| This research to Hyriopsis cumingiifor research object, according to the EST sequences from the mantle transcriptome library construction has been marked, genes full-length cDNA sequence of heat shock protein 90( HcHSP90), activated protein kinase C1(HcRACK1) and leucine repeat gene(HcLRR) have been cloned in the application of cDNA ends(RACE) with its bioinformatics analysis, analyzed the expression of three genes in tissues and in expressing different time points after stress under various stress conditions. The results are as follows:1.Molecular characteristics and expression analysis of HSP90 geneThe full length of HcHSP90 cDNA is 2659bp(Submitted GenBank, accession number: KR633143), an open reading frame(ORF) of 2187 bp, while its 5’- and3’-untranslated region length are 101 bp and 371 bp, of which the 3 ’ untranslated region with AATAAA mRNA tail signals and Poly(a) the oligo adenylic acid. The open reading frame encodes a 728 amino acid,protein molecular 83.7Ku, and an isoelectric point of 5.13. There is no signal peptide and transmembrane structure in thesequence. Amino acid sequence analysis showed that HcHSP90 contained the HSP90 family 5 conserved sequence tags. Homologous alignment analysis showed the highest similarity with the protein encoded by the gene Cristaria mussels(Cristaria plicata) HSP90 amino acid sequence for 83.93%. The resulting amino acidsequence and amino acid sequence of HSP90 in other species multiple sequence alignments, and building the system tree, the results showed that the experiment clones derived from HcHSP90 and has published several mollusks HSP90 together as a team.In non-coercive state, HSP90 gene in various inspection organs(adductor muscle,foot, digestive gland, hemolymph, mantle, gills, gonads) were expressed, highest level ofexpression in the Digestive gland,gonads, and expression was lowest in the adductor muscle.In most tissues, the expression level of HcHSP90 mRNA was significantly induced in different temperature treatments(0, 5, 25, and 35°C)compared to the control(15°C). The expression levelof HcHSP90 mRNA in the adductor muscle and gonad peaked at 5°C(8.89-fold, P<0.01; 2.0-fold, P<0.01, respectively),and in the digestive gland werethey significantly suppressed at 0℃(0.29-fold, P<0.01) and in theother tissues examined the expression levels peaked at 35 C. Were exposed to different concentrations of cadmium(50,100 and 200 μg/l), HSP90 mRNA expression level in hemolymph and gill raised varying degrees, but nocleardose-dependent response.Whencaused by Aeromonas hydrophila infectionlater, and HSP90 mRNA expression in the hemolymph, infected with 36 h peak(12.14, P<0.01), gill, after being infected with 3h expression raise(3.24, p<0.01), and then remained constant until the levels returned to 36 h afterinfection before infection.2. Molecular characterization of the HcRACK1 gene and expression analysisThe full length of HcRACK1 cDNA is 1249 bp, of which the open reading frame957 bp, encoding 318 amino acids. Protein domain analysis showed HcRACK1 contained RACK1 familialy seven WD40 domains. Using fluorescence quantitative PCR technique to analyze the changes of the expression of HcRACK1 in the tissues of non-stress state and under the condition of the infection and the heavy metal cadmium exposure. The results showed, HcRACK1 was expressed in various tissues,and the highest expression tissue was adductor muscle; after Aeromonas hydrophila infected, HcRACK1 expression in hemolymph increased gradually, peaked at 24 h,then decreased; exposure to 100μg/L concentration heavy metals cadmium,HcRACK1 expression in digestive gland gradually increased, peaked at 3d;hemolymph peaked at 2d, then decreased; the expression of HcRACK1 in gill showed no significant changes.3.Cloning and expression analysis of leucine repeat(LRR) geneThe full length of LRR gene cDNA is 3492 bp, containing of 2142 bp ORF,encoding 713 amino acids, including the period of the 25 amino acid signal peptide and a 688 amino acid mature peptide, amino acid sequence analysis showed that the sequence contained a trans- membrane structure, using quantitative PCR to analysis LRR mRNA in normal state and Aeromonashydrophila infection the related tissues expression. The results showed that, under non-stress state, the highest expression of LRR gene is in foot, almost not expressed in other tissues. After Aeromonas hydrophila infection, we saw a significant stimulus in foot and the expression decreased,while other tissues expression peaks appeared at different points, the result showed LRR could be significantly induced. |
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