點-57 bp~48 bp的區(qū)間;預(yù)測的Ikaros基因啟動子區(qū)域具有真核生物啟動子基本結(jié)構(gòu)元件:TATA框、CCAAT框、八聚體元件等。轉(zhuǎn)錄因子結(jié)合位點預(yù)測顯示,在Ikaros基因5'調(diào)控區(qū)-2200 bp~1200bp的序列中存在豐富的轉(zhuǎn)錄因子結(jié)合位點,包含GATA-1、Homeobox、CDP CR3+HD、AP-1等。CpG島分析顯示Ikaros基因具有2個CpG島,分別位于啟動子和第一外顯子上。3.尼羅羅非魚Ikaros基因5'調(diào)控區(qū)序列的SNPs及其與無乳鏈球菌抗性的關(guān)聯(lián)分析采用直接測序法在親本(F0)尼羅羅非魚的Ikaros基因5'調(diào)控區(qū)序列中篩查到5個SNPs,分別將其命名為SNP1(g.562,GA)、SNP2(g.217,GT)、SNP3(g.-53,CT)、SNP4(g.-220,TC)和SNP5(g.-579,TC)。經(jīng)分析發(fā)現(xiàn)這些SNPs均分布在啟動子的各種調(diào)控元件中,可能會對Ikaros基因的精確表達(dá)產(chǎn)生重要影響。通過Snapshot方法對5個SNPs在子一代(F1)尼羅羅非魚易感群體和抗病群體中進(jìn)行基因分型及與無乳鏈球菌抗性進(jìn)行關(guān)聯(lián)分析,結(jié)果發(fā)現(xiàn)SNP2(g.217,GT)、SNP3(g.-53,CT)、SNP4(g.-220,TC)和SNP5(g.-579,TC)的基因型頻率和等位基因頻率在易感群體和抗病群體中存在顯著差異(P0.05),表明這4個SNPs與無乳鏈球菌抗性顯著相關(guān)。連鎖不平衡分析發(fā)現(xiàn),這5個SNPs可形成一個單倍塊和5種單倍型,其中GGCTT單倍型與抗病性顯著相關(guān)(P0.05),GGTCT和GTCCC單倍型與易感性顯著相關(guān)(P0.05)。此外,還發(fā)現(xiàn)SNP2(g.217,GT)和SNP5(g.-579,TC)處于完全連鎖狀態(tài)(r2=1,LOD=57.25,D'=1),可作為羅非魚遺傳育種的標(biāo)簽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

【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S917.4

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