Molecular Cloning And Expression Analysis Of Ikaros Gene From Oreochromis Niloticus And Screening Of Its SNP Markers For Streptococcus Agalactiae Resistance

Tilapia 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 omnivorous feeding habit.However,Streptococcus disease has become the biggest threat for tilapia breeding.Recently,the epidemic and outbreak of tilapia Streptococcus disease caused substantial economic losses to aquaculture industry.Breeding resistant variety in tilapia was an important way to solve the problem of Streptococcus disease.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.However,the research of Ikaros gene in tilapia has not been reported till now.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 thesis,the Ikaros gene of O.niloticus was cloned and characterized,and its structural characteristic and expression patterns were analyzed to understand its biological function.Furthermore,we also detected the SNPs in 5’ regulatory region sequence of Ikaros gene,and performed association analysis of these SNPs with resistance to Streptococcus agalactiae in O.niloticus.The study provided new available molecular markers for research of genetic breeding in O.niloticus.The major results were as follows: 1.Molecular cloning and expression of Ikaros gene in O.niloticusThe cDNA of Ikaros were obtained from O.niloticus through RT-PCR and RACE methods and the genomic DNA of Ikaros were obtained by using PCR and Genome Walking technique.Quantitative real-time PCR was used for analysing the organization distribution and the response of Ikaros to S.agalactiae infection.The genomic DNA was 20545 bp with 7 introns and 8 exons encoding 6 kinds of mRNA splicing isoform via alternative splicing.The deduced amino acid sequences of six splicing isoforms with zinc finger domain had high homology(70.6% ~ 93.7%)with other teleost fish.The Ikaros gene expressed in all of 11 tested healthy tissue or organs,strongly expressed in blood and moderately expressed in thymus,spleen and head kidney.After challenged with pathogenic bacteria S.agalactiae,the Ikaros gene expression levels were up-regulated in blood,thymus,spleen and head kidney,then peaked at 48 hours after challenge.The results suggested that Ikaros gene participated in the immune response of O.niloticus against S.agalactiae infection.2.Cloning and analysis of 5’ regulatory region sequence of Ikaros gene in O.niloticusThe 5’ regulatory region sequence of Ikaros,length of 4178 bp,were obtained through Genome Walking method from O.niloticus.Online bioinformatics software was used to analyze the 5’ regulatory region sequence of Ikaros gene.The predicted transcriptional 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 included basic start of substructure components: TATA box,CCAAT box and octamer.The analysis of transcription factor binding sites showed that abundant of transcription factor binding sites were located at-2200 bp to 1200 bp in 5’ regulatory region sequence 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.3.Identification of SNPs in the 5’ regulatory region sequence of Ikaros gene and their association with resistance to S.agalactiae in O.niloticusFive SNPs in the 5’ regulatory region sequence of Ikaros gene were detected by direct sequencing method from the parents(F0),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 and resistant groups of O.niloticus from the first filial generation(F1).The correlation between SNPs and resistance to S.agalactiae was analyzed.The results showed that four of them(SNP2(g.217,G>T),SNP3(g.-53,C>T),SNP4(g.-220,T>C)and SNP5(g.-579,T>C))were significantly associated with the resistance to S.agalactiae(p<0.05).Linkage disequilibrium analysis shows that the five SNPs could formed one haplotype block and five haplotypes.The haplotypes(GGCTT)were significantly associated with the resistance to S.agalactiae(p<0.05),and two of the haplotypes(GGTCT and GTCCC)were significantly associated with S.agalactiae susceptible(p<0.05).In addition,the SNP2(g.217,G>T)and SNP5(g.-579,T>C)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.