| Megalobrama amblycephala(Wuchang bream)as an important freshwater fish species in China is facing with several severe problems including germplasm resources and aquaculture environment degradations.When comparing with four major Chinese carps,M.amblycephala has poorer environmental hypoxia tolerant capability,which making them hard to adapt to the rapid degradation water,especially hypoxia environment.Additionally,M.amblycephala are able to momentarily and quickly respond to bad environment conditions due to their explosive and easily frightened temper.The above characteristics strongly hinder the sustainable development of M.amblycephala aquaculture.Thus,the ethological,physiological and innate immune responses,the environmental response protein levels of M.amblycephala under different oxygen level and different hypoxia time treatments were studies in the present study.Meanwhile,three members of the phd family were cloned,and the bioinformatics,spatio-temporal expression,DNA methylation levels of phds were analyzed,which will provide fundamental basis for cultivation of hypoxia-tolerant M.amblycephala variety/strain.The main results are as follows:1.The ethological changes of M.amblycephala before and after hypoxia treatmentThe respiration of M.amblycephala increased from average 182/min to 245/min after hypoxia,meanwhile fish swam from bottom and central to the upper space of water conducting aquatic surface respiration(ASR).When the dissolved oxygen(DO)in water consistently decreased and reached nearly to the suffocation point of DO: 0.5 mg/L,some fish lost of equilibrium,and after reoxygenation the above behaviors of M.amblycephala gradually recovered to normal status.Integrally,the spontaneous activity of M.amblycephala increased after hypoxia treatment.2.The physiological and biochemical levels of M.amblycephala before and after hypoxia treatmentWhen M.amblycephala were exposed to different DO including control(DO: 5.5 mg/L)and acute hypoxia(DO: 3.5 and 1.0 mg/L respectively),hemoglobin(Hb),methemoglobin(Met Hb),glucose,Na+,succinatedehydrogenase(SDH)and lactate gradually increased with the decrease of DO level,whereas hepatic glycogen gradually decreased.When M.amblycephala were exposed to different hypoxia time including 0.5 h and 6 h(DO: 3.5 mg/L),and 6 h hypoxia(DO: 3.5 mg/L)to 24 h reoxygenation(DO: 5.5 mg/L),Hb,Met Hb,glucose and lactate increased with the extension of hypoxia time,whereas Na+ and hepatic glycogen decreased,SDH level was unchanged.The above investigated 7 indexes all decreased after reoxygenation.3.The innate immune responses of M.amblycephala before and after hypoxia treatmentInterferon alpha(IFNα)and lysozyme(LYZ)of M.amblycephala increased with the decrease of DO level,whereas albumin level gradually decreased.M.amblycephala IFNα increased with the extension of hypoxia time,whereas albumin and LYZ levels decreased.The above investigated 3 indexes all increased after reoxygenation.4.The environmental response protein levels of M.amblycephala before and after hypoxia treatmentWestern blot assay indicated that Hsp70 expressed two bands in M.amblycephala tissues,and the junior Hsp70 was translated through internal ribosome entry site(IRES).The senior Hsp70 was detected in liver,spleen,brain,gill and kidney,while junior Hsp70 was only detected in liver,spleen and gill.The Hif-1α in liver,spleen and gill were increased with the decrease of DO and extension of hypoxia time.5.M.amblycephala phds c DNA and promoter amplifications and sequence characteristicsAn efficient full-length c DNA amplification strategy based on bioinformatics technology and multiplexed PCR methods was designed and developed.By using this strategy,M.amblycephala phds full-length c DNA and promoter sequences were obtained.Meawhile this strategy was also validated on other fish and shrimp species.Bioinformatics analysis indicated that phds all had 5 exons,the encoded amino acid sequences all had prolyl 4-hydroxylase alpha subunit homologues(P4Hc)and three amino acid residues,H,D and H for binding with irons were identified.The N-terminal of Phd2 had another zf-MYND domain which was different from Phd2 and Phd3.The HRE binding sites were predicted on the promoter of M.amblycephala phd1 and phd3 not phd2.Further dual-luciferase reporter assay indicated that 5 HREs of phd3 promoter responded to Hif-1α,therein HRE1,HRE2 and HRE3 had higher responding activities.6.The expression analysis of M.amblycephala phdsSpatio-temporal expression indicated that phd1 expression reached the maximum level at 17.5 h post fertilization(eye vesicle stage).Phd1 was highly expressed in blood,brain and heart under normoxia condition and down-regulated in blood,muscle,brain,heart and intestine after hypoxia treatment,whereas increased in gill.The results also indicated that phd1 expressed two protein isoforms generated by alternative initiation,and each protein expressed in nucleus,therein the senior protein promoted cells proliferation.Phd2 had higher levels at 2.83 h,4.42 h and 5.17 h post fertilization(16-cell stage,early and mid blastula stages,respectively).Phd2 was highly expressed in muscle and gill in normoxia condition and down-regulated in blood,liver,spleen,muscle and heart after hypoxia.The phd3 expression was detected in the early development stages of embryos and was higher in liver in normoxia conditon.Phd3 was significantly induced in all 9 detected tissues after hypoxia.In addition,phd3 had two m RNA isoforms generated by alternative splicing transcription,and each isoform was induced by hypoxia.7.The touch-down based nest-PCR(TDN-PCR)for M.amblycephala phds DNA methylationPhd1 expression between hypoxia-sensitive and hypoxia-tolerant M.amblycephala sample muscle tissues showed no significant difference,while for phd2 the difference between two groups was significant.Phd3 had higher expression in hypoxia-tolerant M.amblycephala,whereas in hypoxia-sensitive samples phd3 was macroscopic.To analyze the effect of DNA methylation of candidate gene promoter on its expression,Touch-down based nest-PCR(TDN-PCR)was designed and developed,which was more specific and sensitive than the normal PCR.The TDN-PCR in M.amblycephala phds indicated that Cp G regions on phd2 and phd3 promoters from hypoxia-sensitive and hypoxia-tolerant M.amblycephala were unmethylated,while phd1 had two methylated regions on the promoter.To further analyze the methylation levels of phd1 in hypoxia-sensitive and hypoxia-tolerant M.amblycephala groups,the high-resolution melting(HRM)assay based on TDN-PCR was performed,and the result indicated that the DNA methylation level of phd1 promoter was significantly correlated to hypoxia-sensitive M.amblycephala.8.Phd1 DNA methylation level analysis by TDN-PCR in different fishTDN-PCR for phd1 promoter sequencing was conducted in several fish species from cyprinomorpha,percomorpha and ophicoephaliformes taxonomy,and the results indicated that fish with higher methylation in the phd1 promoter had higher suffocation point,which fish was more sensitive to hypoxia.These results indicated that DNA methylation level in the phd1 promoter was positively correlated to suffocation point of fish. |
