虾夷扇贝闭壳肌肌肉分化生长及其基因表达调控研究

Scallop is the important maricultured animals in China. For scallops, the adductor muscle is the main edible part, which has been approved to be delicious in taste and rich in nutrition. Scallop adductor muscle is generally composed of striated and smooth muscle, which are not only responsible for the different contraction mechanism, but also have obvious differentiation in terms of muscle growth and meat quality. As the important economic shellfish in North China, Yesso scallop Patinopecten yessoensis is selected in this project to study the adductor muscle differentiation and growth, and its expression regulation. The histochemical method is carried out to identify muscle fiber types and morphology of striated and smooth muscle. The combination of F-actin fluorescent staining and laser confocal microscopy is performed to investigate the characteristics of muscle fiber differentiation and development; RNA-Seq technology is used to identify the different expressed genes and their enrich pathways between the striated and smooth muscle; The iTRAQ quantitative proteomics will be performed to screen muscle-specific protein expression, and explore the candidate genes of muscle differentiation by integrated mRNA and protein analysis; The spatiotemporal expression patterns of the candidate genes is examined, and their function in specific muscle fiber type is verified by RNA interference. The present results would lay a foundation in revealing the molecular mechanism of adductor muscle growth, and provide the important scientific basis for genetic improvement of cultured scallops.

扇贝是我国重要海产养殖动物，其闭壳肌肉质鲜美、营养丰富，是扇贝的最主要食用部分。扇贝闭壳肌由横纹肌和平滑肌两种肌肉类型构成，两者除运动收缩功能不同外，在生长和肉质方面也存在明显分化。本研究选用我国北方重要经济贝类─虾夷扇贝为研究对象，采用组织化学染色方法查明横纹肌和平滑肌的肌纤维类型及形态结构，结合F-肌动蛋白特异荧光染色和激光共聚焦显微观察揭示虾夷扇贝不同发育时期的肌纤维分化发育特征；利用转录组测序发掘两类肌肉的差异表达基因及其富集信号通路；应用iTRAQ定量蛋白质组学技术筛选两者的差异表达蛋白，结合转录组数据进行mRNA和蛋白质关联分析，发掘肌肉分化生长候选基因；研究肌肉分化生长候选基因的时空表达特征，利用RNAi验证候选基因在肌纤维类型形成中的功能特性。研究结果将为揭示虾夷扇贝闭壳肌生长的分子机理奠定基础，为扇贝优良品种选育提供重要科学依据。

扇贝是我国重要海产养殖动物，其闭壳肌肉质鲜美、营养丰富，是扇贝的最主要食用部分。扇贝闭壳肌由横纹肌和平滑肌两种肌肉类型构成，两者在运动收缩、生长和肉质等方面都存在明显分化，但目前有关横纹肌和平滑肌的肌纤维类型组成、分化生长模式及及其基因表达调控机制尚不明确。本项目通过深入研究虾夷扇贝闭壳肌肌肉分化生长及其基因表达调控机制，取得了如下重要进展：利用组织切片和电镜技术揭示了横纹肌和平滑肌的肌纤维结构特征，横纹肌肌纤维呈长条状，直径大小一致，沿细胞长轴平行排列，有典型的横纹结构，而平滑肌肌纤维呈长梭形，无横纹，肌纤维直径大小不一，有致密体结构。本研究结合F-actin荧光染色和激光共聚焦显微技术，精细描绘了虾夷扇贝不同发育时期横纹肌和平滑肌肌纤维的动态发育过程，系统查明了扇贝原始肌细胞从担轮幼虫开始形成，经历了面盘收缩肌的增大和退化、前闭壳肌的生长和退化的动态发育过程。通过比较转录组分析揭示了两种肌肉的差异表达基因共有9555个，定量蛋白质组学分析揭示平滑肌和横纹肌存在474个差异表达蛋白，主要包括参与肌节结构组装、钙离子信号通路及细胞骨架组装等相关蛋白，利用组学联合分析查明了横纹肌和平滑肌基因在mRNA和蛋白水平存在复杂的转录和转录后调控机制。利用定量PCR和整胚原位杂交等技术手段揭示了肌肉分化生长基因Py-MSTN的组织特异性表达及不同发育时期的表达特征，采用RNA干扰手段查明了靶基因干扰后横纹肌的肌纤维横截面积显著增大，肌纤维数目和细胞核数量显著增多，而平滑肌的肌纤维表型和细胞核数量未出现显著变化，证实Py-MSTN主要通过激活肌肉前体细胞的增殖和合成肌肉核心蛋白组分，以实现对肌肉分化生长过程中的调控作用。本项目取得的研究成果为揭示虾夷扇贝闭壳肌性状形成的分子机理奠定了理论基础，为扇贝闭壳肌肉质性状的遗传改良提供了重要科学依据。