Expression Pattern Of Three Osmoregulation Genes In A High-Salinity Environment From Cypridopsis Vidua(Crustacea,Ostracoda)

Cypridopsis vidua,one of the few global species in Ostracoda,has strong tolerance to various environmental factors.Salinity is known to be a major limiting factor in the distribution of aquatic organnisms.As a freshwater microcrustacean,the C.vidua can be distributed in about 8‰ of natural waters,so the species has become an ideal material for studying the osmoregulation of aquatic animals.In this thesis,we studied the response of three osmoregulation genes to the high-salinity environment,and explored the molecular response mechanism of small crustacean osmoregulation genes with sudden salinity.The results of the study provide a theoretical basis for understanding the adaptive distribution of widely distributed aquatic animalsIn this study,the full length of cDNA of V-type H+-ATPase B subunit gene(designated as CvVHA-B)and Na+/K+/2Cl-cotransporter gene(designated as CvNKCC)was cloned for the first time in C.vidua.CvVHA-B gene was 2178 bp,encoding 498 amino acids,CvNKCC gene was 4001 bp,encoding 1055 amino acids.Molecular phylogenetic studies based on the two genes show that the C.vidua and other "pancrustaceans" are clustered into one brance,indicating that CvVHA-B and CvNKCC genes are conservative in the evolution of "pancrustaceans".On the basis of above work,the expression of CvVHA-B and CvNKCC genes mRNA was further studied by the modified whole mount in situ hybridization technique,preliminarily determined that the inner lamella of C.vidua was the main functional site of osmoregulationThe preliminary salinity gradient experiments(0?4?8?12?16 and 20‰)for this study determined that the high salinity of 24h acute salinity stress for C.vidua was 8‰.On the basis of this result,real-time fluorescence quantitative PCR was performed to investigate the time-dependent changes of mRNA expression levels of Na+/K+-ATPase a subunit gene(designated as CvNKA-?),CvVHA-B,and CvNKCC at 4‰ and 8‰ salinityThe results showed no significant differences in the expression levels of the three genes between the 4‰ salinity and control(0‰)groups(P>0.05).In the 8‰ salinity group,the expression levels of the three genes showed a certain time sequence,that is,the expression of CvNKA-? was significantly lower than that in the control group at 3 h(P<0.05),the expression of CvVHA-B significantly decreased at 12 h(P<0.05),and the expression of CvNKCC showed a significant increase at 24 h(P<0.05).By the combined role of the three genes in the regulation of osmolality in animals,the study suggests that freshwater species C.vidua can downregulate the expression of CvNKA-? and CvVHA-B and upregulate the expression of CvNKCC to adapt to sudden high-salinity environments.This is a good explanation for the reason why freshwater species can be distributed in natural water bodies up to 8‰.