Expression Pattern Of As-trpc4Gene During Diapause Process And Temperature Stress In Artemia Sinica

Artemia sinica is a kind of small crustacean which worldwide distributed in hypersalinewaters and wide temperature, with a strong resilience and tolerance.Under extremeenvironmental conditions, such as low sunshine, high salt and low temperature,the Artemiacan produce the Artemia cysts which is in the diapause period gastrula, to environmentalconditions for growth and development into nauplius.Diapause for Artemia sinica resistanceto extreme environment and to ensure that the embryo survival play a crucial role.TRPC4（transient receptor potential cation subfamily member4）is a kind of nonselectivecation channels with complex accommodation mode and a variety of functions, it mainlymediated the transparent of calcium ions and other cations. Generally, TRP protein family hasthe characteristics of temperature sensitivity. After the diapause of Artemia sinica, embryo bylow temperature stimulation to break dormancy, at this point, the TRP proteins on theembryos of Artemia sinica can be received cold stimulation signal and then be activated,transfer various molecular pathways start work at the same time, in order to achieve the goalof lifting diapause. At the moment,the research of TRPC4for the expression pattern duringdiapause process and in response to stress in Artemia sinica has not been reported, it needfurther in-depth study and careful analysis for providing a theoretical reference for furtherresearch on molecular mechanism of diapause process in Artemia.In this paper, the full-length2388bp cDNA sequence of the As-trpc4gene was clonedfrom A. sinica by RACE technology. As-trpc4cDNA contained an open reading frame of1296bp, and232bp5’-and860bp3’-untranslated regions (UTR). The putative TRPC4protein consists of432amino acids with a DUF3689domain, and has a calculated molecularmass of48.3KDa, with a pI of5.37. And several online services were used to analyzeAs-trpc4phosphorylation sites, signal peptidase, transmembrane region, subcellularlocalization, secondary structure prediction, hydrophilcity and hydrophobility and others.ClustalX2.0and MEGA4.0were used to analyze the evolutionary relationships of the putativeamino acids of this sequence and homologous sequences, The results show the amino-acidsequences of TRPC4between different species are conservative in the long process ofevolutionary.In situ hybridization was performed to determine the expression position of As-trpc4during different development stages of A. sinica. The results show that positive As-trpc4 mRNA signals is widely expressed during different embryonic developmental stages of A.sinica.To determine the level of As-trpc4transcription during different development stages of A.sinica and in response to salinity stress and during embryos and adult stage temperaturestresses, real-time PCR analysis was performed. As-trpc4is widely expressed during differentembryonic development of A. sinica, but the relatively quantitative expression is different.The expression of As-trpc4during embryos period in the ovisacs is much higher than otherperiods. Highest As-trpc4expression was detected at the0h stages, and in embryonic stage(5h,10h,15h) also has a relatively high expression. This is followed by a dramaticdown-regulation and then maintain at very low levels. These findings suggest that As-trpc4plays an important role in diapause termination in Artemia sinica, but also it is an importantregulatory gene during embryonic development which may act as a protective factor. In thetemperature and salinity stress experiments, the expression of As-trpc4gene graduallyincreased with increasing salinity, and in the most extreme200‰has an obvious rise. Whileboth adult and embryos, the expression of As-trpc4gene gradually increased with decreasingtemperature. These findings suggest that As-trpc4may play an important role in overcomingthe extreme environmental stresses, especially it is an significant stress-related gene when inthe temperature stress in Artemia sinica.