Hypoxia-responsive Analysis Of CITED1 And-2 Genes In Blunt Snout Bream (Megalobrama Amblycephala) And The Development Of MSTN Gene Knockout In Blunt Snout Bream

Blunt snout bream(Megalobrama amblycephala),also known as wuchang fish.As an important representative of the freshwater aquaculture economic species(Cypriniformes,Bo subfamily)in our country.Its various good growth characteristics(fast growth,high survival rate,low cost)are increasingly important.From 1960 s,Blunt snout bream became more popular and we have been endeavoring promote the breeding.As a principal herbivorous species in freshwater fish polyculture systems,blunt snout bream(Megalobrama amblycephala)is widely favored as a delicacy in China,whose output is more than 700,000 tons in 2015(FBMA.,2016).In aquaculture,fish usually meet some stress factors(high temperature,crowded,oxygen and bacteria)which resulted in serious losses.,Additionally,except for those outside stress factors,there are also some molecular mechanism of gene regulation limits fish production.Compared with other common farmed fish(carp,crucian carp),Blunt snout bream have not be able to bear or endure low oxygen and break out anoxic death easily which bring losses to aquaculture industry.With the emergence of inbreeding depression,deeper research and responsive mechanism are particularly important to explore.The total target is breeding new strains in Megalobrama amblycephala which resistant to low oxygen has more application value.The CITED proteins belong to a family of non-DNA-binding transcriptional co-regulators that involved in the regulation of various transcriptional responses.Previous studies suggested members of CITEDs could function in response to hypoxia in mammal,however molecular and functional information on CITED genes is paucity in aquaculture fish.Here,we characterized and examined the expression patterns of CITED1 and-2 genes in the hypoxia-sensitive blunt snout bream(Megalobrama amblycephala).Blunt snout bream CITED1 and-2 genes shared a relatively low sequence identity of 45%.The CITED1 and-2 mRNAs were widely expressed in adult tissues.During embryogenesis,CITED1 mRNA was significantly expressed at 4,24,28,40 and 44 hpf,while CITED2 mRNAs were fluctuated from the zygote to the 44 hpflarvae.Whole-mount in situ hybridization demonstrated that CITED1 and-2 mRNAs were detected in the brain at 12 hpf,and at brain and gut at 24 hpf,and at brain at 36 hpf embryos.CITED1 mRNA was also detected slightly at tailbud at 24 hpf.The results of acute hypoxia experiment showed that CITED1 and-2 mRNAs were markedly up-regulated in the kidney and down-regulated in the liver,brain,gill and heart under hypoxia.Embryos in hypoxic conditions at different developmental stages significantly induced the mRNA expression of CITED1 and-2.These results provide new insights into the functions of CITED1 and-2 genes and their responses to hypoxia.Research showed that fish muscle fibers would increase in the whole growth process.Regulation of muscle growth genes including positive control and negative regulatory factors.The Myostatin(MSTN)belong to the TGF-β family.Many studies have confirmed the MSTN mutation or lower transcriptional expression can promote muscle growth and development.Firstly,we have cloned the MSTN a and MSTN b gene in the blunt snout bream.The total length of megalobrama amblycephala MSTNa gene is 2195 bp,encoding 364 amino acids and having high similarity with grass carp(71%)and zebrafish(97%).Then,The total length of megalobrama amblycephala MSTNb gene is 2193 bp,encoding 375 amino acids,sharing higher similarity with grass carp(99%)and zebrafish(97%),respectively.Alignment of the deduced amino acid sequences of Megalobrama amblycephala duplicated MSTNa and MSTNb showed MSTN gene is quite conservative during the evolution and found TGF-β propeptide domain,RXXR hydrolysis sites,TGF-β or TGF-β type domain and conserved cysteine residues.We firstly take the gene editing techniques of CAS9 in order to knock out MSTN gene.According to the design principle of type I and type II in T7 target site,we designed MSTNa target sites in base sequence location of 642,1015,1232 and MSTNb target sites in base sequence location of 466,878,1054,respectively.Cas9/gRNA system consists of Cas9 nuclease and guide RNA(gRNA).Guide RNA which contains main target site sequence is responsible for the identification of target genes.Then Cas9 nuclease is responsible for cutting targets,resulting in double-stranded DNA break(DSB).In the period of 1-2 cell embryonic development in megalobrama amblycephala,we injected gRNA injection(100 ng/ul)and CAS9 protein(900 ng/ul).According to the analysis of T7E1 enzyme testing results in megalobrama amblycephala embryo,we selected and screened higher efficiency MSTNa and MSTNb targets.MSTNa’ s higherefficiency targets is MSTNa-target 3-1232 with enzyme digestion efficiency at about10%.MSTNb’ s higher efficiency targets is MSTNb-target 3-1054 with enzyme digestion efficiency is 26%.The 3-month megalobrama amblycephala progeny test results showed that MSTNa-target 3-1232 knockout efficiency of 9.7%,while MSTNb-target 3-1054 knockout efficiency is 26%.Then we can remove the muscle growth inhibition in blunt snout bream.Afterwards,through the self-cross or gynogenesis of the offspring of double muscle heterozygote F1,we would select significant quality homozygous offspring which is the starting point and the foothold of this experimental design.