Cloning And Expression Analysis Of The Reproduction And Development Related Genes Transformer And Checkpoint Homolog 1 In Daphnia Carinata

The life history of cladocerans is particularly unique, since they undergo both asexual (parthenogenesis) and sexual (bisexual) reproduction. When environmental conditions are favorable, reproduction is generally parthenogenesis. The ova produced by parthenogenesis females can develop into offsprings directly without fertilization. However, if environmental conditions deteriorate, such as the lack of food, too high or low temperature and so on, males will appear, and reproduction switches from asexual to sexual (bisexual) reproduction. The eggs produced by bisexual females withstand the harsh environment and hatch when conditions improve. This reproductive transformation is advantageous for rapid adaptation to different environments and helps to maintain viable populations. The ecology of the reproductive transformation and the environmental influences are well understood, however knowledge of the molecular mechanisms regulating it is lacking, and studys on it remain to be elucidated.In this study, the full-length cDNA of the Transformer gene and the Checkpoint homolog 1 gene from the common freshwater species Daphnia carinata (DcarTra and DcarChkl) were first successfully cloned using primers based on homologous sequences and rapid amplification of cDNA ends (RACE). The full-length DcarTra cDNA was 1620 bp with an open reading frame (ORF) of 1143 bp. The ORF encodes a protein of 380 amino acids with four potential N-glycosylation sites and no signal peptide. The N-terminal region (from Arg3 to Ser85) contains an RS domain. The DcarChkl cDNA was 1817 bp in length with an ORF of 1494 bp. The ORF encodes a protein of 497 amino acids without signal peptide. Moreover, there was a domain with catalytic activity of serine/threonine-protein kinases (S_TKc domain) at from the 21st to 276th positions.Through homology alignment of the amino acid sequences of proteins encoded by DcarTra and DcarChkl, we found that the two genes were poor in evolutionary conservation. Phylogenetic analysis identified low homology with other species. But alignment of DcarTra and homologous sequences revealed a region containing a 26 residue conserved motif that shares a high degree of similarity. Phylogenetic tree analysis showed that in Arthropoda, D.carinata shares the highest homology with its fellow Crustacea D. magna and D. pulex, followed by hymenopteran insects, and is less closely related to coleoptera and diptera homologs. These results conform to the accepted evolutionary relationships.The relative expression of DcarTra and DcarChkl during different reproductive phases were investigated using qPCR. The results showed that DcarTra was expressed in both sexes, but expression was highest in males, intermediate in sexual females, and lowest in parthenogenetic females and prophase sexual females.While DcarChkl was expressed in both females and males, its expression was significantly greater in males than in females, and was greater in sexual females than in parthenogenic females. We also found that the expression of DcarChkl was higher in adults than in larvae, regardless of the sexual phase of the females. These results showed that expression of the two genes both have significant gender differences.The cellular localization of DcarTra and DcarChkl during parthenogenesis and sexual phases were investigated using whole-mount in situ hybridization. The results showed that DcarTra was mainly expressed in the thoracic limbs, ovaries and rectum in parthenogenetic females, and in the joints of second antennae, ovaries, rectum and ventral processes in sexual females. DcarChkl in parthenogenic females was mainly expressed on the surface of the head, on the capillus and on the edge of carapace valve. Meanwhile, in sexual females the expression of DcarChkl was mainly concentrated in the joint of second antenna, on the thoracic limbs and on the capillus.The quantitative expression of DcarTra protein was assessed by Western Blotting. The results showed two differently phosphorylated forms of the Tra protein. When Tra is phosphorylated, DcarTra protein levels were much higher in males than in two females. Otherwise, when Tra is dephosphorylated, the highest DcarTra protein levels were in sexual females, which revealed that D. carinata can control the sexual transition via these two forms. The information on the expression pattern and function of DcarTra and DcarChkl in D. carinata provides a basis for elucidating the molecular mechanisms driving growth and reproductive plasticity in cladocerans.