Expression Analysis Of Diapaus Reproduction Related Genes From Oocytes Of Artemia Parthenogenetica

Artemia (Arthropoda, Crustacea, Branchiopoda, Anostacea, Artemidae, Artemia), commonly known as brine shrimp, is widely distributed in inland salt lakes. They can withstand the widest salinity range of any known metazoan. Embryos of Artemia undergo either oviparous development, producing encysted gastrulae (cysts), or ovoviviparous development, producing swimming larvae (nauplii). The cysts enter diapause, a state of greatly reduced metabolic activity and extreme stress resistance.To better understand the molecular mechanism of diapause regulation, we analyzed the gene expression of oocytes in the oviducts of Artemia parthenogenetica (Gahai Lake, China) by a novel subtractive hybridization method. The main results are listed below: 1) in the aim of developing a full-length cDNA approach, we have improved a PCR-based double-stranded cDNA synthesis method, meanwhile we have systemly analyzed the PCR-suppression effect (PS-effect) and utilized the PS-effect to overcome the biased amplification of short-molecules of PCR; 2) a duplex-specific nuclease-mediated normalization and subtractive hybridization (DNSH) method was developed, by which full-length cDNA of the differentially expressed genes at a rare level of consisting only 0.0001% of the total mRNA can be sufficiently enriched; 3) oocytes from diapause reproduction-destined Artemia was subtracted by the oocytes from nauplii reproduction-destined Artemia using DNSH, a total of 692 cDNA clones were analyzed in which 323 matched GenBank entries. The 692 cDNAs were assembled into 600 contigs, most of which were singletons (529). Real-time PCR analysis of 44 cDNAs (representing 56 clones) revealed that their mRNA levels differed to a maximum of about 15,000 fold. Among them, 11 (21 clones) were significantly (p<0.05) up-regulated and 7 (9 clones) down-regulated in Artemia oocytes that subsequently enter diapause. The results indicated that the reproductive paths of A. parthenogenetica are already determined at the stage of oocytes-filled oviducts. In summary, we have successfully developed a novel subtractive hybridization approach, DNSH, to study the differential transcript profiles in A. parthenogenetic diapause-destined oocytes relative to nauplii-destined oocytes at the filled oviduct stage. Our results showed that many genes whose products commonly regulate metabolism, growth, transcription and stress tolerance are already differentially expressed between diapause- and nauplii-destined oocytes. We could conclude that the reproductive paths of A. parthenogenetica are determined at an early stage of the oocytes in oviducts.