Salt Excretion And Leaf Drop Gene Cloning And Molecular Evolution Of The Great Blue-spotted Mudskipper

The great blue-spotted mudskipper?Boleophthalmus pectinirostris?belongs to the goby fish,which is a warm water,wide-temperature,and wide-salt fish.It lives in the muddy tidal flats and mangrove areas in the intertidal zone of the estuary.It's habitat specificity,the intensity of intertidal salinity change,tolerance to ammonia nitrogen stress and its unique amphibious adaptability in the air,attract scientists to study its physiological and biochemical and adaptive mechanisms.In this study,we studied the cloning,functional analysis,gene expression and molecular evolution of Rhgp and Aquaporin genes,and clarified the correlation between these two types of osmotic pressure-regulated genes in fish and terrestrial animals.Explain its evolutionary choices,in order to achieve the research purpose of the evolution analysis of the salt gene of the large-boiled fish,so as to effectively solve the selection pressure analysis of the salt-discharging genes in the environment of seawater,freshwater,migratory and broad-salt.The great blue-spotted mudskipper is an amphibious teleost that is distinctly adapted to living on beach.They lived in environment with intense salinity change and showed the tolerance to salinity and ammonium stress.Rhgp plays an important role in NH₄⁺transport in fish gills.But their responses to salinity challenge is still unclear in this species.Here,four Rhesus family glycoprotein encoding genes,Rhag,Rhbg,Rhcg1 and Rhcg2 were cloned from the gills of great blue-spotted mudskipper.The coding sequences were 1,308bp,1,386 bp,1,458bp and 1,455bp in length,and encoded 435,461,485 and 484 amino acids,contained 11 and 12 transmembrane domains for Rhag,Rhcg1,Rhcg2 and Rhbg,respectively.The relative expression was significantly decreased for Rhag under low salinity stress while significantly increased for Rhbg under high salinity stress,respectively.The relative expression of Rhcg1 was decreased but not significant under high or low salinity stress,the expression of Rhcg2 increased significantly and then decreased under high salinity stress,but under low salinity stress it decreased significantly.This means that these Rhgp are not only the transporters for NH₄⁺but also are involved in the tolerance to salinity stress for the great mudskipper.Molecular evolutionary analysis detected positively selected sites in fish,suggesting the adaptive evolution for these four genes during fish adaptive radiation to different salinity.This study represents a comprehensive overview of the aquaporin gene family in great blue-spotted mudskipper and provides new insights into the evolution and salinity stress regulation of this gene family.The aquaporin gene family has been systematically characterized in some fish species but not in the great blue-spotted mudskipper.In this study,16 aquaporin genes?BpAQP?were identified in the great blue-spotted mudskipper genome.Six members of the ancestral vertebrate aquaporin genes?BpAQP0,BpAQP1,BpAQP3,BpAQP4,BpAQP8 and BpAQP9?experienced teleost-specific whole genome duplication during evolution,and now each has two copies on different chromosomes.Expression profiles analyses indicated that the transcription of BpAQP genes was detected in 9 different tissues?namely,gill,intestines,skin,liver,brain,spleen,kidney,gonad?female?and muscle?,and the expression pattern also differed among each BpAQP gene.Furthermore,to explore the temporal distribution of four BpAQP genes?BpAQP1a,BpAQP1b,BpAQP3a and BpAQP3b?in gills,great blue-spotted mudskippers were abruptly transferred from 10‰seawater to either hypertonic seawater?30‰?or hypoosmotic freshwater?0‰?.In addition,through the identification,cloning and evolutionary analysis of related functional genes,not only the genomics data of the great blue-spotted mudskipper,but also the experimental research on the physiology of the great blue-spotted mudskipper,especially molecular evolutionary biology and molecular genetics,for the subsequent application of the great blue-spotted mudskipper provide reference for screening important salt-discharging genes for environmental processes.In addition,it will enrich and deepen the understanding of the function and mechanism of the great blue-spotted mudskipper salt-discharging gene in aquatic and terrestrial environments,and will expand the salt cloning and molecular evolution of the whole goby fish,and the results are also expected.It has a major impact in the fields of environmental stress and genetic selection evolution of aquatic animals.