芒果果肉cDNA文库构建及MiCTR1互作蛋白的筛选

Construction of cDNA Libraries of Mango Pulp and Screening of MiCTR1-Interacting Proteins

摘要:

该文旨在鉴定芒果MiCTR1的互作蛋白，深入探究MiCTR1调控芒果果实后熟过程的分子机理。以‘台农1号’芒果为材料，建立芒果果肉的cDNA文库，构建诱饵载体，对其自激活检测后，从文库中筛选与MiCTR1互作的蛋白，并进行回转验证，通过NCBI网站进行序列比对，明确互作蛋白的功能。结果表明：所构建的cDNA初级和次级文库的库容量分别达到了5.7×106和1.2×107 CFU/mL，重组率100%，插入片段长度为1 000~2 000 bp，诱饵载体pGBKT7-MiCTRI不存在自激活。从酵母双杂交文库中筛选到21个初始阳性克隆，经回转验证后最终获得20个与MiCTR1互作的蛋白，主要功能涉及遗传代谢，类叶绿素、维生素E的合成，物质的转运，物质代谢，细胞生长，光合作用。综上，该研究构建的芒果果实cDNA文库质量较高，完整性好，酵母双杂交筛库技术鉴定出多个与芒果MiCTR1互作的蛋白，并且解析其生物功能，为探究MiCTR1调控芒果果实后熟的分子机制奠定理论基础。

Abstract:

MiCTR1-interacting proteins in mango were identified, and the molecular mechanism of MiCTR1 regulating the ripening process of mango fruit was preliminarily clarified. Using Tainong No. 1 mango as the material, cDNA libraries of mango pulp were established. A bait vector was constructed. After the self-activation detection of the bait vector, MiCTR1-interacting proteins were screened from the cDNA library and analyzed by reverse verification. Their functions were identified by aligning with sequences on the NCBI website. The primary and secondary cDNA libraries were 5.7×106 and 1.2×107 CFU/mL, respectively, with a recombination rate of 100% and insert lengths of 1 000 to 2 000 bp, and the bait vector PGBKT7-MiCTRI had no self-activation. A total of 21 initial positive clones were screened in the yeast two-hybrid library, and after rotation verification, 20 MiCTR1-interacting proteins were finally obtained. Their main functions involved genetic metabolism, biosynthesis of chlorophylls and vitamin E, material transport, material metabolism, cell growth, and photosynthesis. In summary, the cDNA libraries of mango pulp constructed in this study were of high quality and complete. MiCTR1-interacting proteins were identified by yeast two-hybrid library screening, and their functions were analyzed. This study lays a theoretical foundation for further clarifying the molecular mechanism of MiCTR1 regulating the ripening of mango fruit.