大鳍鳠dnd基因的克隆与表达特征分析OA

[Objective]This study aimed to investigate the expression patterns of the germ cell-specific marker gene dead end(dnd)during gonadal development in Hemibagrus macropterus.[Method]Tissues in-cluding the heart,liver,spleen,kidney,intestine,brain,gills,and gonads(ovary and testis)were collected from 1-,2-,and 4-year-old male and female H.macropterus.Total RNA was extracted from the ovaries of 4-year-old H.macropterus,which was reverse-transcribed into cDNA,and the full-length cDNA sequence of H.mac-ropterus dnd(Hmdnd)was cloned using RACE technology.Subsequently,sequence analysis was conducted.Using β-actin as the reference gene,real-time quantitative PCR(RT-qPCR)was employed to quantify dnd ex-pression in various tissues of 1-year-old H.macropterus and in gonads at different developmental stages.RNA in situ hybridization(ISH)was applied to localize dnd mRNA within germ cells.[Result]The full-length Hmdnd cDNA sequence was 2 035 bp(GenBank accession:PQ626051),containing an open reading frame(ORF)of 1 131 bp that encodes 376 amino acids.Sequence alignment and phylogenetic analysis revealed the highest protein sequence identity(79.24%)between H.macropterus and Pelteobagrus fulvidraco(yellow cat-fish).RT-qPCR demonstrated that Hmdnd was exclusively expressed in gonads,with significantly higher levels in ovaries than in testes,and no expression in somatic tissues.Hmdnd expression declined markedly during gam-ete maturation.RNA in ISH of gonadal sections revealed that Hmdnd mRNA was predominantly localized in the cytoplasm of germ cells.In ovaries,Hmdnd expression increased sharply as primary oocytes developed from stage Ⅰ to stage Ⅱ,but dropped to stage-Ⅰ-equivalent levels in stage Ⅲ primary oocytes.In testes,the stron-gest Hmdnd signals were detected in spermatogonia and diminished progressively during spermatogenesis,with no expression observed in spermatozoa.[Conclusion]Hmdnd is likely to play a critical role in the early gonadal development of H.macropterus.