| The blunt snout bream, Megalobrama amblycephala, endemic in China, is an important aquaculture species in the fresh water resources and play a significant role in economic productivity. It is widely favored as a delicacy for its low production cost, fast growth, high survival rate, easy catching and good quality of meat. For a long time, the functional exploration of growth and development related key genes have been regarded as the priority work during the fundamental research of aquatic animal genetic breeding. Unfortunately, as a consequence of fast domestication, destruction of their natural habitat and over-fishing, germplasm resources of genus Megalobrama and Parabramis are under threat of recession and mixture due to its artificial breeding. Furthermore, because of the geographical overlapping distribution, similar morphology and life habits, individuals may mate, which is likely to cause the germplasm mixture. Thus, the genetic structure of genus Parabramis and Megalobrama need to be clarified, for the purpose of germplasm resources preservation, genetic variation and germplasm identification.Fibroblast growth factor receptor 1(FGFR1), attached to receptor tyrosine kinases(RTKs), as one of the FGFR family, is involved in regulating cell growth and proliferation, differentiation, cell migration and tissue repair. And it plays an important role in the early embryonic development and organogenesis. Usually, FGFR1 contains four domains, namely, an extracellular ligand-binding region composed of three Ig-like domains(D1, D2, D3), a unique acidic serine-rich region(acid box), a transmembrane domain and a well conserved split tyrosine kinase domain in mammals. Coupling with FGF and heparan sulfate proteoglycan(HSPG), the FGFR1 triggers complex signal transduction, such as protein kinase C(PKC) pathway, PI3K/PKB pathway and the Ras/ERK pathway, to regulate the growth and development and so on.In the first part of the research, 18 pairs of primers which could give stable and polymorphic amplification profiles were screened out from 60 microsatellite loci and used to analyze the genetic structure of Parabramis and Megalobrama species. Meanwhile, the microsatellite DNA fingerprinting in six populations of genus Parabramis and Megalobrama was established based on the results of alleles of microsatellite loci by Popgen1.32 software package and illustrative patterns were constructed by EXCEL office software, including two geographic populations of M. terminalis from Dong River(SJF1) and Qiantang River(SJF2), M. hoffmanni(GDF), M. pellegrini(HHF), M. amblycephala(TTF) and P. pekinensis(CCB). The results showed that a total of 221 alleles were obtained from the six genus Parabramis and Megalobrama populations, and the alleles ranged from 2-20 in each locus. The mean value of number on alleles(Na) of SJF1, SJF2, HHF, GDF, TTF and CCB was 5.17, 6.11, 3.5, 6.56, 5.22 and 5.22, the mean value of expected heterozygosity(He) was 0.6342, 0.7204, 0.5462, 0.6810, 0.6752 and 0.5597, and the average value of polymorphism information content(PIC) was 0.5756, 0.6669, 0.472, 0.6306, 0.6064 and 0.517, respectively. It demonstrated that the genetic diversity of SJF2 was the highest and HHF was the lowest among the six populations. Using Unweighted pair-group method with arithmetic means method(UPGMA) based on their genetic distances, the cluster analysis in six populations showed that SJF2 and TTF first grouped together, genetic distance was 0.5606; then they clustered with SJF1, GDF, HHF, CCB in sequence; the genetic distance between HHF and CCB was 1.7592,which was the farthest. In addition, nine pairs of specific microsatellite markers(TTF1, TTF2, TTF3, TTF7, TTF10, Mam03, Mam25, EST37 and EST66) were screened from microsatellite DNA fingerprinting which could be used to identify most of genus Parabramis and Megalobrama populations. Particularly, microsatellite primers(Mam03 or EST37) could differentiate P. pekinensis(CCB) from genus Megalobrama populations and microsatellite markers(TTF3, EST37, TTF2/TTF10, EST66) combination could completely identify SJF1, HHF and GDF populations among Megalobrama spp.. However, as a result of the relative consistency of the genetic characteristics, specific SSR markers, which could distinguish M. terminalis from Qiantang River(SJF2) and M. amblycephala(TTF), were not found. Overall, these results could provide a theoretical basis for germplasm conservation, species identification and genetic breeding of genus Parabramis and Megalobrama in China.In the second part of the research, we report the isolation and characterization of duplicated fgfr1 genes in blunt snout bream(M. amblycephala). We first isolated a 4572 bp c DNA clone for the blunt snout bream fgfr1 a that includes a complete 5’ untranslated region(5’UTR) 702 bp, a 2,457-bp open reading frame(ORF), and a 413-bp 3′-UTR with a poly(A) tail, with 818 amino acid residues encoded by fgfr1 a ORF. The size of the fgfr1 b c DNA was 3117 bp, including a 469-bp 5′-UTR, a 2250-bp ORF, and a 398-bp 3′-UTR with a poly(A) tail. The fgfr1 b ORF encoded 749 amino acid residues. Blunt snout bream fgfr1 a and-1b c DNAs were found to share a relatively high sequence identity of 82%. During embryogenesis, both fgfr1 a and-1b m RNAs were highly detected at zygotes but gradually decreased and constantly expressed after 16 hpf, besides a strong expression for fgfr1 b m RNA at 12 hpf. Whole-mount in situ hybridization demonstrated that fgfr1 a m RNA was transcribed at the eyes, mid-hindbrain boundary(MHB), brain, posterior somites and tailbud at 16 hpf, while fgfr1 b m RNA was only detected at the eyes and posterior somites at the same period. At 28 hpf embryos, both fgfr1 a and-1b m RNAs were expressed in the eyes, brain, pharyngeal arches and tailbud, and in the eyes, brain, pharyngeal arches and notochord at 55 hpf. In adult fish, fgfr1 a m RNA was strongly expressed in the gill, gonad, brain and midgut, but examined relatively low in the skin and kidney. In contrast, fgfr1 b m RNA was highly detected in the brain and liver and quite low in the skin, gill and kidney. During starvation, both fgfr1 a and-1b m RNAs were significantly up-regulated in the intestine and liver, but down-regulated in the nervous system. Moreover, duplicated fgfr1 m RNAs were differentially inhibited in tissues with exogenous recombinant h GH. Our results suggest that two fgfr1 genes play important roles in regulating blunt snout bream growth and development.In the third part, the Tgf2 transposon insertion copies and distribution of deletion were studied by southern blotting and PCR in goldfish under natural condition. Two to six copies were found in the genomes and differences were existed among strains and individuals. PCR amplification revealed that different internal deletions(from ORF1 to ORF2 and from ORF2 to ORF4) were commonly existed in the transposon, with deletion type I 7.78%, deletion type II 73.33%, and entire deletion type 13.33%. Complete Tgf2 transposon merely accounts for 5.56%. Our present study provided a new insight into further study of Tgf2 transposition mechanism. |
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