吉富尼罗罗非鱼Ikaros基因5'调控区的克隆、序列分析及抗无乳链球菌相关SNP位点筛选

摘要

为获得尼罗罗非鱼(Oreochromis niloticus)抗链球菌病相关的单核苷酸多态性(single nucleotide polymorphism, SNP)标记,本研究通过染色体步移法克隆了尼罗罗非鱼Ikaros基因5'调控区序列,长度为4178 bp,并使用生物信息学软件对 Ikaros基因的启动子和转录调控元件进行预测和分析.利用PCR产物直接测序法从亲本(P0)尼罗罗非鱼Ikaros基因5'调控区中筛查到5个SNPs,分别为SNP1(g.562, G>A)、SNP2(g.217, G>T)、SNP3(g.–53, C>T)、SNP4(g.–220, T>C)和SNP5(g.–579, T>C).利用Snapshot技术对子一代(F1)尼罗罗非鱼易感群体和抗病群体进行相应 SNPs 的基因分型, 分析两个群体遗传多样性参数, 结果显示多态信息含量(polymorphism information content, PIC)值在0.0872~0.3747之间,表明Ikaros基因5'调控区中所有SNPs的多态性均较低.Ikaros基因5'调控区SNPs与抗链球菌病性状关联分析结果表明, SNP2、SNP3、SNP4和SNP5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(P<0.05).连锁不平衡分析结果显示Ikaros基因5'调控区SNPs可形成1个单倍块和5种单倍型, 其中GGCTT单倍型与抗病性显著相关(P<0.05), GGTCT和GTCCC单倍型与易感性显著相关(P<0.05).此外, 还发现SNP2和SNP5处于完全连锁状态(r2=1, LOD=57.25, D'=1), 可作为罗非鱼抗链球菌病遗传育种的标签SNP.综上所述,在Ikaros基因5'调控区筛选到4个抗链球菌病相关SNPs和1个单倍型(GGCTT),均可作为尼罗罗非鱼分子育种的候选分子标记.%Tilapia(Oreochromis)is one of the most important fishery species in the world and has been introduced and cultured widespreadly because of its fast growth rate, strong reproductive capacity, good adaptability and om-nivorous feeding habit. However, Streptococcus disease has become the biggest threat for tilapia breeding. Re-cently,the epidemic and outbreak of tilapia Streptococcus disease caused substantial economic losses to aquacul-ture industry.Breeding resistant variety in tilapia was unimportant way to solve the problem of Streptococcus dis-ease. The molecular marker assisted selection (MAS) has become an efficient breeding method for selecting and breeding tilapia, and will accelerate genetic improvement and increase selection intensity for disease resistance. The single nucleotide polymorphism (SNP) markers are used in many genetic and breeding studies because they are abundant in genomes, and can be genotyped easily. Ikaros is a kind of transcription factor with zinc finger structure that is essential to the development of lymphocyte and to the maintenance of normal immune function. Therefore, to obtain large amount of effective SNP molecular genetic markers and to perform MAS for disease resistance in tilapia,it is essential to study immune related candidate Ikaros gene in Oreochromis niloticus and to examine whether the SNPs in the gene are associated with disease resistance. In this study, the 5' regulatory region sequence of Ikaros,length of 4178 bp,were obtained through Genome Walking method from O.niloticus.Bioin-formatics software was used to analyze the 5'regulatory region sequence of Ikaros gene.The predicted transcrip-tional start site (TSS) was in the initiation codon (ATG) upstream of 931 bp, and the core promoter regions was located at –57 bp to 48 bp when the TSS was specified as 1. The predicted promoter regions of Ikaros gene in-cluded basic start of substructure components: TATA box, CCAAT box and octamer. The analysis of transcription factor binding sites (TFBS) showed that abundant of TFBS were located at –2200 bp to 1200 bp in 5' regulatory region of Ikaros gene, such as GATA-1, Homeobox, CDP CR3+HD and AP-1. The analysis of CpG islands showed that two CpG islands were in 5' regulatory region sequence of Ikaros gene, one of which was located in promoter regions and the other of which was located in the first exon. Five SNPs in the 5' regulatory region of Ikaros gene were detected by direct sequencing method from the parents (P0), which are named SNP1 (g.562, G>A), SNP2(g.217, G>T), SNP3(g.–53, C>T), SNP4(g.–220, T>C) and SNP5(g.–579, T>C). The five SNPs were sited in various regulatory elements in promoter regions which could have major implications for exact expression of Ikaros gene. Based on Snapshot method analysis, the frequencies of alleles and genotypes were calculated in susceptible groups and resistant groups of O.niloticus from the first filial generation(F1).The polymorphisms and genetic parameter of the SNPs in resistant groups and susceptible groups were calculated by software Popgen 32 and PIC.The result showed that the polymorphism information content(PIC)of the 5'regulatory region of Ikaros SNPs was 0.0872~0.3747, suggesting that all the SNPs had moderate of intermediate polymorphism. The correla-tion between SNPs and resistance to S. agalactiaewas analyzed by software SPSS 17.0, the results showed that four of them (SNP2, SNP3, SNP4and SNP5) were significantly associated with the resistance to S. agalactiae(P<0.05).Moreover,All of the SNPs in the 5'regulatory region of Ikaros could formed one haplotype block and five haplotypes from the prediction of linkage disequilibrium analysis used software Haploview 4.2. The haplo-types(GGCTT)were significantly associated with the resistance to S.agalactiae(P<0.05),and two of the haplo-types(GGTCT and GTCCC)were significantly associated with S.agalactiae susceptible(P<0.05).Furthermore, the SNP2 and SNP5 were completely linked with each other (r2=1, LOD=57.25, D'=1), which could be selected as tag SNP for research of genetic breeding in O. niloticus. The results suggest that the four SNPs (SNP2, SNP3, SNP4 and SNP5) and the haplotypes (GGCTT) in the 5' regulatory region of Ikaros could be potential genetic markers for future molecular selection of O.niloticus resistant to S.agalactiae.