| Sulfotransferase plays a vital role in catalyzing the transfer of sulfonic acid groups in the processes of glycosaminoglycan synthesis, and glycosaminoglycans consist of many copies of negatively charged sulfonic acid groups participate in the nucleation process of biomineralization. In this study, we have cloned the full length sequences of five sulfotransferase genes using the RACE technology, studied their expression patterns by real-time PCR. The potential functions of these five sulfotransferases in nacre formation explored by RNAi technology, and studied the effect of sulfotransferases in the synthesis of glycosaminoglycans at the same time. The results are as follows:1. Two keratan sulfate sulfotransferase PmCHST1 were cloned, they were named PmCHST1a(P. fucata martensii Carbohydrate sulfotransferase 1a) and PmCHST1b(P. fucata martensii Carbohydrate sulfotransferase 1b).PmCHST1a: The full length was 1 385 bp, encoded for a protein of a 366 amino acids. PmCHST1 a carried with a Sulfotransferase domain(Sulfotransfer-3 domain), a signal peptide and a transmembrane domain which made the protein located in the Golgi apparatus. Real-time PCR results showed that PmCHST1 a was significantly highly expressed in the central zone and gill(p<0.05) of P. fucata martensii. After the RNAi of PmCHST1 a, the expression of PmCHST1 a significantly decreased in central zone(p<0.05), and also with the significant reduction of the concentration of glycosaminoglycans in the extrapallial fluid(p<0.05). Whearas the shell nacre crystallized irregularly, blurred the boundaries between the sheets, and merged with each other. These results showed that PmCHST1 a could affect the formation of nacre through the influence of glycosaminoglycans concentration in the extrapallial fluid.PmCHST1b: The full length was 1 342 bp, encoded for a protein of 408 amino acids. PmCHST1 b carried with a Sulfotransferase domain(Sulfotransfer-3 domain) without signal peptide and transmembrane domain and belonged to cytoplasmic sulfertransferase. PmCHST1 b was expressed in the central zone, adductor muscle, hepatopancreas and hemocytes. After the RNAi with PmCHST1 b, the expression of PmCHST1 b gene significantly decreased in central zone(p < 0.05) and the concentration of glycosaminoglycans in the extrapallial fluid was significantly reduced(p<0.05). SEM results indicated that the shell nacre crystallized with no obvious layers structure, hollow surface, irregular sheets and blurred the boundaries between them. The results showed that PmCHST1 a could affect the formation of nacre through influence of glycosaminoglycans concentration in the extrapallial fluid.2. Two chondroitin sulfate sulfotransferase PmCHST11 were cloned, designated as PmCHST11a(P. fucata martensii Carbohydrate sulfotransferase 11a) and PmCHST11b(P. fucata martensii Carbohydrate sulfotransferase 11b).PmCHST11a: The full length was 1 467 bp, encoded for a protein of 438 amino acids. PmCHST11 a carried a Sulfotransferase domain(Sulfotransfer-2 domain), a signal peptide and a transmembrane domain which made the protein located in the Golgi apparatus. Real-time PCR results indicated that the expression of PmCHST11 a was highly expressed in the central zone and gill. After RNAi of PmCHST11 a, the expression of PmCHST11 a was significantly decreased in the central zone(p < 0.05), and the concentration of glycosaminoglycans in the extrapallial fluid was significantly reduced(p<0.05). Further more the shell nacre crystallized irregularly with rough surface and appeared obvious C axis growth on the sheets. These results showed that PmCHST11 a could affect the formation of nacre by affecting the concentration of glycosaminoglycans in the extrapallial fluid.PmCHST11b: whose full length was 1 540 bp, coded 392 amino acids. PmCHST11 b carried with a Sulfotransferase domain(Sulfotransfer-2 domain), a signal peptide and a transmembrane domain which made the protein located in the Golgi apparatus. The method of real-time PCR was applied to detect the expression of the gene in different tissues of P. fucata martensii and found that PmCHST11 b was significantly highly expressed in the central zone(p<0.05). After the RNA interference experiments, the expression of PmCHST11 b gene significantly decreased in central zone(p<0.05), the concentration of glycosaminoglycans in the extrapallial fluid was significantly reduced(p<0.05). The shell nacre crystallized irregularly with rough surface and blurred the boundaries between the sheets. All of above showed that PmCHST11 b could affect the formation of nacre through influence the concentration of glycosaminoglycans in the extrapallial fluid.3. We have cloned the full length cDNA of PmCHST9 and studied its expression pattern in different tissues. Using rapid amplification of cDNA ends technology, the full length of PmCHST9 cDNA was 1 388 bp, which obtained a 5’ UTR of 122 bp, a 3’ UTR of 192 bp and a 1 074 bp of the open reading frame(ORF) which encoded 357 amino acids. The predicted molecular weight of PmCHST9 was 42 889.2 Da, the isoelectric point was 9.28, the aliphatic index was 81.32 and the grand average of hydrophobicity(GRAVY) was-0.493. Amino acids sequence analysis showed that PmCHST9 had a typical sulfotranferase-2 domain and a transmembrane domain. Multiple sequence alignments results showed that PmCHST9 shared low sequencing homology to CHST9 of other species. In addition, PmCHST9 expression was significantly higher in the gill(p<0.05). The results suggested that PmCHST9 may be involved in the adjustment of immune function and may offer new information on the PmCHST in the innate immunity of P. fucata martensii. |
