利用酵母双杂交系统筛选猪Calsarcin-1基因相互作用蛋白

 【目的】利用酵母双杂交系统研究与猪Calsarcin-1相互作用的蛋白,了解Calsarcin-1在猪骨骼肌纤维类型形成和信号通路的调控功能。【方法】首先用长白猪Calsarcin-1基因构建既无自激活性又无毒性的pGBKT7-CS1诱饵载体;然后从猪的骨骼肌组织样中提取mRNA，利用SMART技术合成并纯化双链cDNA;最后通过酵母双杂交系统的共转化法筛选与Calsarcin-1相互作用的蛋白，在GenBank中比对分析相互作用基因，分析Calsarcin-1在骨骼肌中的功能。【结果】构建了pGBKT7-CS1诱饵载体，获得具有完整3′端的猪骨骼肌单链cDNA，并合成双链cDNA。筛选出4个与Calsarcin-1相互作用的蛋白，经与人的基因比较分析，发现Calsarcin-1与α-1肌动蛋白和α-3辅肌动蛋白互作，与骨骼肌Z线附近结构形成有关;与肌集钙蛋白-1和肌钙蛋白T3互作，影响钙离子的调控。【结论】分析所筛选出的蛋白功能，推测Calsarcin-1通过结合这些蛋白，维持细胞结构的稳定，影响相关蛋白的钙离子结合能力，从而参与钙离子调控;而钙离子浓度的变化将影响到其它控制肌纤维分化的因子，或其它调控通路，共同调节快慢肌纤维的形成与转化。

Screening for Porcine Calsarcin-1 Interacting Proteins by Yeast Two-Hybrid System

【Objective】 The function of Calsarcin-1 was studied in the formation of skeletal muscle fiber and signaling pathway regulation through the screening for porcine Calsarcin-1 interacting proteins by yeast two-hybrid system. 【Method】 The pGBKT7-CS1 bait vector without toxicity or auto-activation was constructed with the Calsarcin-1 gene of Landrace pig, firstly. Then the mRNA was isolated from the skeletal muscle of the adult Landrace and purified ds-cDNA by SMART technology. Ds-cDNA was amplified by long distance PCR. At last, the Calsarcin-1 interaction proteins were screened through co-transformation with bait vector and ds-cDNA prey library. With the alignment of interacted gene in GenBank, the function of Calsarcin-1 was analyzed.【Result】 In this study, pGBKT7-CS1 bait expression vector was constructed. The ss-cDNAs of the porcine skeletal muscle with full length 3’ end were isolated and the ds-cDNAs were synthesized. Four proteins interacting with Calsarcin-1 were identified. The Calsarcin-1 showed the important function in maintaining the structure of Z line through interacting with ACTA1 and ACTN3, while involved to calcium ion regulation through CASQ1 and TNNT3 possibly.【Conclusion】 According to the result, it is presumed that the Calsarcin-1 affected the formation of Z-line and the calcium binding by combination with these identified proteins and participated in the calcium ion regulation and maintaining the cytoskeleton stabilization. By this way, the calcium ion concentration also affected other factors and signaling pathways related to the slow and fast muscle fiber formation and transformation.