菠萝体细胞胚酵母单杂交cDNA文库及AcSERK1启动子相关诱饵载体的构建

Construction of Somatic Embryo Yeast One-hybrid cDNA Library and Bait Vector Related to AcSERK1 Promoter of Pineapple

为筛选与菠萝AcSERK1启动子中胚性细胞特异性表达调控区段(-983~-881 nt)的结合蛋白,本研究利用MatchmakerTM Gold Yeast One-Hybrid System构建菠萝体细胞胚酵母单杂交cDNA文库以及胚性细胞特异性表达调控区段(SE103)的诱饵载体。建立的菠萝体细胞胚酵母单杂交cDNA文库库容量为1.25×107CFU,其插入片段平均长度约1.5 kb,重组率为100%。通过检测,AbA最低浓度为600 ng/mL时,构建的诱饵载体pAbAi-SE103在Y1HGold酵母菌株中的自激活活性被有效抑制,表明该菠萝体细胞胚酵母单杂交cDNA文库以及诱饵酵母菌株Y1HGold(pAbAi-SE103),可用于AcSRKE1启动子中胚性细胞特异性表达调控区段互作蛋白的筛选实验,为胚性细胞特异性转录因子的分离鉴定提供数据参考。

In order to screen binding proteins associated with embryogenic cell-specific expression regulatory segments(-983~-881 nt)in AcSERK1 promoter,MatchmakerTM Gold Yeast One-Hybrid System was used to construct the somatic embryo yeast one-hybrid cDNA library and bait vectors for the embryogenic cell-specific expression regulation segment(SE103)of pineapple.The capacity,average length of insert fragment and recombination rate of constructed somatic embryo yeast one-hybrid cDNA library of pineapple were 1.25×107 CFU,1.5 kb and 100%,respectively.The constructed bait vector pAbAi-SE103 had no self-activating activity and no toxicity in Y1HGold yeast strain.The constructed somatic embryotic yeast one-hybrid cDNA library of pineapple and the bait-yeast strain Y1HGold(pAbAi-SE103)can be used for the screening of embryogenic cell-specific interacting protein with AcSRKE1 promoter,which laid a foundation for the isolation and identification of embryogenic cell-specific transcription factors.