Molecular Cloning And Expression Analysis Of Trx And Grp78cDNAs From Freshwater Pearl Mussle

Trx and Grp78cDNAs were cloned from freshwater mussle, Cristaria plicata and Hyriopsis cumingii, using rapid amplification of cDNA ends and nested PCR.The expression of Trx mRNA in the tissue of hemocytes, hepatopancreas, gill, mantle and muscle in Cristaria plicata was measured by fluorescent real-time quantitative RT-PCR. The expression level of Trx mRNA after PBS and Staphylococcus aureus stimulation were also detected by RT-PCR. The tissue expressions of Grp78in Hyriopsis cumingii were measured by semi-quantitative RT-PCR, as well as cold and hot shock induced.1. The full-length cDNA sequence of CpTrx was1169bp (GenBank accession number:KC209545), consisting of a5’-terminal untranslated region (UTR) of11bp, a3’-terminal UTR of840bp with a poly (A) tail, a tailing signal (AATAAA), and the open reading frame of318bp encoding105amino acids with a predicted molecular mass of11.6kDa, and a theoretical isoelectric point of5.38, no signal peptide was found. The deduced amino sequence of Trx contains a structural motif of T3-K104with a conserved activity site CGPC. Sequence homology comparison results showed that the consistency of Trx mRNA sequence from Cristaria plicata compared with Crassostrea gigas and Mytilus galloprovincialis were56%and52%, respectively. The predicted space structure of Trx was composed of5beta folding and4alpha helices, alpha helice surrounded beta folding and form a tight sphericity which was quite similar to human Trx. CpTrx mRNA was expressed in hemocytes, hepatopancreas, gill, mantle and muscle, the highest in gill, hepatopancreas followed. Trx expression level increased in all tissues after Staphylococcus aureus stimulation compared with PBS induction. In hemocytes Trx reached highest expression at12h and then decreased gradually, while still higher than control group at48h. Trx continuously rose in hepatopancreas from12to48h until reaching its peak and then declined. In mantle, the expression reached the lowest at12and24h, and then increased and reached its peak at36h, while reduced again at48h. Trx from muscle appeared obviously increased and got the highest expression at12h, after that it declined clearly until under the control group.2. The full-length cDNA sequence of HcGrp was3721bp, with a5’-terminal untranslated region (UTR) of148bp, a3’-terminal UTR of1578bp with a poly (A) tail, a tailing signal (AATAA), and the open reading frame of1995bp encoding664amino acids. The predicted molecular mass of HcGrp was73.27kDa, and the theoretical isoelectric point was5.38. The N-terminus had the features consistent with a signal peptide as defined by SignalP software with a putative cleavage site located after position22. The44-51,232-245and369-383amino acids were three Signature sequence of HSP70family, they were IDLGTTYS、VFDLGGGTFDVSLL and VVLVGGSTRIPKVQQ. HcGrp contained16Serine phosphorylation sites,8Threonine phosphorylation sites and4Tyrosine phosphorylation sites. Sequence homology comparison showed that Grp78in Hyriopsis cumingii was highly conserved than other species, the consistence of92%. Grp78expressed in muscle, mantle, gill, hepatopancreas and hemocytes. The expression after4℃stimulation showed that no significant variation compared with20℃stimulation. After40℃induction the expression in hepatopancreas increased sigmificantly, and also were distinctly in hemocytes and gill, while without clear change in muscle.