Hypoxia Response Analysis Of Heme Oxygenase (HO)2 Repeated Gene In Megalobrama Amblycephala And Insertion Mutagenesis Of PB-Tgf2 Transposition Systems

Blunt snout bream(Megalobrama amblycephala),cyprinid fish,is an important economical freshwater cuture fish in China.With the advantages of low cost of breeding,high survival rate,fast growth and so on.As shanghai ocean university has cultured Pujiang No.1 that has a clear growth advantage,but it is an extremely hypoxia-nontolerant fish compared with Cyprinoid and Carassius auratus.And it is often caused death because of hypoxia,which is harmful to the blunt snout bream’s farming.Therefore,it would be great significance for cultivating a new variety of blunt snout bream resistant to hypoxia and aquatic animal genetic breeding by exploring hypoxia regulation mechanism and characterizing related key genes.Heme oxygenase(HO),a rate-limiting enzyme in heme catabolism,decomposes heme to carbon monoxide(CO),ferrous iron and form biliverdin,which is subsequently reduced to bilirubin by biliverdin reductase.In this study,HO-2a and HO-2b genes were successfully cloned by rapid amplification of cDNA ends(RACE).The embryos and adult fish were treated with hypoxia at different times and the expression of embryos and adult tissues in different developmental stages were analyzed.Sequence analysis showed that the full-length of HO-2a and HO-2b genes were 1380 bp and 1390 bp,respectively,encoding 305 amino acids(aa)and 317 aa,and shared a relatively low sequence identity of 67%.The HO-2a and-2b mRNAs were widely expressed in adult tissues.During embryogenesis,HO-2a mRNAs was significantly upregulated at 16 hpf and then maintained with high lever,while HO-2b mRNAs was gradually increased at 12 hpf and then reduced significantly.Whole-mount in situ hybridization demonstrated that HO-2a and-2b mRNAs mainly detected in brain and eyes at different embryonic stages.The results of acute hypoxia experiment showed that both HO-2a and-2b mRNAs have significant changes in different tissues.Both HO-2a and-2b mRNAs were significantly up-regulated in the brain,but down-regulated in the gill and liver during hypoxia.Under hypoxia condition,HO-2a mRNA in the heart was significantly increased while HO-2b mRNA was decreased.Embryos in hypoxic conditions at different developmental stages strongly induced the mRNA expression of HO-2a and-2b.These results provide new insights into the functional conservation and divergence of HO-2 genes and improve our understanding of HO-2 responses to hypoxia.At the same time,with the widely application of transposon in recent years.It has unique advantages in the acquisition and research of functional genes.The donor vector plasmids pPBs-Tgf2-RPrmP-eGFP and the helper plasmid pCS2-PBTP were constructed by our laboratory.We injected the PiggyBac-Tgf2 transposition system with PB transposase mRNA into the fertilized eggs together by microinjection to explore the transposition efficiency of the PB-Tgf2 transposition system and the data of the transgenic blunt snout bream with carrying the eGFP gene.The results showed that the integration efficiency of PB-Tgf2 transposition systems was 62.26%.It indicated that the transposable efficiency of the PB-Tgf2 transposition system was significantly higher than that of the PiggyBac transposon and Tgf2 transposon in the blunt snout bream.Our data make great significance to the further study for insertional mutagenes of blunt snout bream.