Studies On The Sequences And Polymorphism Of Growth Hormone Gene In Three Fishes Of Siniperca

Siniperca chuatsi, Siniperca kneri and Siniperca scherzeri were studied while long PCRand T-A cloning technique were used to clone GH (growth hormone) gene containing completereading frame of three fishes of siniperca. Then sequence structure of GH gene, amino acidsequence encoding by DNA, difference between sequences and phyletic evolution were studying.Polymorphism detections of GH gene and studies of hereditary constitution of population wereundertaken in wild populations coming from Eastern Dongting Lake using PCR-SSCP,PCR-RFLP, SSR, polymorphism detection of intron and sequencing. These studies revealed themolecular hereditism background of growth difference between three fishes of siniperca whichwere farmed in China as well as established foundation for molecular breeding of mandarinfish.The main results were as follows:1. Three mandarinfish's complete GH genes were cloned successfully using long PCR.Recombined clones were sequenced. The length of GH gene of S.chuatsi,S.kneri and S.scherzeriwere 5548bp, 5483bp and 5789bp respectively. These sequences have been verified and givenaccession numbers after they were submitted to GenBank. Their Accession Numbers wereEF205280, EF205281 and EF441623 respectively. Long PCR displayed good prospects ingenomic study in post genomic period as it could be used to clone long gene from genomic DNAdirectly.2. Main difference were found in the 1st, 2nd and 3rd intron while three GH genes werecompared. The 4th and 5th intron had little difference. These difference came from repeats ofnear sequence, deletions or insertion of DNA fragment and single nucleotide polymorphismmainly.3. Amino acid sequences were deduced from DNA sequences of GH gene, and comparisonswere done. Interspecific difference of amino acid sequence couldn't be found betweenS.chuatsi's GH and S.kneri's while one difference of amino acid existed at locus 150 betweenthem and S. scherzeri. S.scherzeri had methionine instead of leucine which the other two hadleucine at this locus.4. Microsatellite whose core sequence was AG was found among all 2nd intron of GH geneof three mandarinfish. Detection among populations proved that the locus had highpolymorphism. One microsatellite locusing in main functional gene was discovered in fishes ofsiniperca. This locus had possible to correlate with economic character.5. One conservative sequece existed in boundary between 3rd intron and 4th exon whoselength was 37 bp had one repeat at the end of 3rd intron of all three GH genes. Other 10 GHgenes of vertebrate sepcies downloaded from GenBank hadn't the repeat while the barramundi(L.calcarifer) had one partial repeat.6. Polymorphic detections were done among three wild populations from S.chuatsi, S. kneriand S.scherzeri respectively using PCR-SSCP, PCR-RFLP, SSR, length polymorphic detectionof introns and sequecing. 38 haplotypes had been found at 7 locuses. 31 haplotypes werepossessed only by one species among these haplotypes. Distribution of haplotypes betweenspecies had strong specificity.7. It was found that the length of the 1st, 2nd and 3rd intron had specificity between specieswhen sequence comparisons and polymorphic detections among populations were undertaked.8. S.scherzeri had 6 polymorphic locuses, and the other two had 5 polymorphic locusesrespectively at all 7 locuses. S.scherzeri, S.kneri and S.chuatsi had 2.43, 2.43 and 1.86 average alleles, and had 1.5, 1.35 and 1.36 average effective alleles respectively. S.scherzeri'spolymorphic index was 0.44, higher than S.chuatsi which was 0.38, approximate to S.kneri's0.35.9. The analysis about distribution of genetic polymorphism in three wild mandarinfishpopulations indicated that intraspecies polymorphism and interspecies polymorphism accounted70.3 percent and 29.7 percent in whole polymorphism respectively. GH gene polymorphismdisplayed as intraspecies polymorphism mainly. Average Fst (species differentiation index) was0.68. Average Nm(gene flow) was 0.12. Three species had strong differentiation and littleexchange of gene between them.10. Phylogenetic tree were constructed basing on sequence comparison as well as geneticdistance between populations which were calculated according the relult of polymorphicdetection. The trees' topology were identical. S. kneri and S.chuatsi clustered before cluster withS.scherzeri in the trees. It indicated that S.scherzeri differentiated from common ancester earlierthan S.kneri and S.chuatsi.