Analysis Of The Genomic DNA Methylation Between Mohe Tilapia "Guangfu No.1" And Its Parents

The Mohe tilapia “Guangfu No.1”(Oreochromis mossambicus?×O.hornorum?)could be cultured in 0‰-30‰ salinity water,which has an obvious heterosis in the salt-tolerant traits compared with the parents.In this study,the DNA methylation differences between the Mohe tilapia “Guangfu No.1” and its parents were analyzed and the relation between the methylation differences and the heterosis was discussed.Using methylation differential fragment analysis,the prkaca gene was obtained,and the sequence structure of prkaca gene and gene expression under saline-alkali stress were analyzed.Furthermore,the relationship between the methylation level of prkaca gene promoter and the gene expression was analyzed by BSP technique.The results may be important for screening the molecular markers of the Mohe tilapia “Guangfu No.1” and study on the functional mechanism of genes related to saline-alkali stress.1.Analysis of genomic DNA methylation differences between Mohe tilapia “Guangfu No.1”and its parentsThe method of methylation sensitive amplified polymorphism(MSAP)was used to analyze the DNA methylation difference and the change of methylation pattern in eleven tissues of Oreochromis mossambicus,Oreochromis hornorum and Mohe tilapia “Guangfu No.1”,such as skin,muscle and gill etc.The results showed that there were differences in the methylation levels among these different tissues of the same species and among different species of the same tissues;the average methylation level of these tissues from O.mossambicus,O.hornorum and Mohe tilapia “Guangfu No.1” was 38.03%,32.21% and 29.77%,respectively.The total methylation level of hybrid tilapia was lower than the parents.75.08% methylation patterns of cytosine in Mohe tilapia “Guangfu No.1” were similar to its parents,while 24.92% cytosine demethylation and hypermethylation were found in the genome of Mohe tilapia “Guangfu No.1”,the number of demethylation sites(E type,15.73%)was more than that of hypermethylation sites(D type,9.19%).In addition,the fragmentes displaying DNA methylation differences between the hybrid progenies and parental progenies were sequenced and the prkaca gene obtained was used as the target gene in the next step.2.Molecular cloning and expression of prkaca gene in Mohe tilapia “Guangfu No.1” and its parentsProtein kinase A(PKA),a holoenzyme exists as a tetramer,is comprised of a regulatory(R)subunit dimer and two catalytic(C)subunits.Upon binding of two molecules of the second messenger cAMP to each R subunit,a conformational change in the PKA holoenzyme occurs to release the C subunits and play a biological role.The prkaca gene is a member of the protein kinase A(PKA)family,which encodes a catalytic subunit of PKA.The primers for the amplification of the prkaca gene indentifed in the methylation differential fragment analysis were designed according to the prkaca gene sequence of Nile tilapia(Oreochromis niloticus).The results showed that the complete cDNA sequences of prkaca gene of O.mossambicus,O.hornorum and Mohe tilapia “Guangfu No.1” were 4975 bp,including 1056 bp open reading frame,encoding 351 amino acids.Amino acid domain prediction analysis showed that Prkaca amino acid sequence includes a serine/threonine protein kinase catalytic domain(S_TKc)and a serine/threonine protein kinase elongation domain(S_TK_X).Multiple alignment and homological analysis revealed that the Prkaca amino acid sequence of the three tilapias is similar and shared high levels of amino acid identity with Prkaca amino acid sequence from Oreochromis niloticus(100%),Maylandia zebra(100%)and Haplochromis burtoni(100%),as well as the identity at 93.7-99.4% with Prkaca amino acid sequence of other fishes.Real-time quantitative PCR(qPCR)results demonstrated that there was a significant difference in the expression level of prkaca gene in 10 tissues of O.mossambicus and O.hornorum.The expression level of prkaca gene in brain tissue was highest and was significantly higher than those in other tissues(P <0.01).The expression change of prkaca gene of muscle,gill,kidney and brain tissue at different time points(0 h,24 h,48 h,72 h and 96 h)were measured after saline-alkali stress(salinity 10,alkalinity 3 g/L,S10A3)in O.mossambicus,O.hornorum,Mohe tilapia “Guangfu No.1” and O.niloticus.Change of prkaca gene expression in the four tilapia showed the same trend that it firstly increased significantly(P<0.05),then decreased significantly and returned to the original level.The expression of prkaca gene in Mohe tilapia “Guangfu No.1” was significantly up-regulated at 24 h,while the parents were significantly up-regulated at 72 h.It means that the prkaca gene is highly responsive to saline-alkali challenge in the four tissues and involved in the osmotic regulation of tilapia,and the Mohe tilapia “Guangfu No.1” has a fast response mechanism to maintain the osmotic pressure balance in saline-alkali changed water environment.3.Analysis of DNA methylation level of prkaca gene in Mohe tilapia “Guangfu No.1” and its parentFirstly,the genomic sequences of prkaca gene were cloned.The length of prkaca genomic sequence amplified by PCR was 21207 bp,containing 11 exons and 10 introns.The possible CpG islands were analyzed by MethPrimer and JASPAR software online and the 416-bp(-544 to-129 region)promoter sequence containing four transcription factor binding sites was selected as the target fragment for methylation analysis.On the one hand,the methylation level of prkaca gene promoter of the muscle and kidney tissue in Mohe tilapia “Guangfu No.1” was detected using Bisulfite sequencing PCR(BSP)methods,and the relationship between methylation level and gene expression was analyzed.The results showed that the methylation level of the target fragment in the muscle tissue decreased significantly at 24 h(P<0.05)and increased significantly at 72 h(P<0.05).The degree of methylation of the target fragment in kidney at 48 h was higher significantly than that at 0 h(P<0.05).The results indicated that the DNA methylation level of prkaca gene promoter was negatively correlated with gene expression level.On the other hand,the differences in methylation level between Mohe tilapia “Guangfu 1” and its parents in the prkaca gene promoter region were analyzed.There was no significant difference in the methylation level of prkaca gene promoter in the muscle tissue between Mohe tilapia “Guangfu No.1” and its parents(P>0.05).While in the kidney tissue,the methylation level of the prkaca gene promoter of Mohe tilapia “Guangfu No.1” was significantly lower than its parents(P<0.05).The forms of prkaca gene methylation may be one of the manifestations of lower methylation levels of genomic DNA in Mohe tilapia “Guangfu No.1”.