Construction Of Ssh Library For Litopenaeus Vannameiexposed To Cold Stress And Cloning And Characterization Of Cold-induced LvTSPAN8 Gene

The study adopted Suppression Subtractive Hybridization(SSH)to construct the cDNA libraries of Litopenaeus vannamei in cold stress,and confirmed some cold-related genes by dot blot hybridization,ESTs sequence and analysis,and Real-time Quantitative PCR.Furthermore,full-length of the most abundant Tetraspanins gene from the forward library was cloned and characterized.The results are as follows:1.Construction and analysis of SSH libraries for Litopenaeus vannamei exposed to cold stressThe study constructed two gene's differential expression libraries for the low-temperature processing and the room-temperature by Suppression Subtractive Hybridization(SSH)technique,named forward library and reverse library.The result of dot blot hybridization showed that 152 of 768 clones from the forward library were up-regulated at the low-temperature group,and 331 of 768 clones from the reverse library were up-regulated at the room-temperature group.Thirty nine contigs were obtained after assembling 100 ESTs sequenced from the forward library,including 29 known genes,and eighteen contigs were obtained after assembling 50 ESTs sequenced from the reverse library,including 17 known genes.2.RQ-PCR confirmation of cold-induced genesThe expression level of 4 high abundance genes from the forward library(2 known genes TSPAN8 and HSPB1,and 2 unknown genes NGT4 and NGT7)had been confirmed by RQ-PCR.The results showed that all the 4 genes presented an up-regulated expression when stressed in the low temperature of 18?,and the expression level of TSPAN8,NGT7 and HSPB1 reached the peak at 15'C,the expression level of NGT4 reached the peak at 13?.Although the expression level of the genes was down when the temperature further lowered,they were still much higher at 11? than at the room-temperature..The expression level of TSPAN8 gene in different tissues of cold exposure(13oC)shrimp was further estimated using room-temperature shrimp as control.The results showed that expression of TSPAN8 gene was up-regulated in heart,gill and hepatopancreas and down-regulated in the muscle at the low temperature(13?).3.Cloning and bioinformatics analysis of LvTSPAN8 geneThe most abundant tetraspanin gene from the forward liabrary contains an open reading frame(ORF)of 720b preducing to a 239 aa peptide with predicted MW of 25.61 kDa The tetraspanin gene isolated from L.vannamiei exhibited 83.7%,52.7%and 48.5%identity with the reported F.chinensis TSPAN8 protein,Scylla pararriamosain tetraspanin protein and CD53 protein,respectively.The multiple sequence alignment revealed a 22 amino acids domain that was conserved among the various tetraspanin proteins,indicating some conserved function for tetraspanin family genes.The phylogenetic tree analysis showed the presence of L.vannamei TSPAN8 and F.chinensis TSPAN8 in the same clade,confirmed that the tetraspanin gene was TSPAN8 of L.vannamei(LvTSPAN8).Analyzed by TMHMM software,the deduced LvTSPAN8 protein in this study have a classical 4 trans-membrane segments,and both the N-and C-termini lie on the intracellular side of the membrane and conserved CCG motif and cysteine residue patterns present in large extracellular loop region of various tetraspanin proteins.