Gene’s Cloning And Select Related To Osmoregulation Of Litopenaeus Vannamei And Portunus Trituberculatus

The osmoregulatory mechanisms of aquatic crustacean animasl has been a hotresearch.By SSH and the clone of gens’related to osmoregulation， this articlestudied the salinity stress on osmotic related genes’expression，preliminary discussedthe crustacean animals’osmotic adjustment mechanism。The article mainly includesthree aspects:（1）Litopenaeus vannamei’s differentially expressed genes’ screening andidentification, expression analysis after the change of salinity;（2） two carbonic anhydrase gene cloning and expression analysis of Litopenaeusvannamei;（3） three two carbonic anhydrase gene cloning and expression analysis of Portunustrituberculatus. The main results are as follows:1. Litopenaeus vannamei’s differentially expressed genes screening andidentification, expression analysis after the change of salinity.Using the technique of suppression subtractive hybridization，we obtained the cloneof differentially expressed genes after the change of salinity，and by real timefluorescent quantitative PCR technique we detected the genes’ expression changesafter salinity changes.were have mainly obtaine phosphofructokinase, adhesionfactor, a putative protein S18₈73₀036, putative protein BCW_A0010, cytochromeoxidase subtype I, elongation factor2, pyridine dehydrogenase and shrimp whitespot disease virus gene，et al.2. Litopenaeus vannamei’s differentially expressed genes screening andidentification, expression analysis after the change of salinity.In this study, the cDNAs of CAc and CAg were cloned from the gills ofLitopenaeus vannamei using rapid amplification of cDNA ends （RACE） approaches. The full-length cDNA of LvCAc was of1161bp with an open reading frame （ORF）of810bp and a mature polypeptide of270amino acids with the predicted molecularmass of29.15kDa. The length cDNA of LvCAg was of908bp with an open readingframe （ORF） of778bp bp encoding a22amino acids signal peptide and apolypeptide of268amino acids.Using RT-PCR, CAc mRNA was expressed at the highest level in gills andmuscle, hepatopancreas, and low in hemocytes have no expression in heart. CAgmRNA was expressed at high level in gills and muscle, and low in hepatopancreas,have no expression in hemocytes and heart. The prediction of protein structureshows that,80-100amino acidswas the Zn2+binding sites, CAg105-120aminoacids was the Zn2+binding sites, there is a hydrophobic pocket includes Val, Leu,Tyr and Leu and by the Thr and His consisting of a proton transfer channel. Prolineis the highest content amino acid, and there is no disulfide bond. His-94, His-96andHis-119is conserved and very important in the combining of Zn2+.Using real-time fluorescence quantitative PCR analysis we searched two kindsof CA gene expression changes of Litopenaeus vannamei under salinity change. Theresults show that, CAc expression increased when the salinity is decreased, whileCAg only showed the salt sensitive, when the salinity is low.3. Three two carbonic anhydrase gene cloning and expression analysis ofPortunus trituberculatus.In this study, the cDNAs of CAc and CAg were cloned from the gills ofPortunus trituberculatus using rapid amplification of cDNA ends （RACE）approaches. The length cDNA of LvCAc was of669bp with an open reading frame（ORF） of618bp and a mature polypeptide of206amino acids. The length cDNA ofLvCAg was of782bp with an open reading frame （ORF） of699bp encoding a22amino acids signal peptide and a polypeptide of233amino acids.Using RT-PCR, CAc mRNA was expressed at the highest level in gills andmuscle, hepatopancreas, and low in hemocytes have no expression in heart. CAg mRNA was expressed at high level in gills and muscle, and low in hepatopancreas,have no expression in hemocytes and heart.The CAc expression is higher than CAg.Using real-time fluorescence quantitative PCR analysis we searched two kindsof CA gene expression changes of Portunus trituberculatus under salinity change.The results show that, CAc expression increased when the salinity decreased, whileCAg only showed the salt sensitive when the salinity is low.