中国水产科学2018，Vol.25Issue(2)：237-250,14.DOI:10.3724/SP.J.1118.2018.17248
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
Molecular cloning analysis of the 5' regulatory region of Ikaros gene from Oreochromis niloticus and screening of its SNP markers for Streptococcus agalactiae resistance
摘要
Abstract
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.关键词
尼罗罗非鱼/Ikaros基因5'调控区/单核苷酸多态性/无乳链球菌Key words
Oreochromis niloticus/5'regulatory region of Ikaros gene/single nucleotide polymorphism/Strepto-coccus agalactiae分类
农业科技引用本文复制引用
陈昆平,卢迈新,刘志刚,高风英,曹建萌,张德锋,李庆勇,胡欣欣..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[J].中国水产科学,2018,25(2):237-250,14.基金项目
现代农业产业技术体系专项资金项目(CARS-46) （CARS-46）
广东省自然科学基金项目(2016A030313146) （2016A030313146）
国家自然科学基金项目(31502205). （31502205）
