杜氏盐藻环盒子1相互作用蛋白的酵母双杂交法筛选

Screening of proteins that interact with RBX1 from Dunaliella salina using yeast two-hybrid system

目的:构建杜氏盐藻环盒子1(RBX1)的酵母双杂交诱饵载体p GBKT7-RBX1,筛选与RBX1相互作用的蛋白。方法:采用PCR技术扩增杜氏盐藻RBX1的开放阅读框,测序分析后插入酵母表达质粒,构建诱饵载体p GBKT7-RBX1。将酶切鉴定正确的诱饵载体用PEG/Li Ac法分别转化到酵母菌Y187和AH109中,通过表型筛选检测RBX1对酵母菌有无自激活和毒性作用。转化诱饵质粒的Y187与文库菌AH109杂交,待三叶草形状的合子形成后用营养缺陷型培养基和α-半乳糖苷酶活性实验筛选阳性克隆并测序。结果:成功构建了p GBKT7-RBX1,它对Y187和AH109两种酵母菌既无自激活又无毒性作用。杂交后筛选得到两个阳性克隆,阳性克隆1与莱茵衣藻和拟南芥中氧化还原酶铁硫蛋白亚基的同源性为38%和39%,阳性克隆2与莱茵衣藻中转化抑制剂蛋白的同源性为57%。结论:诱饵载体p GBKT7-RBX1可用于酵母双杂交。成功筛选得到两个阳性克隆,可能是与RBX1相互作用的蛋白。

Aim： To screen the proteins that could interact with ring-box 1（ RBX1） in Dunaliella salina,a bait vector p GBKT7-RBX1 for RBX1 from Dunaliella salina was constructed. Methods： As a model organism,the ORF of RBX1 from Dunaliella salina was obtained by PCR amplification and confirmed by sequencing,and then inserted into the yeast expression plasmid p GBKT7 to construct a recombinant bait vector p GBKT7-RBX1. After being identified by double digestion,the constructed bait vector was respectively transformed into the yeast strains Y187 and AH109 using PEG / Li Ac method.Subsequently,the self-activation and toxicity were respectively examined by phenotype assay. Y187 transformed with bait vector was hybridized with AH109 transformed with the Dunaliella salina c DNA expression library. After the formation of the clover shaped zygote,positive clones were screened in auxotrophic medium by the active experiment of alpha galactosidase.Then the screened clones were sequenced. Results： The constructed bait vector of p GBKT7-RBX1 was successfully transformed into yeast strains Y187 and AH109. The expression products of the recombinant bait vector had no self-activation or toxic effects on neither Y187 nor AH109 strains. Sequence analysis on screened positive colonies showed that they were respectively homologous with ferredoxin thioredoxin reductase subunit from chlamydomonas reinhardtii and arabidopsis thaliana（ 38% and 39%） and translational inhibitor protein from chlamydomonas reinhardtii（ 51%）. Conclusion： The bait vector p GBKT7-RBX1 could be used for yeast two-hybrid system,and two positive colonies were obtained.