酵母双杂交系统筛选新橡胶延长因子HbREF3互作蛋白

Studies on the Candidate Proteins Interacting with a Novel HbREF3 of Hevea brasiliensis Using Yeast Two-hybrid System

橡胶延长因子(rubber elongation factor,REF)等橡胶粒子蛋白可能通过相互作用形成蛋白复合体在橡胶生物合成发挥作用。为鉴定REF家族中一新成员Hb REF3的互作蛋白,以橡胶树Hb REF3基因的c DNA序列为基础,分别构建了筛选Hb REF3相互作用蛋白的3种诱饵载体p GBKT7-Hb REF3-F(含全长ORF序列)及p GBKT7-Hb REF3-N(含N端序列)和p GBKT7-Hb REF3-C(含C端序列),并分别转化到Y2H Gold酵母菌株中,进行诱饵表达载体的自激活性与毒性检测。实验证明,3种诱饵载体转化的酵母菌能在SD/-Trp平板上正常生长,而在SD/-Trp/-His/-Ade平板上不能生长,因此,诱饵载体本身没有自激活性,它们的表达产物也没有毒性,不影响转化酵母菌Y2H的生长,满足作为诱饵质粒的条件,并用于橡胶树胶乳c DNA酵母表达文库的筛选。结果表明,用Hb REF3基因的全长ORF编码产物作诱饵,没有筛选到任何候选互作蛋白,而用Hb REF3的N端和C端区域分别作诱饵,均筛选到了可能与Hb REF3具有相互作用的候选蛋白,其中包括REF家族的其他成员和膜联蛋白D4等。

Rubber elongation factors （REFs） together with other rubber particle proteins, play their roles in rubber biosynthesis of rubber trees possibly through the formation of protein complexes. To identify the proteins inter- acting with HbREF3, which was a novel member of the REF family, three bait vectors of pGBKTT-HbREF3-F, pGBKTT-HbREF3-N and pGBKT7-HbREF3-C were constructed based on the cDNA sequence of HbREF3, and then transformed into Y2H Gold yeast strains to verify its self-activation and toxicity by yeast two-hybrid system. The results demonstrated that the transformed Y2H gold cells could grow well on the SD/-Trp plate, but not on the SD/-Trp/-His/-Ade plate, which indicated that the three bait vectors had no activities of self-activation. In addition, the expression products of these bait vectors had no toxicity, and did not affect the growth of the transformed yeast Y2H cells. Therefore, the three bait vectors pGBKT7-HbREF3-F, pGBKTT-HbREF3-N and pGBKT7-HbREF3-C could be further used to screen the latex cDNA library using yeast two hybrid. The presented data demonstrated that no candidate proteins interacting with HbREF3 could be screened by vector pGBKTT-HbREF3-F, in which the whole ORF sequence of HbREF3 gene was cloned and used as the prey. While several candidate interacting proteins were detected by both pGBKT7-HbREF3-N and pGBKT7-HbREF3-C, respectively, in which the partial N-end or C-end sequence of HbREF3 was used as the prey. The candidate proteins interacting with HbREF3 included rubber elongation factor, small rubber particle proteins, annexin D4 and so on.