Observation On The Mantle, Cloning And Expression Analysis Of The Calmodulin CDNA From Hyriopsis Schlegeli

For a high quality introduced freshwater pearl shell, Hyriopsis schlegelii has been wildely applied in our country's freshwater pearls industry. The mantle of shellfish is the most important position for cultivating pearls. However, there was no report about mantle's histological structure of H. schlegelii, as well as genes which participate in and regulate the pearl formation. In our study, the technologies of paraffin section and H.E staining were used, and the thickness, shape and size of different protuberances were studied. The calmodulin(CaM) cDNA of mantle was cloned and its expression characterization was analysed.1. Observation on the Mantle of H. SchlegeliThe histological structure of the mantle in 4 regions of H. schlegeli was observed. The typical structure of the mantle consists of a middle zone and a marginal zone. Three protuberances are observed in the marginal zone, which are shell protuberance, sensory protuberance and velum protuberance. The structures of the mantle in different regions are similar, composed of inner epithelium, outer epithelium, connective tissue and muscular fiber. Three kinds of secretory cells are distributed in the inner epithelium and connective tissue, which are basophilic mucous cells, big secretory granulosa cells, and small secretory granulosa cells. While one kind in the outer epithelium, which was vacuoles secretory cells.The are great differences in the shape and size among the three protuberances in various regions The thickness of the main part of mantle marginal zone is not the same, while the central and the posterior parts are thicker.2. Cloning and expression analysis of Calmodulin cDNA from Hyriopsis Schlegeli.Calmodulin is a small molecular calcium-binding protein, participating in several cellular processes by binding with Ca²⁺, and interacting with target enzymes. In mollusca, as the member of Ca²⁺ channel and pivotal regulator of Ca²⁺-ATPase, CaM plays an important role in Ca²⁺ uptake and transport in order to form shell. The degenerate oligonucleotide primers used for amplification were designed based on the conserved regions of CaM nucleotide sequence from Genbank, and partial sequence of CaM cDNA was cloned by RT-PCR technology. After sequencing and identification, four specific primers were designed. The regions of 5'-UTR and 3'-UTR were obtained by SMART RACE technology. After splicing the overlapping sequence, the full length of CaM cDNA was acquired. It was 855 bp, including a 70 base 5'-untranslated sequence, an open reading frame consisting of 447 bp, a TGA stop, a 309 base 3'- untranslated sequence, and a poly(A) tail of 26 nucleotides. The deduced CaM protein consists of 149 amino acids with a calculated molecular mass of 16.8 kDa and an isoelectric point of 4.14. Like vertebrate and other mollusk, CaM in the H. schlegelii contains 4 EF-hand domains, and each domain contains 2α-helix and one Ca²⁺-binding loop. Multiple protein sequence alignment analysis showed that the CaM sequence shares high similarity with other CaMs isolated from invertebrate and vertebrate animals.CaM mRNA expression in different tissues of the shell was examined by real-time PCR. The results showed that CaM mRNA was expressed in mantle, gill, gonad, foot, and adductor muscle, but different level with each other. Adductor muscle's expression level was set as calibrator, and there was highest expression level in the gill, which was 106 times of the calibrator. CaM mRNA expression in foot and mantle were aslo high, 60-80 times of the calibrator; and the level of gonad was higher than calibrator.In the same time, real-time PCR was also used to test CaM mRNA expression level in mantle tissues among 1-5 ages of H. schlegelii. The results suggested that there was highest expression in the mantle of four-year old shellfish, followed by five-year old shellfish, and there was no significant difference among one-year old, two-year old and three-year old shellfish.