Screen for stage-specific expression genes between tail bud stage and heartbeat beginning stage in embryogenesis of gynogenetic silver crucian carp

Abstract

A systemic study was initiated to identify stage-specific expression genes in fish embryogenesis by using suppression subtractive hybridization (SSH) technique. In this study, we presented a preliminary result on screen for stage-specific expression genes between tail bud stage (TBS) and heartbeat beginning stage (HBS) in gynogenetic silver crucian carp ( Carassius auratus gibelio). Two SSH plasmid libraries specific for TBS embryos and HBS embryos were constructed, and stage-specific expression genes were screened between the two stages. 1963 TBS positive clones and 2466 HBS positive clones were sampled to PCR amplification, and 1373 TBS and 1809 HBS PCR positive clones were selected to carry out dot blots. 169 TBS dot blot positive clones and 272 HBS dot blot positive clones were sequenced. Searching GenBank by using these nucleotide sequences indicated that most of the TBS dot blot positive clones could not be found homologous sequences in the database, while known genes were mainly detected from HBS dot blot positive clones. Of the 79 known genes, 20 were enzymes or kinases involved in important metabolism of embryonic development. Moreover, specific expressions of partial genes were further confirmed by virtual northern blots. This study is the first step for making a large attempt to study temporal and spatial control of gene expression in the gynogenetic fish embryogenesis.

Identification of a novel C2 domain factor in ovaries of orange-spotted grouper (Epinephelus coioides)

“…An SSH library between embryos at blastula and tail bud stage was constructed as described previously (Shi et al, 2002;Xie et al, 2003;Zhang et al, 2003). Briefly, double strand cDNAs synthesized from 2 μg polyA + RNA of blastula and tail bud stage embryos were subjected to blunt-end digestion by RsaI.…”

“…Dot-blot hybridization was carried out as described (Xie et al, 2001;Shi et al, 2002;Zhang et al, 2003). Secondary PCR products were inserted into pGEM-T vector (Promega) and transformed into Top-10 of Escherichia coli.…”

Molecular characterization and IFN signal pathway analysis of Carassius auratus CaSTAT1 identified from the cultured cells in response to virus infection

“…Virtual Northern blotting can obtain the identical information provided by standard Northern analysis for identification of differential expressed genes, because the difference is just that Northern blotting uses the RNA samples derived from virally induced cells and mock-infected cells but virtual Northern blotting uses the SMART cDNAs made from the same RNA samples, and virtual Northern blotting is very convenient if the RNA sample is difficultly available and limited [26,44,45]. Due to using the cDNA samples for hybridization, all procedures are similar to Southern blotting [46].…”

Identical sequences but different expression patterns of Hira gene in gynogenetic and gonochoristic crucian carps

“…At present, the molecular mechanisms underlying the reproductive strategies of gibel carp are poorly understood, making this fish a promising study model for developmental biology (Gui 1996;Zhou et al 2000b;Xie et al 2001Xie et al , 2003. It has been proposed that the reproductive modes of this species may subject to some regulatory factors during oocyte maturation and fertilization (Wang et al 2000;Fan et al 2001;Shi et al 2002).…”