The Construction Of Phylogenetic Tree And The Study Of Genetic Diversity Of Growth Hormone Gene In Takifugu

Growth hormone (GH) has visible effect in promoting foodintake, boosting feedstuff conversion rate, incentiving and promoting growth, participating growth metabolic regulation in fish. The GH gene of Takifugu obscurus was amplificated with two pair of primers based on the study of GH gene in livestock and fish and the sequences of GH gene of other homology species in GenBank , and the phylogenetic tree of GH gene was constructed using UPGNA method. In addition, High size and nucleotide polymorphisms of GH gene was checked in 82 individuals from Takifugu obscurus, Takifugu rubripes and Takifugu niphobles with PAGE,SSCP and sequence analysis. The results showed that:1. There were 6 exons and 5 introns in GH gene in Takifugu obscurus, which codes 196 amino acids.2. The mRNA and amino acids sequences of GH gene of 24 vertebrate species were gained from GenBank., and the phylogenetic tree of GH gene was constructed with MEGA 3.1 using UPGMA method. In UPGMA consensus tree, 25 species were divided into two clusters. The first cluster, including Sus scrofa, Canis familiaris, Felis catus, Rattus norvegicus, Mus musculus, Ovis aries, Bos Taurus, Gallus gallus, Anas platyrhynchos, Ambystoma barbouri, Anguilla anguilla, Acipenser gueldenstaedtii and Homo sapiens, were subdivided as follows: mammalians(Sus scrofa, Canis familiaris, Felis catus, Rattus norvegicus, Mus musculus) constructed one group firstly, which were subsequently joined by ruminant(Ovis aries, Bos taurus), poultry(Gallus gallus, Anas platyrhynchos), reptiles(Ambystoma barbouri), Acipenser gueldenstaedtiihuman and Homo sapiens in order, Anguilla anguilla was joined last. The second cluster, including Takifugu obscurus, Takifugu rubripes, Danio rerio, Ctenopharyngodon idella, Verasper variegates, Oncorhynchus sp, Betta splendens, Cottus kazika, Carassius auratus gibelio, Perca flavescens, Cyprinus carpio and Pangasianodon gigas, were subdivided as follow: Cottus kazika clustered with Perca flavescens firstly, followed by Betta splendens and Takifugu in order, formed group 1; Danio rerio and Ctenopharyngodon idella, Carassius auratus gibelio and Cyprinus carpio were clustered together respectively, which was subsequently joined by Oncorhynchus sp and Pangasianodon gigas, formed group 2.3. There were nine size variants (A, B, C, D, E, F, G, H, I) in intron 2 of GH gene. Results showed that there were nine length variants (A~I) in these fishes, ranging from189 -209 bp, with a frequency of variation of 24.22%. Sequence analysis of the 9 length variants showed the average percentages of the four bases (A, T, G and C) were 17.15%, 20.77%, 37.38% and 24.70%, respectively, and the difference between GC content (45.47%) and AT content (54.53%) was not remarkable. Length variation was mainly due to the variable number of microsatellite TCTG repeats (N=20-40). Further sequence comparison revealed 4 substitution sites: a C-A transversion at nucleotide 103 and 3 transitions 83(C-T), 101(A-G) and 296(G-A), respectively. Phylogenetic analysis of these length variants placed DD and II into one group first, which was subsequently joined by GG and AA. BB and CC, EE and HH were then clustered together. FF was joined last. Thus, intraspecific variations were much smaller than interspecific variations for the intron 2 of the GH gene.4. There were four size variants AA(353bp), BB(360bp), CC(337bp), DD(327bp) in intron 5 of GH gene in three Takifugu, and the sequences analysis showed that there were 7 mutations. The frequency of variation was 1.77%. The length variants was mainly due to the deletion at 28(A), 193-198(ATGATG), 244(G), 274-284(TGGTGAAGATG), 312-320(ATGATGATG). Of the 7 mutations, 1 was transversion site, at 251(T-A), and 6 were transition sites, at 181, 190, 251, 262, 268, 271(C-T), and the frequency of transversion(14.29%) were lower than that of transition(85.71%).5. There were two size variants in 3′-UTR of Takifugu GH gene, 320bp and 317bp respectively, which was different from the sequence (316bp) in the GenBank (accession number: FRU63807). And 6 genotypes aa, bb, ab, bc, cd and dd were found, the frequency of variety was 4.36%; aa, bb, ab and bc was found in Takifugu obscurus , cd and dd can only be found in Takifugu niphobles and Takifugu rubripes respectively. Of the 7 mutations,1 was of transversion site, at 212(T-G), and 6 were of transition sites, at 120, 180, 227, 265, 287(C-T) and 199(A-G) respectively.