Construction Of A SSH CDNA Library, CDNA Cloning And Expression Of Glutamine Synthetase In Litopenaeus Vannamei, Boone Induced By Hypo-osmotic Stress

A subtractive cDNA library of hepatopancreas in L.vannamei was constructed bysupression subtractive hybridization (SSH) induced by acute low salinity stress. cDNAclones were selected from library and sequenced. These sequences were analysed usingBLASTX software and classied for the results. Moreover, full-length cDNA of glutaminesynthetase (GS) of L.vannamei was cloned, and the sequence was analysed. Theexpression of LvGS was analysed in different tissues induced by acute low salinity, theexpression of LvGS, GS activity, glutamine in hepatopancreas and nitrogen metabolismin hemolymph were analysed induced by acute low salinity stress after different times.The results provided scientific basis for deep insight to molecular mechanism ofL.vannamei dadaption to low salinity water. The content of studies and results are asfollowings:1. Subtracted cDNA library construction and sequences analysis162expressed sequence tags (ESTs) were obtained by randomly sequencing, and131unigenes including21contigs and110singlets after CP3online matching were gained byclustering and assembling. The results of sequence homology search showed that thetewere41known with significant homology to Genbank database sequences that wereidentified.The clusters together in7processes according to their functions. They are proteinsynthesis and processing, carbohydrate metabolism and energy production, transport, cellgrowth and apoptosis, cell defense and homeostasis, signal transduction and others. Thepercentage of cDNA sequences of protein synthesis and processing was the highest inobtained unigenes, followed by transport and carbohydrate metabolism and energyproduction. cell defense and homeostasis, signal transduction were low proportion. Resultsshowed that the expressions of protein metabolism, carbohydrate metanolism and transportrelated genes, especially protein metabolism related genes from hepatopancreas wereup-regulated induced by acute low salinity stress.1. Cloning and sequence analysis of glutamine synthetase (GS) of L.vannameihepatopancreas Based on the sequence of glutamine synthetase EST obtained from subtractive libraryof L.vannamei hepatopancreas. The full-length cDNA of GS was obtained using5′-RACEand3′-RACE, and named LvGS. GS cDNA sequence was analysed using bioinformatics,The full length of GS is1455bp, containing56bp5′-untranslated region,298bp3′-untranslated region and1101bp open reading frame (ORF). It encoded a peptide of366amino acids. The calculated molecular weight is40.92kDa and theoretical isoelectric pointis6.34. The results of multiple alignment showed that LvGS was similar by93%withFenneropenaeus chinensis,86%with Panulirus argus and73%with Crassostrea gigas. Aphylogenetic tree analysis of GS sequences indicated that LvGS clustered with theinvertebrate group. Evolutionary relationship was very closely, and was far with vertebrate.2. Effects of acute low salinity on expression of LvGS, GS activity, glutamine andnitrogen metabolism in L.vannameiQuantitative RT-PCR analysis showed that LvGS was expressed in in muscles,stomaches, gills, intestines, hearts, hepatopancreas and eyestalks, and its profile wasup-regulated in hepatopancreas, intestines, stomaches, gills, down-regulated in hearts,eyestalks and muscles post acute low salinity stress. The expression of LvGSsignificantly(P<0.05) increased in hepatopancreas post-transfered to low salinity for6h,12h,1d and2d followed decreased. Hepatopancreas GS activity, hepatopancreas glutamine,hemolymph urea and uric acid had an increased followed decrease trend post-transfer from3h to20d. Hemolymph total protein and ammonia decreased then increased post-transferfrom3h to20d. No significant difference was occurred in hepatopancreas GS activity,hepatopancreas glutamine, hemolymph total protein and hemolymph ammonia.Hemolymph urea significantly(P<0.05) increased post-transfered to low salinity for3h and12h. Hemolymph uric acid significantly(P<0.05) increased post-transfered to low salinityfor3h,6h,12h,2d and3d. The LvGS expression, heapotancreas glutamine concentration,glutamine synthetase activity, hemolymph urea, ammonia, urea acid was trend to normallevel post-transferred to low salinity for30d.