Molecular Structure And Functional Mechanisms Of Hypoxia Response Genes In Megalobrama Amblycephala

Blunt snout bream (Megalobrama amblycephala) is an extremely hypoxia-ntolerant fish. It will cause mass death and great economic losses under hypoxicconditions, which restricts the improvement of its production. To maintain survivalunder hypoxia condition，present genes about hypoxia repondent have been found infish, two of them are IGFBP-1and EPO. But their molecular structure and expressionpatterns is still unclearly. So research on the structure and function of IGFBP-1andEPO is important to explain molecular mechanism of bream response to hypoxia.In this study, we cloned the full-length cDNA of the bream IGFBP-1and EPO byRT-PCR and RACE methods. The sequence of IGFBP-1and EPO is1135bp and903bprespectively, the open reading frame is789bp and552bp, encoding263and183aminoacids. Sequence analysis showed that IGFBP-1N terminal contain IGFBP motif, whichwas this family mark. IGFBP-1has high similarity in both N and C terminal domain;EPO is a glycoprotein and contains three N-linked sugar chains as well as one O-linkedsugar chain.its signal peptide has been assigned.On the one hand,IGFBP-1and EPO mRNA expression patterns of bream in differentstages of embryonic development and tissues of adult fish by RT-PCR and whole embryo in situ hybridization.In the normoxic state, the expression pattern of breamIGFBP-1was increased with the development of embryonic. The expression pattern ofbream EPO in different stages of embryonic development was relatively constant;IGFBP-1expression were detected in all tissues of bream adult fish in the normoxicstate. IGFBP-1has high expression level in the liver, intestine and heart; Except othertissue the brain，liver and heart tissues have expression of EPO. After hypoxic stress theIGFBP-1mRNA in skin,liver,kidney,spleen,intestine and heart were23.3,21.0,32.7,26.10,19.0and32.0as control;EPO mRNA expression were significantly increased1980%,2010%and3940%respectively in brain, liver and heart, it also have expressionin kidney; but EPO mRNA levels in the corresponding tissues did not changesignificantly;When recovered the value of water dissolved oxygen,both of genestranscriptional level was down to normal.These results implied that bream IGFBP-1andEPO play different roles in the process of growth and development under hypoxicresponse. This study provide an experimental basis for exploring the molecularmechanisms of bream physiological adaptation to hypoxic environment.On the other hand, we also amplification and identification the flanking sequencesand copy number of Tgf2transposon in goldfish of different strains. Found naturalactive Tgf2transposon in Carassius auratus individuals. by inverse PCR amplification,the pearl Tgf2transposon flanking sequences;Both the left and right arm,we found at theend of thearms,there has8bp repeated base sequence. In the pearl, glossy and butterflystrains, its have six, four and four different8bp repeated base sequences respectivelyand their flanking sequences were identified. The results showed that there were sixkinds of copies in the Tgf2transposon at least, and different strains have differentflanking sequences and8bp repeated sequences. Contrast with piggyBac transposonstend to insert TTAA and Tc1/mariner transposon family preference for AT sites, Tgf2transposon just random insertion, which may establish material basis of genomeinsertion mutagenesis for cultured fishes.