Cloning Of CCT-α,CCT-β And CCT-η CDNAs And Their Expression Responding To Cold Stress In Pinctada Maxima

Pinctada maxima is well-knowned for producing large pearls. Pinctada maxima can grow faster in water at 25~30℃, and can’t resistant to low temperature. It is important to understand the mechanism for Pinctada maxima response to low temperature.In this study, CCT-α,CCT-β and CCT-η c DNAs in Pinctada maxima(Pm CCT-α, Pm CCT-β, Pm CCT-η) were cloned and analysised and the expression pattens of CCT-α,CCT-β and CCT-η in different tissues and under cold stress condition were studied. The results showed as follows:1. cloning and analysising of CCT-α, CCT-β and CCT-η c DNAsThe c DNA sequences of Pm CCT-α,Pm CCT-β and Pm CCT-η were cloned by rapid amplification of c DNA ends methods(RACE), and bioinformatics analysis of the three CCT genes was conducted. The full-length c DNA of Pm CCT-α was 1803 bp and contained an 1608 bp ORF encoding 535 amino acids which was less 20 aa than other species. Three CCT signature sequences(36KSSLGPVGLDKML48,57ITNDGATILKLLEVEHP73, 85QDQEVGDGT94), three functional structure domains(Equatorial domain, Apical domain, Intermediate domain), nine ATP binding sites(at 39-41, 91, 95, 137, 377, 395, 434, 486 and 488 amino acid residues)and six protein binding sites(at 1-3, 256, 457-461, 463, 525 and 527-535 amino acid residues) was found in the peptide. Homology analysis indicated that the deduced amino acid sequence of Pm CCT-α had 80.7%, 77.1% and 75.4% consensus with CCT-α of Aplysia californica, Danio rerio and Homo sapiens, resepectively.Two Pm CCT-β full-length c DNA was obtained and contained same 5’UTR of105 bp, an open reading frame(ORF) of 1593 bp encoding 530 aa and two different3’UTR of 591 bp and 131 bp. Three CCT signature sequences(38KSTLGPKGMDKI L50, 58VTNDGATILKSIGIVNP74,86QDDEVGDGT95), three functional structure domains(Equatorial domain,Apical domain,intermediate domain), nine ATP binding sites(at 42-44, 92, 96, 160, 387, 405, 445, 488 and 490 amino acid residues) and three protein binding sites(at 1-4, 467 and 526-530 amino acid residues) was found in thepeptide.In addition, Pm CCT-β had a DEAD box motif(377ILDEADRSL385) that was not found in othes species. Homology analysis indicated that the deduced amino acid sequence of Pm CCT-β had 90.4%, 76.8% and 75.8% consensus with CCT-β of Crassostrea gigas, Danio rerio and Homo sapiens, resepectively.Pm CCT-η full-length c DNA was 1895 bp and contained an 1895 bp ORF encoding 543 amino acids which containded three CCT signature sequences(37RTTLGPRGMDKLI49, 58ISNDGATILKTLDIVHP74, 86QDAEVGDGT94), three functional structure domains(Equatorial domain, Apical domain, Intermediate domain), nine ATP binding sites(at 40-42, 92, 96, 161, 391, 409, 449, 492 and 494 amino acid residues) and three protein binding sites(at 1-4, 250 and 543 amino acid residues). Homology analysis indicated that the deduced amino acid sequence of Pm CCT-η had 83.2%, 81.2%, 80.1% and 79.3% consensus with CCT-η of Crassostrea gigas, Aplysia californica, Danio rerio and Homo sapiens, resepectively.2. CCT-α, CCT-β and CCT-η gene tissues expression analysisExpression of Pm CCT-α, Pm CCT-β and Pm CCT-η gene in eight tissues(heart,adductor muscle, mantle, gill, hepatopancreas, stomach, labial palps, pleopod) was studied by real-time PCR. The results showed that, Pm CCT-α,Pm CCT-β and Pm CCT-η gene expressed in all tissues. Pm CCT-α gene expression level was very high in gill, mantle and adductor muscle, followed by in the stomach, labial palps and pleopod, while Pm CCT-α gene expression level was lowest in the heart and hepatopancreas; Pm CCT-β gene expression level was very high in gill and adductor muscle, followed by in the labial palps and pleopod, while Pm CCT-β gene expression level was lowest in the mantle, heart,hepatopancreas and stomach; Pm CCT-ηexpression level was the highest in the adductor muscle and was lowest in the he heart and hepatopancreas. The high expression level of three gene in the adductor muscle and gill indicate that the three gene play an important role in maintaining the normal physiology condition of adductor muscle and gill. Especially,Pm CCT-α may have novel functions in mantle.3.Effect of cold stress on the transcription of CCT-α, CCT-β and CCT-η geneDetermination of the changes of CCT-α, CCT-β and CCT-η gene m RNA expression levels after exposed to low temperature water at 20℃ was studied by real-time PCR. The results showed that the expression of three genes under cold stress appeared same trend which increased firstly and decreased subsequently. The Pm CCT-α expression quantity of adductor muscle,hepatopancreas and heart increasedsignificantly at 6h then still keep on a high level at 12 h, and decreased significantly to the same level of 0h after 12 h later. In the gills and mantle, the expression quantity of Pm CCT-α gene markedly up-regulated at 12 h and decreased significantly to the same level of 0h at 24 h in the gills and mantle. The Pm CCT-β gene expression quantity of heart and hepatopancreas increased to the top at 6h then still keep on a high level at12 h, and decreased significantly to the same level of 0h after 12 h later. In the adductor muscle, gill and mantle, the Pm CCT-β gene expression quantity significantly rise at12 h, and decreased to the same level of 0h at 24 h. The Pm CCT-η gene expression quantity of adductor muscle peaked at 12 h then fell below the level of 0h. In hepatopancreas and heart, the Pm CCT-η gene expression quantity peaked at 6h, and decreased significantly at 12 h to the same level of 0h, then kept on this level at 24 h.Under cold stress condition, the expression quantity of CCT-α, CCT-β and CCT-η gene increased significantly in the pinctada maxima, and different tissues have distinct expression profiles responding to cold stress, which heart and hepatopancreas were more sensitive than the other tissues. It indicated that CCT-α, CCT-β and CCT-ηmay play a particularly important role in protecting hepatopancrea, heart and other internal organizations of pinctada maxima.