A Study On Genes Correlated To Salt-tolerance Of Black-chin Tilapia( Sarotherodon Melanotheron)、Nile Tilapia (Oreochromis Niloticus) And Their Hybrids

Black-chin tilapia (Sarotherodon Melanotheron) was natively distributed in estuaries and lagoons of West Africa from Ivory Coast to Senegal, which has a character of strong salt-tolerance. New Gift is a new tilapia variety by mass selection from nile tilapia (Oreochromis Niloticus) which has the advantage of rapid growth in fresh water but with low salt-tolerance. The two fishes are in different genus of Sarotherodon and Oreochromis. They adopt different reproductive strategies and it is impossible for natural hybridization between them. To take full advantage of the rich salt water and sea water resources in China, we obtained their hybrids through artificial crossbreeding which have both rapid growth and the ability to withstand high salinity water. The genes of sodium / potassium - transporting ATPase alpha-1 subunit (NKAa1), sodium / potassium / chloride transporters 1alpha subunit (NKCC1a), insulin-like growth factor–I b subunit (IGF-Ib) and prolactin I (PRL-I) are supposed to be closely related with the fish osmoregulation. In this study, we performed a chronic salt-tolerance test for Black-chin tilapia, Nile tilpia, hybrid F1 and F2, and then analyzed the sequence compositions, protein spatial structures and mRNA differential expressions of the four genes above. Our aims are exploring salt-tolerance mechanism of Black-chin tilapia, Nile tilapia and their hybrids, providing essential theoretical support and technical foundation for selecting and promotion of the new salt-tolerance tilapia. The major results are as following:1. Comparative study of salt tolerance among S.melanotheron, O.niloticus and their hybridsIn experimental conditions, a study on the acclimation to the salt water was conducted for Black-chin tilapia, Nile tilapia, and their hybrid F1 and F2. Batches of 10 juveniles (10 to 14 g) of the four genotype tilapias reared in freshwater were subjected to gradual increases in salinity until 100% mortality with a daily increase of salinity of 8. The mean cumulative survival (CS) and median lethal salinity (MLS) are analyzed to compare their salt resistance levels. Thy results showed: (1) The MLS of Black-chin tilapia is significantly higher than that of Nile tilapia with the values are 125.78±1.66 and 54.22±2.51, respectively. The MLS values of hybrid F1 and F2 are 77.33±1.89 and 73.73±1.32, respectively. Which are also significantly lower than that of Black-chin tilapia but are still significantly higher than that of Nile tilapia. There is no significant difference of MLS between hybrid F1 and F2. (2) The first mortality of Black-chin tilapia, Nile tilapia and their hybrids appeared from a salinity of 96, 40 and 56, respectively. While the last dead of fish was observed at a salinity of 136, 80 and 96, respectively. The numbers of the total duration in days of mortality in relation with the daily increase in salinity of 8 are 6 days, 5days and 5days, respectively. The results showed no significant difference between each other.2. Cloning, sequencing and different mRNA expression of S.melanotheron, O.niloticus and hybrid F1 NKAa1 geneAccording to the sequences of Mozambique tilapia NKAa1 gene from the GenBank, we designed one pair of primers using premier primer 5.0. Firstly, we gain the part conserved sequences of NKAa1 of Black-chin tilapia, Nile tilapia and hybrid F1 through polymerase chain reaction (PCR), then we designed another six pairs of primers for 3` and 5` Rapid Amplification of cDNA Ends (3`RACE and 5`RACE) followed by the conserved sequences. Splice the sequencing results in Vector NTI 8.0 we got the full sequences of cDNA of NKAa1 gene for the three genotype tilapia. Also, a pair of primers for quantitative reverse transcriptase PCR (qRT-PCR) was designed to test the differential mRNA expressions of NKAa1gene using SYBR fluorescent dye in iQ5 PCR system. The results are showed as follows: (1) The full cDNAs of NKAa1gene of Black-chin tilapia, Nile tilapia and hybrid F1 are 3 227 base pairs (bp), 3 257bp and 3 223bp, respectively. Pairwise alignments for nucleotides among them show 95.76% similarity from Black-chin tilapia to Nile tilapia, and 97.73% similarity from Black-chin tilapia to hybrid F1, and 93.92% similarity from Nile tilapia to hybrid F1. The open reading frame (ORF) of the three genotype tilapias is about 3 072bp coding 1 023 amino acids with 98.3% similarity from Black-chin