Cloning And Expression Analysis Of Heat Shock Protein And Toll-like Receptor Genes In Pinctada Martensii

In this paper, full-length cDNA sequences of heat shock protein60(PmHSP60), heatshock protein90(PmHSP90), Toll-like receptor4(PmTLR4), Toll-like protein (PmTLP)were cloned from mantle of P.martensii using rapid-amplification of cDNA ends(RACE)technology with the unigenes of HSPs and TLRs selected from transcriptome library ofpearl sac and six digital gene expression profiles of pearl sac after the nucleus insertedsurgery constructed by our laboratory,and then charactereized by bioinformatic methods.the organizations expression of four genes and expression of different time after stresswere analyzed. The results were as follows:1.Cloning and sequence analysis of four immune-related genes: The full-lengthHSP60cDNA of Pinctada martensii(PmHSP60) was comprised of2495bp, containing a1734bp ORF, which encoded577amino acids；The full-length cDNA of PmHSP90consisted of2584bp, with a2160bp ORF encoding719amino acids. The full-lengthcDNA of PmTLR4is composed of3138bp, with an open reading frame of2697bpencoding899amino acids. The full-length cDNA sequence of PmTLP is consisted of2214bp with a readable coding frame encoding681amino acids. Homology analysis showedthe PmHSP60and PmHSP90shared highly homologous with other mollusks. PmHSP60shares78%similarity with Hyriopsis cumingii, PmHSP90shares89%similarity withHSP90of Pacific oysters，Crassostrea gigas. However, PmTLR4and PmTLP share lowsimilarity with other species. the PmTLR4shares34%similarity with toll protein fprecursor sequence of Mytilus galloprovincialis; the PmTLP shares38%similarity withtoll protein sequence of Pacific oysters，Crassostrea gigas. Amino acid sequence analysisfound PmHSP60amino acid sequence had typical characteristics of mt-HSP60sequence,C-terminal typical repeat motifs of GGM and an ATP binding domain. PmHSP90aminoacid sequence had five typical characteristics of HSP90sequence: NKEIFLRELISN[A/C/S]SDALDKIR，LGTIA [K/R] SGTIG QFGV GFYSAYLVA[E/D]， IKLYVRRVFI，GVVDSEDLPLNISRE和MEEVD of C terminal；PmTLR4and PmTLP have typicaldomain of toll like protein family: TIR domain and LRR domain2. Expression analysis in different tissues of these four immune-related genes: Anapplication of Real-time PCR technology was developed to detect the mRNA expression of the immune-related genes of PmHSP60, PmHSP90, PmTLR4and PmTLP, in differenttissues of Adductor muscle, mantle, hemolymph, Hepatopancrea, gonads and gills. Theresults showed these four genes were expressed in various tissues and appeared higherlevels of expression in the liver and gills, while a lower expressed in the adductor muscleand other non-immune tissues.3. Sequential expression patterns at three different conditions of stress (ie LPSstimulation, nucleus inserted surgery and thermal stimulation). In the LPS experiment, fourimmune-related genes were upregulated after LPS injection, which PmHSP60arrived themaximum expression levels at6h after LPS injection, while the other three immune genesPmHSP90, PmTLR4and PmTLP is3h after LPS injection. Overall, these four genes canresponse to LPS promptly, gene expression profiles of these genes were the fast responsesignificantly and then gradually decreased; Different days of Nucleus inserted surgery,expression level of PmHSP60increased at first days, followed by reduction and a reboundat the third day, the highest level of expression appeared at5d, while PmHSP90andPmTLR4expression level began to rise at first day, and reached the maximum expressionlevel at second day. Under the heat stress, expression level of HSP60and HSP90mRNA inhemolymph tissue significantly increased. PmHSP60mRNA expression levels achievedmaximal expression amount at12h after thermal stimulation, which was a12.28times ofthe control group (0h)(P<0.05); the expression of PmHSP90reached the maximumthermal stimulus at3h which was28.89times of the control group (P<0.05),then graduallydecreased, and reached the normal levels of expression after24h.4. The prokaryotic expression of PmHSP60. We constructed the PET28a-HSP60prokaryotic expression plasmid and transfer into E.coli BL21(DE3). By using IPTGinduction, we obtain a size of about61.8kDa protein bands. After the expressionconditions optimization, we found that recombinant protein appeared highest solubleexpression at37℃,10h and0.1Mm IPTG. Above all, the results provide a reliablereference data for investigating the regulated mechanism and defensive mechanisms ofP.martensii immune related genes; It will be great of guiding significance for the furthercomprehensive and understanding of the molecular mechanisms of shellfish immunedefenses, thereby guiding the nurturing of disease strains and genetic improving of P.martensii.