A Study On Salt Tolerance Related Genes Of Oreochromis Mossambicus, O. Hornorum And Their Hybrids

Tilapias belong to the family Cichlidae in the order Perciformes. Most of them areeuryhaline species, which not only can be cultured in fresh water but also can grow inbrackish water after domestication. The mechanisim of salt tolerance of tilapia is verycomplex, involving various aspects e.g., physiology, ecology, environmental andgenetic factors. TRPV4, prolactin Ⅰ (PRLⅠ) and Na+-K+-ATPase alpha1subunit(NKAα1) and some other genes play important roles in fish osmoregulation. In thisstudy, we performed an acute salt-tolerance test for the hybrids of O. mossambicus andO. hornorum, and then analyzed the sequence compositions, protein spatial structuresand mRNA differential expressions of the three genes above. Our aims are exploringsalt-tolerance mechanism of O. mossambicus, O. hornorum and their hybrids, providingessential information for breeding and promotion of new varieties. The major results areas following:1. Comparative study of salt tolerance on the hybrids of O. mossambicus and O.hornorumThe salt-tolerance differences on the hybrids of O. mossambicus and O. hornorumare compared by acute salt-tolerance test. The results showed that: The median lethalsalinity-96(MLS-96) of crossbeed and reciprocal cross were29.80±3.03and29.38±4.48, there was no significant difference between them. There is heterosis andthe salt tolerance of hybrids is higher than that of their parents.2. Cloning and sequence analysis of three salt tolerance related genes of O.mossambicus, O. hornorum and their hybridsThe TRPV4, PRLⅠand NKAα1genes from O. mossambicus, O. hornorum andtheir hybrids were cloned by homology cloning approach. The amino acid sequencesdeduced from their cDNA sequences were analyzed. The results showed that the lengthof TRPV4, PRLⅠand NKAα1genes from the four different species of tilapia is3198bp,798bp and3165bp, including open reading frame (ORF) of2613bp,639bp and3072bp,encoding putative protein of870,212and1024amino acids, respectivly. The sequencesof TRPV4gene are exactly the same among the four tilapias. The alignment of aminoacid sequences deduced from PRLⅠ cDNA sequences showed that there is only oneamino acid residue difference between O. mossambicus and O. mossambicus at the31stsite, where a homologous fragment of both the hybrids is leucine at31st site as in O. mossambicus, and the other homologous fragment is phenylalaline as in O. hornorum.The sequences of NKAα1from O. mossambicus and O. hornorum are exactly the same,but there is one amino acid difference in a homologous fragment from reciprocal crosshybrid compared to between the two parents and crossbeed hybrids. Phylogenetic treeof TRPV4and PRLⅠ gene by MEGA5.1showed that the four tilapias were clusteredinto one group with other tilapias, and then the group clustered into one group withother Perciformes fish, reflecting the evolution of creatures to some extent. Thephylogenetic relationship of NKAα1from O. mossambicus, O. hornorum and theirhybrids is closer to Mozambique tilapia souce from genbank. The three freshwaterspecies from Synbanchiformes and Cypriniformes were clustered into one group,suggesting that the evolution of gene from fish may be related to salt tolerance.3. Different mRNA expression of salt tolerance related genes of O. mossambicus,O. hornorum and their hybrids3.1Different mRNA expression of TRPV4geneTRPV4gene expression level of O. mossambicus, O. hornorum is relatively high inthe pituitary, liver, skin, gill and heart. The expression of TRPV4is generally low in alltissues and all of them are less than0.005. The expression of TRPV4from O.mossambicus, O. hornorum and their hybrids reduced in the blackish water (22), andsuch changes were more evident in gill and kidney. O. mossambicus, O. hornorum andtheir hybrids TRPV4mRNA level rebounded in seawater (35). TRPV4mRNA levelin kidney of O. hornorum and muscle of O. mossambicus was even higher in seawaterthan the level in fresh water. It is shown that TRPV4gene expression is related to theenvironment osmolarity.3.2Different mRNA expression of PRLⅠ genePRLⅠ transcript was detected at high level in pituitary and low in other tissuesand organs, suggesting that the pituitary gland is the main expression site of PRLⅠ intilapia. The expression of PRLⅠ was downregulated significantly after exposure tosalinity stress in pituitary. The changes of PRL Ⅰ expression in muscle of O.mossambicus, O. hornorum and their hybrids were similar to pituitary, but thesechanges were more moderate. These results suggested that PRLⅠ plays an importantrole in tilapia osmoregulation.3.3Different mRNA expression of NKAα1geneO. mossambicus and O. hornorum NKAα1mRNA level is high in kidney andintestine in freshwater. The expression of NKAα1in gill is low than in kidney andintestine, but it increased most obviously after salinity stress. It is shown thatNa+-K+-ATPase is closely related to fish osmoregulation.