硒蛋白W和硒结合蛋白1调控肌纤维类型的作用及机制研究

It is well known that the composition of skeletal muscle fibers is one of the key factors determining meat quality. Xiang pig, which contains considerably higher levels of oxidative muscle fibers than does Large White pig, our previous studies have confirmed that Selenoprotein W (SELW) gene expression was significantly higher in Large White pig than Xiang pig, whereas that of the Selenium-binding protein1 binding protein (SBP1) was lower than Xiang pig. In this project, we aim to investigate the effects and mechanisms of SELW and SBP1 on the development of muscle fiber types. Firstly, the correlation of the development of muscle fiber types and SELW, SBP1 expression and will be revealed by Q-PCR. Next, the techniques, such as gene overexpression, gene knock down and immunohistochemistry will be performed to verify how the SELW and SBP1 regulate muscle fiber types in pig cells and muscle tissues. Furthermore, the key MicroRNA, proteins and signaling pathway involved in SELW and SBP1 regulating muscle fiber types will be analyzed and validated by RNA-seq and proteomics. Finally, we will explore protein interaction between the SELW, SBP1 and the protein on the key signaling pathway based on co-immunoprecipitation and Dual Luciferase Reporter Gene Assay System. In that way, the new functions of SELW and SBP1 will be investigated. This project could provide new insights and fundamental theory for the animal industry to improve meat quality.

肌纤维类型是决定肉品质的重要因素。申请人前期比较研究了香猪和大白猪背最长肌转录组，发现硒蛋白家族中的硒蛋白W（SELW）在酵解型肌纤维含量较多的长白猪中高表达，而硒结合蛋白1（SBP1）在氧化型肌纤维含量较多的香猪中高表达。为了探寻这两个硒蛋白调控猪肌纤维类型的功能及其作用机制。本研究拟通过体内和体外实验，首先利用Q-PCR技术研究SELW和SBP1时空表达与肌纤维类型发育变化的关系；进一步利用超表达、siRNA、免疫组化等技术明确SELW和SBP1在猪骨骼肌细胞和组织中调控肌纤维类型的功能；接着应用RNA-seq、蛋白组学技术筛选出关键参与调控的小分子RNA、蛋白和信号通路；并通过免疫共沉淀和双荧光素酶报告基因技术阐明SELW和SBP1与关键信号通路上蛋白的互作机制。本研究结果可为猪肉品质改善提供新的思路和新的理论依据。

肌纤维类型是决定肉品质的重要因素。前期比较研究了香猪和大白猪背最长肌转录组，发现硒蛋白W（SELW)在长白猪中高表达，而硒结合蛋白1（SBP1）在香猪中高表达。为了探寻这两个硒蛋白调控猪肌纤维类型的功能及其作用机制。我们首先研究了猪背最长肌中SELW、SBP1和肌纤维类型基因和蛋白的时空表达模式，相关性分析发现SELW基因的表达与MyHCIIB的表达呈显著的正相关。进一步成功构建了SELW和SBP1慢病毒超表达和基因沉默载体，转染猪骨骼肌卫星细胞并筛选获得稳定表达细胞，诱导分化7天后测定肌纤维类型基因和蛋白的表达，在体外明确了SELW基因主要调控猪细胞酵解型肌纤维MYHCⅡB型的表达，SBP1基因主要调控猪细胞氧化型肌纤维MYHCⅡA型的表达。在生长育肥猪日粮中添加不同浓度的硒建立高硒和低硒动物模型，研究发现硒沉积规律为脂肪→血清→背最长肌→肾脏→肝脏，0.3-1mg/kg时SBP1达量最高，1-3mg/kg时SelW表达量最高，氧化型肌纤维蛋白IIA和I在0.3mg/kg时表达量最高，而IIB型蛋白表达呈现剂量依赖；同时背肌脂质组学筛选出高硒和低硒组50个差异脂质分子；16S测序发现硒显著提高了肠道益生菌属，降低了异丁酸和异戊酸的浓度。进一步应用TMT蛋白组学技术，通过体内外实验筛选出了SELW关键调控信号通路5条，SBP1基因关键调控信号通路2条。最后利用CO-IP和LC-MS/MS测定分析，筛选到SELW互作蛋白28个，Score最高为Eif4a1和Eif4a2。SBP1互作蛋白96个，Score最高的为Moesin，并绘制了SELW和SBP1互作蛋白网络图。本研究明确了SELW和SBP1调控肌纤维类型转化的功能并探寻了作用机制。研究结果可为猪肉品质改善提供新的思路和新的理论依据，同时本研究还拓展了硒对肠道微生物的影响研究，为后期肠道-肌肉轴的研究奠定了一定的基础。