Cloning And Expression Characterization Analysis Of AQP1 And MTOR Genes Related To Salt-alkaline Tolerance In Tilapia

Tilapia is a warm water fish belonging to Perciformes, cichlids. Tilapia is an important freshwater culture fish currently due to the high growth rates, and easy reproduce. Saline-alkali in water is one of the important factors affecting the osmotic balance of fishes, which cause organic damage and thereby affect the osmotic balance of fishes through the high p H, high salinity, high alkalinity. AQP1 and m TOR are associated with salt-alkali tolerance genes in fish. In this study, acute alkali stress was performed and the characterizations of salt-alkaline tolerance were analyzed in Oreochromis mossambicus, O. hornorum and their hybrids(O. mossambicus♀×O. hornorum ♂). The AQP1 and m TOR genes were cloned and the gene expression characterization on salt-alkali tress in the tissues was studied using real-time quantitative PCR between the three tilapias and Nile tilapia. The research results will provide references for study the mechanisms of salt-alkali tolerance in tilapia and fish breeding in future researches.1 The preliminary study of the acute alkali stress of O. mossambicus, O. hornorum and their hybrids(O. mossambicus♀×O. hornorum♂) In order to assess the alkalinity(Na HCO3) tolerance of the three tilapias, we determined the 96 h median lethal concentration(LC50) of Oreochromis mossambicus, O. hornorum and the hybrids(O. mossambicus♀×O. hornorum♂). The results of acute alkali stress show that the 96 h LC50 of O. mossambicus, O. hornorum and the hybrids(O. mossambicus♀×O. hornorum♂) are 3.99 g/L, 6.67 g/L and 4.96g/L under the conditions of p H 9.3-9.5, respectively. The result shows that the capacity of acute alkali resistance of O. hornorum is the strongest among the three tilapias, the hybrids is medium, and O. mossambicus is worst.2 Cloning and sequence analysis of AQP1 gene in O. mossambicus and m TOR gene in O. hornorum Using the total RNA of O. mossambicus and O. hornorum respectively as the templates to reverse transcription, the sequence of c DNA of AQP1 and m TOR were got through the PCR amplification of 5’race and 3’race. The length of AQP1 c DNA sequence in O. mossambicus is 2612 bp, including 1760 bp 5’ non-coding region, 774 bp open reading frame(ORF), and 75 bp 3’ non-coding region, coding 257 amino acid; the DNA sequence of O. mossambicus AQP1 is 3215 bp including two intron and three exon; the AQP1 has the two NPA conserved domains and a potential cysteine residues binding site TGCG that can inhibit the cell permeability and has six transmembranes analysised by online software TMHMM 2.0. The c DNA sequence of O. hornorum m TOR is 8193 bp, including 158 bp 5’ noncoding region, 7551 bp ORF, and 484 bp 3’ non-coding region, coding 2515 amino acid which contains 300 negative electricity amino residues(Asp+Glu) and 281 positive electricity amino residues(Arg+Lys). The isoelecric point is 6.53. Through amino acid sequence alignment BLAST, the sequence homology of AQP1 of O. mossambicus and m TOR of O. hornorum is 99% with those of O. niloticus. Phylogenetic tree constructed by MEGA5.1 indicate that AQP1 of O. mossambicus and m TOR of O. hornorum have a high evolution level as Nile tilapia. The AQP1 has the high conversed domain NPA and the amphiphilic channel; the m TOR contains the ATP binding site, catalytic loop, activation loop and polypeptide binding site.3 The expression of AQP1 and m TOR in the tissue of gill、kidney、intestine of tilapia under salt-alkali stress 3.1 AQP1 expression characterisation under salt-alkali stress The expression of AQP1 in gill tissue of O. mossambicus, O. hornorum, the hybrids(O. mossambicus♀×O. hornorum♂) and O.niloticus in freshwater is higher than that of those fishes under the salt-alkali stress and there is a significant difference(p<0.05); the AQP1 expression in kidney tissue of the four fishes with the increase of salt-alkali stress is on a downward trend and the difference was significant(p<0.05). The results indicated that the AQP1 is related to the salt-alkali stress. 3.2 m TOR expression characterisation under salt-alkali stress The expression of m TOR in gill tissue of O. mossambicus, O. hornorum, their hybrids and O. niloticus in freshwater is higher than that of those fishes under the saltalkali stress. With the salt-alkali stress time increasing, the expression of m TOR reduced and then stabilized. The difference is significant(p<0.05); the m TOR expression in kidney tissue shows a trend that firstly drop, then rise and decline, and the difference is significant(p<0.05); the m TOR expression in intestine of the four kinds of tilapia shows a downward trend, and m TOR expression is different significantly at 0h compared with other time points(p<0.05). It is indicated that the m TOR gene is closely related to the salt-alkali stress and participates in the osmoregulation.