厚壳贻贝几丁质提取及几丁质相关基因筛查

Chitin extraction and chitin-related genes screening in Mytilus coruscus

几丁质是贝类贝壳的重要组成成分,在贝壳形成和生物矿化中具有重要作用。本研究通过对厚壳贻贝的贝壳和外套膜边缘三层褶皱进行形态和组织学观察,发现贻贝角质层壳膜是从外褶皱和中褶皱之间的壳膜沟中分泌,而外套膜三层褶皱间组织形态、细胞类型都存在较大差异。此外,本研究还从厚壳贻贝的角质层和贝壳层中提取并鉴定到β-几丁质成分,并构建了外套膜内褶皱(IF)、中褶皱(MF)、外褶皱(OF)的转录组文库,筛查出几丁质合成、降解、结合相关功能的三类几丁质相关基因,对其结构域和表达模式进行进一步分析,发现几丁质合成酶基因以及β氨基己糖苷酶、二-N-乙酰壳二糖酶等几丁质酶基因主要在内、中褶皱表达,而壳三糖苷酶类的几丁质酶基因和几丁质结合蛋白基因则主要在外褶皱表达。在三褶皱差异表达基因的GO富集分析中,进一步发现内、中褶皱主要参与几丁质的生物合成,而外褶皱则主要参与调控几丁质结合、代谢、角质层色素沉着等过程。本研究揭示了外套膜三褶皱在几丁质合成、代谢、结合等功能方面的差异,其中外褶皱可能在厚壳贻贝的贝壳角质层的形成和色素沉着中发挥更重要的作用。本研究有助于了解贻贝贝壳中的几丁质成分以及几丁质相关基因,为软体动物贝壳形成与生物矿化的研究提供理论参考。

Chitin is an important component of mollusk shell and plays a key role in shell formation and biomineralization.In this study,we analyzed the structure of the shell and the three folds of mantle edge of the hard-shelled mussel Mytilus coruscus by physiological and histological observation.It was found that the periostracum was secreted from the periostracum groove between the outer fold and the middle fold.Also,there were great differences in the histological morphology and the epithelial cells among the three mantle folds.According to powdery products extracted from periostracum and shell,it was found that these two products had characteristic absorption peaks of chitin by Fourier Transform infrared spectroscopy(FTIR).Additionally,we further constructed RNA-seq libraries of inner fold(IF),middle fold(MF)and outer fold(OF).Based on the three-fold mantle edge transcriptome data,three types of chitin-related genes were identified,including chitin synthase(CHS)genes,chitinase(CHIA)genes and chitin-binding protein(CBP)genes.According to the domain structures and expression patterns of these chitin-related genes,the genes of chitin synthases,beta-hexosaminidases and di-N-acetylchitobiase were found to be mainly expressed in the inner and middle folds,while acidic-mammalian-chitinase genes and chitin binding protein genes were mainly expressed in the outer fold.In GO enrichment analysis of differentially expressed genes in the three folds,the inner and middle folds were witnessed to be mainly involved in the biosynthesis of chitin,while the outer fold was mainly involved in regulating chitin binding,chitin metabolism,and chitin-containing cuticle pigmentation.Therefore,this study suggests that there are significant differences in functions such as chitin synthesis,metabolism and binding among the three folds of mantle,and the outer fold may play a more important role in the shell periostracum formation and pigmentation.The result will contribute to the understanding of the chitin composition and chitin-related genes in mussel shell,and will provide theoretical reference for the study of mollusk shell formation and biomineralization.