Chitin is the main component of mollusk shell organic scaffold and its metabolism is important for shell mineralization.Chitinase with the function of chitin hydrolysis plays a key role during chitin metabolism process and is related to the formation process of shell organic scaffold.In this study,by the mass spectrum analysis of shell protein,it was proved that chitinase had an abundant amount in the shell of Chlamysfarreri.The cDNA sequence ofgene chitinase was obtained by RACE and its full length is 1587 bp,coding 439 amino acid residues.Analysis of chitinase protein sequence with bioinformatics software shows that it has a conservative function domain existing in chitinase protein family with the function of chitin hydrolysis.Chitinase in C.farreri had a much higher expression level in the mantle tissue than in other tissues,especially the area of mantle edge.In the process of shell regeneration after shell notching,the expression level of chitinase showed a decreasing tendency indicating that chitinase responded as a negative regulatory factor during shell regeneration.After knocking down the expression of chitinase,the structure of the mineral slices was irregular and fused with a fuzzy shape and unclear boundary.In conclusion,it is suggested that chitinase in C.farreri might be related to the formation of shell organic scaffold with its function of chitin hydrolysis,which is also important during the process of shell biomineralization.%为研究栉孔扇贝贝壳形成机理,首先应用质谱分析的方法,研究了几丁质酶在栉孔扇贝贝壳的蛋白质组中所占的比例.进一步利用RACE技术克隆获得栉孔扇贝几丁质酶的cDNA,全长共1587 bp,可编码439个氨基酸残基.氨基酸序列的功能结构域分析表明,该几丁质酶具有保守的几丁质酶特征结构域.组织表达分析表明,栉孔扇贝几丁质酶在外套膜组织中表达量最高,并且在外套膜边缘的表达量高于外套膜中心,推测其参与了贝壳的形成.应用实时定量PCR方法,检测贝壳损伤修复过程中基因表达水平,结果显示,几丁质酶基因表达水平呈现下调趋势,暗示其作为负调控因子参与贝壳修复过程.利用RNAi技术降低外套膜组织中几丁质酶基因的表达水平,同时应用扫描电子显微镜观察贝壳内表面的结构,发现贝壳矿物晶体片层的轮廓呈现不规则沉积.综合以上实验结果,推测几丁质酶通过水解几丁质来保证有机框架的有序性,从而对贝壳形状和外观起到调控作用.研究几丁质酶的基因组织表达规律及其生物学功能,将有助于揭示贝壳生物矿化的机制.
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