Zhu Huaping

Effect of low temperature on genomic DNA methylation in Nile tilapia(Oreochromis niloticus)

In this study,using the methylation sensitive amplified polymorphism(MSAP) method,we globally assessed the effect of low temperature adaptation on the level and variations of cytosine methylation alterations at CCGG sites of genomic DNA in Nile tilapia(Oreochromis niloticus) strains with good coldresistance characters developed by the successive and directional selection breeding of multi-generations. Total of 849 fragments were identified by 18 pairs of se1ective primer combinations of which 411 and 438 were detected in cold-resistance tilapia strain and the control,respectively. Among these fragments,the methylation sites were 72 and 104,and the corresponding total-methylation levels w ere at 17.52% and 23.74%,respectively. The full methylation sites at the internal Cs were 37 and 65,and the corresponding full methylation levels were at 9. 00% and 14. 84%,respectively,and the hemi-methylation sites at the external Cs were 35 and 39,and the corresponding hemi-methylation levels were at 8.52% and 8.90% in coldresistance tilapia strain and the control,respectively. Further analysis of DNA methylation indicated that a statistically decrease in the overall level of total-methylation levels and both methylation types was detected in cold-resistance tilapia strain compared with the control(decrease at 6.22%,5.84% and 0.38%, respectively),and the changes of DNA methylation pattern were recognized mainly in the full methylation sites. The level of decrease in DNA methylation suggested that the levels of genomic DNA methylation were changed during the successive cold stress of multi-generations in tilapia,and it also indicated that DNA methylation alteration in tilapia treated with cold stress was mainly through de-methylation that occurred in some CCGG sites. All these results implied that the change of DNA methylation was closely associated with cold tolerance of tilapia. The present findings are valuable to further explore the application potential of DNA methylation alteration in tilapia genetic improvement and provide a new method and theoretical basis for fish stress resistance breeding.

Molecular cloning, tissue distribution of two estrogen receptor β cDNAs of blue tilapia (Oreochromis aureus) and effects of estrogen on their expression.

Two sub-type estrogen receptor β (ERβ1/β2) cDNAs were obtained from blue tilapia,Oreochromis aureus by Reverse Transcription Polymerase Chain Reaction (RT-PCR) and RACE. The full length cDNA sequence of ERβ1 was 4 262 bp in length,containing a 5 Untranslated Regions (UTR) of 239 bp,3 UTR of 2 349 bp and an open reading fame (ORF) of 1 674 bp which encoded a putative protein of 557 amino acids with an estimated molecular weight of 62 ku. The ERβ2 cDNA was 2 506 bp in length with 393 bp of 5 UTR,109 bp of 3 UTR and an ORF of 2 004 bp which encoded a putative protein of 668 amino acids with an estimated molecular weight of 74 ku. The deduced amino acid sequence of ERβ1 of blue tilpia possessed 99.1% similarity with the counterpart of nile tilapia,O. niloticus,and similarities with other Perciformes fishes were from 82.6% to 94.2%. The ERβ2 amino acid sequences of blue tilpia shared 98.7%,81.8%,76.3%,64.7% and 55.0% identities with nile tilapia,Micropterus salmoides,Oncorhynchus mykiss,Fundulus heteroclitus and Danio rerio respectively. Blue tilpia ERβ1/β2 were clustered with nile tilapia ERβ1/β2 respectively in the phylogenetic tree. ERβ1/β2 mRNA expressions between male and female were compared by Real-Time PCR (RT-PCR). ERβs were expressed ubiquitously and differently in ten tested tissues. ERβ1 was significantly expressed in ovary,testis,liver,kidney and intestinal tissues compared with other tissues (P0.05). ERβ2 was highly expressed in testis,liver and kidney (P0.05). In ovary,testis,intestine and hypothalamus,expression of ERβ1 was significantly higher than that of ERβ2,and it was 164 times of ERβ2 in ovaries. After 17 beta-estradiol (E2) was injected for 24 h,ERα/β1 mRNA in hypothalamus of male blue tilpia was increased,of which ERα increased significantly. Expression of ERα in hypothalamus increased significantly in 7.5 mg/kg E2 treated group,while no significant change was observed for ERβ2. In pituitary,expression of ERα/β1/β2 in the low and middle E2 concentrations (2.5,5.0 mg/kg) was down-regulated,but up-regulated in high E2 concentration group (7.5 mg/kg),and significant difference was found for ERα (P0.05). ERβ2 expression was increased in testis significantly (P0.05) in middle and high E2 concentration groups (5.0,7.5 mg/kg). No significant change was observed for ERα/β1 expression. The different distribution of the ERβ1/β2 mRNAs and different regulation of ERα/β1/β2 mRNA in hypothalamus,pituitary and testis by E2 indicated that various biological functions existed among these three ER subtypes in blue tilpia.