核蛋白筛选系统(NTT)的建立及鉴定

Construction and Identification of a Nuclear Transportation Trap (NTT) System

为了研究植物核蛋白的组成及其在各种环境条件下的动态变化,利用核蛋白的核定位信号(nuclear localization signals,NLS)筛选编码核蛋白的基因,建立了一套快速、省力、低成本的核蛋白筛选系统(nuclear transportation trap,NTT)。通过鉴定发现,将合成的SV40蛋白大T抗原的核定位信号序列插入筛选载体的多克隆位点,转化酵母EGY48,插入核定位信号的筛选载体转化酵母后可以在选择性培养基(SD/His-/Leu-)上生长,而没有插入核定位信号的筛选载体转化酵母后不能在选择性培养基上生长,从而证明此核蛋白筛选系统有效。比较实验表明,本研究构建的核蛋白筛选系统与已报道的系统相比筛选效率更高。利用此系统从cDNA文库中分离编码核蛋白的基因,对于研究核蛋白的动态组成、了解核蛋白基因在调控细胞发育及其环境胁迫条件下的作用机制具有重要的意义。

To study the composition of nuclear proteins and its dynamic variation under various environmental conditions, a high-efficiency nuclear transportation trap （NTT） system was constructed to clone genes encoding nuclear proteins that contain all kind of nuclear localization signals （NLS） sequence in this research. To identify the nuclear transportation trap system, NLS sequence of the large T antigen of SV40 protein was synthesized, inserted into multi-cloning site of selective vector of NTT system, and transferred into yeast strain EGY48. The results showed that yeast transferred with selective vector with NLS sequence could grow on SD/His-/Leu- selective medium, whereas yeast without NLS sequence couldn＇t grow, which indicated that NTT system could work. Moreover, comparing analysis showed that the selective efficiency of the vector constructed in this study is higher than that of the vectors reported. The NTT system will be helpful in the screening of genes encoding nuclear proteins from cDNA library, which is very important to study the dynamic changes of nuclear proteins and the modulating network of all genes in nuclear.