tilapia to hybrid F1, 97.65% similarity from Nile tilapia to Black-chin tilapia and to hybrid F1. (2) The results of nucleotides and amino acids clustal analysis indicate that hybrids F1 is more closely related to Black-chin tilapia than to Nile tilapia on NKAa1 gene. (3) The mutant residue Ser519 of Black-chin tilapia results the secondary structure of NKAa1 gene changed comparing to Nile tilapia and hybrid F1. The putative spatial structure of NKAa1 protein is composed of eight transmenbrane domains(TM) in which the TM1~4 and TM7~8 are highly conserved in all of the homologue species, while the distributions of the TM5~6 of Nile tilapia are variety to Black-chin tilapia and hybrid F1 which may be due to the two residues` mutation of Phe916 and Thr917 of Nile tilapia. (4) In fresh water, the highest mRNA expression of Nile tilapia NKAa1 gene is in the liver and the lowest is in intestine. The mRNA expressions of Black-chin tilapia NKAa1 gene in gill, intestine and kidney are significantly higher than that in liver. For hybrid F1, the lowest expression of NKAa1 gene is in intestine, but there is no significant differences compared to gill, liver and kidney. In lethal salinity water, the mRNA expressions of Nile tilapia NKAa1gene in gill and intestine are significantly increased, on the contrary, the expressions in liver and kidney are significantly decreased. The increase in salinity is very significantly affected the expression in gill of Black-chin tilapia with about 20 times by expression of 19.821±3.951 in lethal salinity water to that of 1.054±0.185 in fresh water. The mRNA expressions in gill, liver, intestine and kidney of hybrid F1 are increased with the increased salinity but showed no significant differences.3. Cloning, sequencing and different mRNA expression of S.melanotheron, O.niloticus and hybrid F1 NKCC1a geneNa+/K+/2Cl?cotransporter 1-αsubunit (NKCC1α) is one of the key ion cotransporters localized in gill chloride cells which has been associated with the maintains of osmotic homeostasis. The transport process mediated by NKCC1αis characterized by electroneutrality with a stoichiometry of 1Na:1K: 2Cl. The results of study on NKCC1a gene are as follows: (1) The RT-PCR and RACE methods were used to identify the full cDNA of the NKCC1αof 3 832bp, 3 819bp and 3 827bp for Black-chin tilapia, Nile tilapia and hybrid F1, respectively. The ORF of NKCC1a gene is about 3 456bp which contains 1 151aa. Amino acid multiple alignments and phylogenetic analysis showed that the isoforms we got in this study are very similar with NKCC1a isoforms in Oreochromis mossambicus, Salmo salar and Anguilla anguilla, and there is the highest homologous with that of Mozambique tilapia (>99%). (2) The predicted protein secondly structures of NKCC1αof Black-chin tilapia, Nile tilapia and hybrid F1 contain 10 transmenbrane domains, which were highly conserved in sequences and distributions relatively to other bony fishes. (3) The quantitative real time polymerase chain reaction (qRT-PCR) assay was developed to estimate the mRNA expression levels in gill, liver, intestine and kidney. In freshwater, the results showed a tissue-specific model. The mRNA expression of Black-chin tilapia in gill is significantly higher than those in liver, intestine and kidney, for Nile tilapia and hybrid F1, the relatively expressions of NKCC1a in kidney are very significantly higher than in other tissues. Furthermore, the salinity significantly affects the relative expression level of NKCC1αmRNA in gill of Black-chin tilapia with a 4.9 times higher in lethal salinity water than that in fresh water. For Nile tilapia and hybrid F1, the ratios reach to 13.9 and 8.8 times higher, respectively. The results suggest that the NKCC1αis very closely related to salt tolerance in the three genotype tilapias. (4) In intestine, the relative mRNA expression in Nile tilapia increased significantly followed the increase of salinity but opposite in hybrid F1. The results show that Nile tilapia and hybrid F1 may take different osmotic adjustment mechanisms in hypertonic environment with high salinity and Nile tilapia adopt an osmoregulation pattern similar to freshwater fishes. (5) The mutant residues Ser252, Leu398 and Met409 of Black-chin tilapia and hybrid F1 to Nile tilapia changed the secondary structures of NKCC1a protein. We can conclude that these residues may closely relative to high salt tolerance of Black-chin tilapia and hybrid F1.4. Cloning, sequencing and different mRNA expression of S.melanotheron, O.niloticus