| Environmental stimuli initiate a switch in the reproductive strategy of Daphnia pulexfrom asexual to sexual reproduction; however, occasionally, changes in environmentalconditions will not lead to transition. So study genetic responses to environmental stimuli andthe molecular basis for environmental sex determination and control the switch betweenreproductive modes are urgently needed. Therefore, we isolated and sequenced a D. pulexdoublesex1gene (Dpdsx1) and analyzed its expression and location by quantitativepolymerase chain reaction (qPCR) and whole-mount in situ hybridization in D. pulex in theswitch between reproductive modes. Then using RNA interference technique to suppress Dpdsx1mRNA expression and detecting changes at spatial and temporal expression with interfered differentreproductive daphnias. We aim to identify functional gene in switch reproductive modes of D.pulex andprovides a theoretical basis for the molecular mechanisms of regulating switch. Specific findings are asfollows:(1) The cDNAof dsx1was cloned by the RT-PCR and rapid amplification of cDNAends (RACE). TheDpdsx1full-length cDNA was1639bp in length (GenBank accession number: KC470706),and included a5’ UTR of169bp, a3’UTR of458bp, one canonical AATAAA signal and anopen reading frame (ORF) of1008bp encoding335amino acids. The theoretical isoelectricpoint (pI) of the DpDsx1protein was8.84, and its predicted molecular weight was37.27kDa.An analysis of the SignalP4.0server revealed no signal peptides. A domain structure analysisshowed that the DpDsx1protein had two functional domains: an N-terminal DM domain anda C-terminal oligomerization domain. Homology analysis indicated DpDsx1had77%,61%and54%identity to the Dsx1of D. magna, D. galeata and C. dubia, respectively.Phylogenetic analysis showed D.pulex had a closed relationship with D. magna, D. galeata, C.dubia, A. florea, A. mellifera and so on. The topology of the phylogenetic relationship for Dsxorthologs was in very good agreement with taxonomic relationships of insects. (2)Real-time PCR showed that Dpdsx1expression decreased significantly (P<0.05) indifferent reproductive modes in the following order: male, parthenogenetic female, ephippialfemale, resting egg and juvenile female. Whole-mount in situ hybridization revealed thatDpdsx1expressed in the first antennae, first thoracic limb and compound eye in males,whereas expression levels in the corresponding sites of parthenogenetic and ephippial femaleswere relatively weak. Dpdsx1could not be detected in the gonads of males or ephippial andparthenogenetic females. Dpdsx1gene expression showed male-biased condition in D. pulex.These suggested that Dpdsx1may play a significant role in switching between reproductivemodes and environmental sex determination.(3)Analysis changes of expression levels in different reproductive daphnias with interfered Dpdsx1mRNA. Using Real Time PCR and Whole-mount in situ hybridization detected the overall Dpdsx1mRNAexpression changes in the characteristics after the interference. Real time-PCR results showed that theDpdsx1mRNAexpression decreased in male, parthenogenetic daphnia (with summer eggs), sexual daphnia(with winter eggs) expression, which declined most in the parthenogenetic daphnia, followed by male, sexualdaphnia (with winter eggs).Whole-mount in situ hybridization results showed that the overall interfered Dpdsx1mRNAreducedits parts and expression significantly compared with uninterfered, which were similar to Real time-PCRresults, but the amount of sites expression decreased more. In parthenogenetic daphnia expression was notdetected in any sites. In sexual daphnia expression was only a litter in the first antenna portion. In maledaphnia expression site also detected some parts of the first antennae.Compared with uninterfered daphnia itsexpression and sites reduced significantly and becomes weaker.As a conclusion, combinng with Real time-PCR and Whole-mount in situ hybridization results inexpression and location, Dpdsx1expression and localization are specific differences in the different. In themale characteristic organ-first antennae, there were significant differences before and after interference.According to the above results we believe Dpdsx1involved in reproduction switch and maintenance ofmale morphology process. |
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