【摘要】 OsBP-73是用酵母单杂交系统,以水稻蜡质基因(Wx)的顺式作用元件为诱饵,从水稻cDNA表达文库中筛选获得的转录因子。本文以OsBP-73基因为例介绍了用"下拉"(pull-down)方法筛选转录因子靶基因的一般步骤。将含有该基因DNA结合功能域的cDNA片段构建到原核表达载体上,并在大肠杆菌中诱导表达获得其蛋白p73。用纯化后的p73蛋白通过"下拉"实验对OsBP-73靶基因进行初步筛选,获得了22个阳性克隆,为进一步研究转录因子OsBP-73参与的水稻转录调控网络提供依据。更多还原

【Abstract】 Previous data showed that a 31-bp(from -840 bp to-810 bp)DNA fragment located at the 5’ upstream region of rice waxy gene could inter- act with nuclear protein extracted from develop- ing endosperm of rice.When this 31 bp DNA se- quence was used as a bait to screen a rice cDNA library with a yeast one-hybrid system,three groups of cDNA clones were isolated.One of them is pC73,the correspondent rice gene of pC73 was named as OsBP-73(Oryza sativa binding protein). A pull-down assay was made to identify the target genes of transcription factor by using genomic DNA and recombinant p73 protein.The cDNA fragment containing DNA-binding domain of OsBP-73 was cloned into expression vector pET28-c(+)(Fig.1)to produce protein p73,fused with a his6-tag,from E.coli BL21(DE3)(Fig.2). The p73 was purified with Ni-NTA under native condition(Fig.3).The target genes of p73 were identified in rice genome-wide by using a pull- down assay,and 22 candidate genes were obtained (Figs.4 and 5,and Table 1).The obtained results show that putative light-repressible receptor pro- tein kinase and GAMYB-binding protein could serve as targets of the OsBP-73,suggesting that OsBP-73 might be involved in light signal transduction.更多还原