番茄SlWD1基因的克隆及SlWD1与DDB1的相互作用

Cloning of SlWD1 Gene and Interaction of SlWD1 with DDB1 in Tomato

在真核生物中,CDT2作为一个WD40家族蛋白,是CUL4-DDB1 E3泛素连接酶复合体的重要组成部分.其主要功能是将CUL4-DDB1 E3泛素连接酶和靶向蛋白连接起来,从而实现靶蛋白的泛素化修饰.利用RT-PCR技术和3’RACE技术,本研究在番茄中克隆到一个与人类CDT2同源的基因,命名为SlWD1.通过对番茄SlWD1蛋白序列与其它模式生物的蛋白序列进行氨基酸保守结构域分析和同源性比对,发现SlWD1具有2个非常保守的WD40家族的DWD结构域.酵母双杂交和免疫共沉淀实验都证明,SlWD1与DDB1具有相互作用;实时定量RT-PCR分析证明,SlWD1基因在各个组织中都表达,为组成型表达,但是,在生殖器官中的表达量要明显比其它的组织高.

In eukaryotes, WD40 family protein CDT2 is an important component of CUL4-DDB1 E3 ligase complex. It serves as a linker between CUL4-DDB1 and its target protein, then achieves degradation of the target protein. With 3＇RACE and RT-PCR technique, we cloned a tomato gene named SIWD1, which is homologous of HsCDT2 in Homo sapiens. Amino acid sequence alignment of S1WD1/CDT2 proteins from different species showed that two conserved DWD Box domains were contained in SIWD1. Yeast two-hybrid and CO-IP assays both suggested the interaction between tomato DDB1 and S1WD1. Real-time PCR analysis indicated that SIWDI gene expressed in all tested tissues, suggesting the expression pattern of SIWD1 was constitutive expression. However, the expression level of SIWD1 was much higher in reproductive organs than in other tissues. Fig 7, Tab 1, Ref33