To elucidate the regulation mechanism of gondadotropin releasing hormone receptor (GnRHR) in sexual differentiation and gonad development, the sequence, phylogenetic pattern and expression of GnRHR gene in yellow catfish Pelteobagrusfulvidraco were analyzed by reverse transcription PCR, multiple alignment of amino acid sequence, phylogenetic tree and real-time quantitative PCR analysis. It was found that the yellow catfish GnRHR cDNA was cloned to be 2 894 bp in length, containing a 1 155 bp open reading frame (ORF) encoding a putative protein of 384 amino acids, a 239 bp 5′untranslated region (UTR) and a 1 500 bp 3′UTR. Protein conserved domain prediction showed that the yellow catfish GnRHR protein contained seven transmembrane domains (TMs). Multiple sequence alignment analysis of amino acid sequence of yellow catfish GnRHR homologues revealed that GnRHR shared high similarities with GnRHRs of other fishes, including channel catfish Ictaluruspunctatus (96%), zebrafish Daniorerio (87%), common carp Cyprinuscarpio (83%) and crucian carp Carassiusauratus (82%). However, yellow catfish GnRHR had low similarities with mammalian GnRHRs ranging from 42% to 44%. Phylogenetic analysis indicated that yellow catfish GnRHR were clustered with type ⅡB GnRHRs of other teleost. Real time quantitative PCR analysis demonstrated that yellow catfish GnRHR transcript was mainly expressed in telencephalon, mesencephalon, hypothalamus, pituitary and testis, without expression in ovary and other tissues. Furthermore, yellow catfish GnRHR mutants were generated by the CRISPR/Cas9 system and several different yellow catfish GnRHR F0 mutants were obtained. The findings indicated that yellow catfish GnRHR played potential role in male development of yellow catfish, and that provided important information for further research of the function of yellow catfish GnRHR and the molecular mechanisms underlying yellow catfish reproduction.
LI Shizhu, FANG Wenyu, LUO Mingfei, LU Jianguo.
Molecular cloning, and expression of GnRHR gene and establishment of GnRHR mutant by CRISPR/Cas9 in yellow catfish Pelteobagrus fulvidraco[J]. Journal of Dalian Fisheries University, 2021, 36(3): 383-392 https://doi.org/10.16535/j.cnki.dlhyxb.2020-120
中图分类号：
S 917.4
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