Cloning And Functional Research Of Dermatopontin And Toll Like Receptor3of Pearl Oyster, Pinctada Martensii

Pinctada martensii is cultured for production of pearls in China. In our previousresearch, using RNA-seq technology and bioinformatics method, we have analyzed thetranscriptome of the pearl sac from Pinctada martensii and found51biomineralization-related unigenes and268immune-related unigenes. In this research,we mainly focus on one biomineralization related genes-dermatopontin (DPT) and oneimmune related genes-Toll-like receptor3(TLR3). Through RACE technique toobtain full-length of these two genes and qRT-PCR to analyse the expression ofthem; RNA interference (RNAi) to verify they functionality.DPT was identified as a major component of the shell matrix proteins. In thisresearch, we have got the full length of DPT from Pinctada martensii by rapidamplification of cDNA ends (RACE). Sequence analysis indicated that the amplifiedproduct was about797bp, containing open reading fragment (ORF) of537bp encoding apolypeptide of178amino acids with an estimated molecular mass of21.369kDa and atheoretical isoelectric point of5.94,5’-UTR of11bp,3’-UTR of249bp which included18bp poly (A) tail and a signal sequence with a cleavage site between the position22and23of the peptide. The sequence of Pinctada martensii DPT showed40.1%identity toHaliotis diversicolor supertexta sequence,36.3%to Haliotis discus discus,29.9%toSuberites domunculaand and28.8%to Biomphalaria glabrata described previously. Sixpotential phosphorylation sites were identified in its sequence. One glycosylation site,which was inferred to be responsible for biomineralization, was also found at position50of the peptide. Meanwhile, in the sequence, there were eight cysteine residues, whichmediated protein cross-linking. This result indicated that DPT could form intermoleculardisulfide with other proteins and construct the nacre organic matrix framework.Meanwhile, we performed qRT-PCR to analyze the expression patterns of DPT in severaltissues of adult Pinctada martensii. Results showed DPT was highest expressed in pearlsac and mantle and lowest expressed in gills and adductor muscle. After reducing DPTexpression using RNAi method, compared with the control, the prismatic layer of theshells did not have significant changes, while, the nacre layer showed a disorderedgrowth and some regions appeared dissolved status. So, these results suggested thatcloned DPT in our research was a matrix protein and play important role in the nacre formation by constructing the nacre organic matrix framework with other proteins.The TLR family was one important class of pattern recognition receptors (PRRs).They played crucial roles in the natural immune response, which participated in defensesfor a variety of disease. In this study, we cloned partial cDNA of Toll-like receptor3(TLR3) gene from total RNA of pearl sac in Pinctada martensii by qRT-PCR and RACE(rapid amplification of cDNA ends). Sequence analysis indicated that the amplifiedproduct was about894bp fragment including a3’-UTR of385bp, and encoding a polypeptide of168amino acids with a poly (A) tail of13A. qRT-PCR assays demonstratedthat the amplified TLR3were constitutively expressed in various tissues in Pinctadamartensii (Adductor muscle, Gills, Pearl sac, Mantle, Hepatopancreas, Gonad, Foot,Blood), with predominant level in Blood, lowest expression in Gills. Furthermore,polyI:C could significantly induced the expression of TLR3and NF-κb. Meanwhile,reducing TLR3expression using RNAi technology could significantly inhibit theexpression of NF-κb which was activated by PolyI:C. these results indicate that theamplified fragment of TLR3was important in the NF-κb pathway which was activated bypolyI:C.