and hybrid F1 IGF-Ib geneWe cloned partial cDNA fragments of insulin-like growth factorIb subunit (IGF-Ib) from gill extracts through 3′RACE methods. The results demonstrated that: (1) The partial IGF-Ib gene cDNAs of Black-chin tilapia, Nile tilapia and hybrid F1 are about 1 076bp,1 075bp and 1 079bp, respectively, and each comprises a stop codon,a 546bp ORF encoding 182 amino acid peptides,and the 3′-untranslated region (3′-UTR).The deduced amino acid sequence of IGF-Ib precursor is composed of signal peptide,mature peptide (including conserved B,C,A and D domains consisting of 29-aa,10-aa,21-aa and 8-aa respectively) and E domain of 70aa. Results of the predicted secondary structures were typed as“mixed patterns”. (2) The amino acid multiple alignment results of the three genotype tilapias` IGF-Ib genes with other vertebrate animals` IGF-Ib genes showed highly homologous by 75.8% to 100% identity. The B and A domains are highly conserved in all of the allied species. There are 2aa-deletion between No.82 and 83 sites in C domain,3aa and 1aa-deletion next to No.131 and No.159 site of tilapia IGF-Ib gene,respectively. (3) There is an Ala/Pro substitution occurred at No.133 of E domain of Black-chin tilapia compared with Nile tilapia and hybrid F1, which is identical to Oreochromis mossambicus and Oreochromis urolepis hornorum, as they are believed to be the other two euryhaline teleosts which can adapt to high salinity water. We presumed that the differences of salt-tolerance property of Black-chin tilapia, Nile tilapia and hybrid F1 may be due to the alterative splicing of the IGF-Ib E domain. (4) In freshwater, real-time PCR results showed IGF-Ib is expressed mainly in liver of the three genotype tilapias. In lethal salinity water, the highest relative expression of Nile tilapia IGF-Ib appears in intestine, it is very significantly higher than those in gill, liver and kidney. High salinity affected the mRNA expressions in gill, liver and kidney and decreased significantly while the salinity of water increased. Meanwhile, the expression quantity increased significantly in gill of Black-chin tilapia and hybrid F1 but decreased significantly in liver, intestine and kidney of hybrid F1.5. Cloning, sequencing and different mRNA expression of S.melanotheron, O.niloticus and hybrid F1 PRL-I geneGeneral cloning and RACE was used to gain partial cDNAs of PRL-I gene of Black-chin tilapia, Nile tilapia and hybrid F1. The results of sequence and mRNA expression analysis are showed as follows: (1) The nucleotide sequences of the three genotype tilapias` PRL-I gene are 1 444bp, 1 449bp and 1 499bp in length, respectively. The PRL-I gene is composed with a short 5`-UTR, a conserved coding region and a long 3`untranslated region. The ORF is 639bp in length coding 212aa which including the signal peptide of 24aa and mature peptide of 188aa and a stop coden (TAA). (2) Amino acid alignments of the three genotype tilapias show that Black-chin tilapia and hybrid F1 are exactly the same and there are 99.5% similarity compared to Nile tilapia. The mutant residues Arg2 and Ser46 of Nile tilapia may correlate to the differences of salt tolerance among the three genotype tilapias. (3) Homologue alignments show that there are four cysteine residues are highly conserved in all vertebrate, and the residues Glu29, Met132, Lys140 and Leu186 are specific to tilapias. (4) In freshwater, PRL-I mRNA expression can be detected in gill, liver, intestine and kidney in the three genotype tilapias. For Nile tilapia, the minimum expression value is appeared in intestine but there is no significant differences among the four tissues. The mRNA expressions in liver and intestine of Black-chin tilapia are significantly higher than those in gill and kidney. For hybrid F1, the mRNA expression in liver is very significantly higher than those in gill, intestine and kidney. (5) In lethal salinity water, the relative expressions in intestine of Nile tilapia is about 30 times higher than that in freshwater and the expressions in liver increased significantly, too. Lethal salinity increased the expressions in gill of Black-chin tilapia. There was no significantly difference between freshwater and lethal salinity water in gill of hybrid F1, but the expressions in liver and intestine decreased significantly, on the contrary, the expression in kidney is increased significantly. In conclude, the three genotype tilapias may adopt different osmotic adjustment modes correlated to PRL-I in freshwater and salt water.