Cloning, Expression And Function Analysis Of Doublesex Gene In Daphnia Carinata

Daphnia carinata is an order of small, primarily freshwater crustaceans commonly known as water fleas. These small-bodied organisms are an important component of the zooplankton and are fundamental to the structure, function and biological productivity of freshwater ecosystems. Healthy cladoceran populations make a significant contribution to the quality and productivity of freshwater fisheries, being rich in protein, fat, carbohydrates and vitamins.Doublesex gene is an important regulator of sex, originally discovered in Drosophila melanogaster. In the three cascade path of sex determination, Doublesex locates after Sex-lethal (Sxl) and Transformer (Tra). Doublesex gene is required for the produce of any kind of sex phenotype, and its mutation can change the expected phenotype, such as making a XX embryo developing into a male individual or a XY embryo into a female individual. Found in Bombyx mori and Drosophila melanogaster, according to the different stages of pre-mRNA, by female-specific cutting, Doublesex will be expressed differentially, and transcribed into gender specific mRNA isoforms.D. carinata displays alternate modes of reproduction in response to fluctuating environmental conditions. When conditions are favorable they reproduce parthenogenetically, which enables rapid population growth. As conditions deteriorate, sexual reproduction is initiated and both males and females are produced. Eggs fertilized by males develop into resting eggs which lie dormant until conditions improve, thus ensuring the continued survival of the population. So far, research into the initiation of the different phases of reproduction has focused primarily on the ecological factors, while few studies have attempted to elucidate the molecular mechanism involved.This study successfully cloned two full-length cDNA sequences of the gene Doublesex, designated DapcaDsxl (Genbank accession number:KF753235) and DapcaDsx2 (Genbank accession number:KF753236). The full-length cDNA of DapcaDsxl is 1618 bp, and the 1218bp open reading frame (ORF) encoded a protein of 405 amino acids. The amino acid sequences encoded by DapcaDsxl were compared with 15 equivalent amino acid sequences in GenBank. The results showed that the highest homology was with D. magna, D. pulex, and D. galeata. D. carinata clustered together with D. magna, D. pulex, D. galeata finally. Via sequence contrasting and splicing, we obtained a 1108 bp full-length cDNA of DapcaDsx2. It also contained an 849 bp ORF, which encoded a protein composed of 282 amino acids. The highest homology of the DapcaDsx2 protein sequence was shared with D. magna, followed by D. pulex and D. galeata. A phylogenetic tree was reconstructed. In terms of molecular evolutionary distance, the DapcaDsx2 cDNA was closely related to crustacean sequences, and more distantly related to insect or other sequences.Expression of DapcaDsxl was compared between the different reproductive phases of D. carinata using qPCR. Significantly different levels of DapcaDsxl expression (P< 0.05) were detected between juvenile females, parthenogenetic females, prophase sexual females, sexual females and resting eggs, with the highest levels of difference observed in prophase sexual females, followed by sexual females, parthenogenetic females and resting eggs; the lowest range of expression was observed in juvenile females. DapcaDsx2 was most abundantly expressed in prophase sexual females and sexual females, followed by juvenile females and parthenogenetic females. Lowest expression was detected in the resting eggs. Expression levels of DapcaDsxl and DapcaDsx2 in asexual reproduction and sexual reproduction were significantly different. Taken together, the results clearly show that DapcaDsx2 mRNA has specific expression during the different reproductive stages.The digoxin-labeled RNA riboprobe was used to investigate DapcaDsxl and DapcaDsx2 expression and cellular localization in individual organisms during different growth and reproductive phases. DapcaDsxl was expressed mainly in the thoracic limbs, the second antennae, and several sites on the ventral carapace. The level of expression was higher in sexual females than in parthenogenetic females. DapcaDsx2 was mainly expressed in the second antennae and several sites of the ventral carapace. In contrast, expression level in sexual females was higher than parthenogenetic females. In addition, the sense probe detected no signal in the corresponding sites. These findings are consistent with the quantitative expression results, which indicated specific expression profiles of DapcaDsxl and DapcaDsx2 mRNA in different reproductive stages. The marked expression in such important organs indicates that DapcaDsxl and DapcaDsx2 may play a role in regulating growth and development. Further research is required to determine why it is expressed in these areas and how it functions.Using the technology of RNA interference (RNAi), the molecular mechanism and function of DapcaDsxl in D. carinata was studied preliminarily. The results showed that, the method by constructing RNAi vectors, feeding bacteria containing double-stranded RNA (dsRNA), could achieve better effects of RNA interference. In the RNAi phenotype of D. carinata, the first antennae became shorter, the expression of DapcaDsxl became decreased, the activity of alkaline phosphatase (AKP) and acid phosphatase (ACP) became decreased. This indicated that, DapcaDsxl may affect the appearance and metabolism of D. carinata, may play an important role in growth and development.