Cloning And Expression Of The Sorbitol Dehydrogenase Gene During Embryonic Development And Temperature Stress In Artemia Sinica

Artemia sinica lives in the hypersaline waters. It mainly distributes in the interiorhypersaline lakes. Artemia has a strong ability resisting extreme environments, the diapauseeggs that laid in the extreme environment could explore under hypersaline, low temperature,low light for a long time.Sorbitol dehydrogenase (SDH) catalyzes the interconversion of polyols and ketoses,using zinc and NAD+as cofactors. SDH converts sorbitol into fructose and plays an importantrole in the sorbitol metabolic pathway. In the early embryonic development of manyinvertebrates, sorbitol usually accumulates in diapause embryos of insects to protect theembryos from frostbite, which indicates the vital function of SDH in the diapause anddiapause-termination stages of embryo development.In this study, RACE PCR was used to amplify the full length cDNA of As-sdh; In situhybridization was used to detect the distribution of As-sdh during different stage ofdevelopment; Real-time PCR was used to detect the expression level during different stage ofdevelopment and under temperature stress; softwares such as MEGA4.0and Clustal, andother online softwares were used for informational analysis.In this study, a1,311bp full-length cDNA of As-sdh, including a28bp5’ UTR and a59bp3’ UTR, was cloned from Artemia sinica. As-sdh encoded a348amino acid protein.Bioinformatic analysis revealed that this gene was highly conserved in zinc binding site and coenzyme binding site. In situ hybridization analysis showed that As-sdh was expressed in awidely dispersed pattern before incubation, but was mainly concentrated on the body surfaceand the inner wall of the alimentary tract after nauplius stage. Expression pattern analysis ofdifferent development stages indicated that As-sdh was expressed at relatively high levelsduring the0h embryonic stage, while quite high in5and7day-old embryos. Expressionpattern analysis of low temperature stress showed that when the embryos were exposed totemperatures ranging from25–15°C, the level of As-sdh transcript increased dramatically withthe temperature decreasing; when the temperature was below15°C, the expression of As-sdhwas significantly reduced.Our results suggested that As-sdh was highly conserved in zinc binding site andcoenzyme binding; As-sdh mainly distributed in the inner wall of the alimentary tract andbody surface after nauplius stage; the result of Expression pattern analysis indicated thatAs-sdh was integral to the process of diapause and diapause termination process in A. Sinica,and was very important to the process of adult individuals fight against low temperature.