马氏珠母贝磺基转移酶基因的克隆及功能

摘要

硫酸角质素具有丰富的携带大量负电荷的磺酸基团,参与生物矿化的成核过程.磺基转移酶催化磺酸基团的转移,对硫酸角质素的生物合成起决定性作用.本研究利用RACE技术克隆马氏珠母贝磺基转移酶PmCHST1a全长,并通过RNA干扰技术检测PmCHST1a对硫酸角质素合成及贝壳珍珠层形成的影响.结果显示,PmCHST1a基因全长1385 bp,编码366个氨基酸;含有磺基转移酶结构域,具有跨膜结构和信号肽,定位于高尔基体上.组织差异表达分析发现,PmCHST1a在中央膜显著高表达.注射Pm-CHST1a的RNAi探针后,PmCHST1a在中央膜的表达量显著下调,并且外套膜外液中硫酸角质素的浓度显著降低;SEM检测发现珍珠层结构紊乱.综上所述,PmCHST1a可能通过影响外套膜外液中硫酸角质素的合成,参与珍珠层的形成.本研究为进一步探讨磺基转移酶及其参与合成的糖胺聚糖硫酸角质素在马氏珠母贝生物矿化中的作用提供依据.%Biomineralization is the process by which mineral crystals are deposited in an organized fashion in the matrix (either cellular or extracellular) of living organisms and the deposition of precise arrays of inorganic crystals in many organisms involves controlled nucleation at interfaces between the crystals and substrate macromolecules. Mollusc shells are used as a model for studying "organic-matrix-mediated" biomineralization, in which crystals are nucleated and grow in a pre-formed structural framework composed of proteins and polysaccharides. Acidic matrix macromolecules glycosaminoglycans were also associated with biological crystal growth and keratan sulfate widespread in animal tissues had a potential role in the process of biomineralization. Sulfotransferase plays a vital role in catalyzing the transfer of sulfonic acid groups in the processes of glycosaminoglycan synthesis, and glycosaminoglycans keratan sulfate with copious amounts of negatively charged sulfonic acid groups participate in the nucleation process of biomineralization. In this study, we cloned the sulfotransferase gene PmCHST1a (Pinctada martensiicarbohydrate sulfotransferase 1a) which participated in the synthesis of keratan sulfate and explored the function of sulfotransferase and keratan sulfate in the biomineralization. The full length sequence of PmCHST1a gene was obtained using the RACE technology, and we detected its expression pattern by real-time PCR. RNAi technique was used to study the potential functions of PmCHST1a in nacre formation, and we detected the effects to the synthesis of glycosaminoglycans keratan sulfate at the same time. The results showed that PmCHST1a, whose full length was 1385 bp, encoded a protein of 366 amino acids.PmCHST1a carried with a typical Sulfotransferase-3 domain (Sulfotransfer-3 domain), a signal peptide and a transmembrane domain which made the protein located in the Golgi apparatus. The results of real-time PCR showed that PmCHST1a was highly expressed in the central zone of mantle. After the RNA interference experiments, the expression of PmCHST1a significantly decreased in the central zone of P. martensii, in union with the significant reduction of the concentration of glycosaminoglycans keratan sulfate in the extrapallial fluid. And the shell nacre crystallized irregularly compared with two control groups. These results showed that PmCHST1a may affect the formation of nacre through affecting the concentration of glycosaminoglycans keratan sulfate in the extrapallial fluid. This study provided the fundamental basis for further research of sulfotransferase and glycosaminoglycans in the formation of nacre.