Trovato M, Cianfriglia M, Filetici P, Mauro M L, Costantino P
Dipartimento di Genetica e Biologia Molecolare, Università di Roma La Sapienza, Italy.
Gene. 1990 Mar 1;87(1):139-43. doi: 10.1016/0378-1119(90)90506-m.
Expression of the rolB gene of Agrobacterium rhizogenes TL-DNA is sufficient to trigger root differentiation in transformed plant cells. To investigate the role of RolB in differentiation, a large portion of rolB, comprising about 90% of its C-terminal coding sequence, was cloned into vectors pEX34 and pEA305 in frame with the truncated N termini of the pL-MS2 phage DNA polymerase and, respectively, the ptac-c Its phage lambda repressor gene. Hybrid proteins were expressed from both fusions and the one from pMTBEX1 was utilized to raise antibodies. These antibodies specifically recognize the RolB moiety in both pL-MS2-rolB and ptac-cI-rolB fusions. Unfused, complete RolB protein was obtained by in vitro translation in a rabbit reticulocyte system of a transcript obtained by in vitro transcription of rolB. RolB protein is specifically immunoprecipitated by the antibodies raised against the hybrid protein MS2-RolB.
发根农杆菌TL-DNA的rolB基因的表达足以在转化的植物细胞中引发根分化。为了研究RolB在分化中的作用，将rolB的大部分（约占其C端编码序列的90%）与pL-MS2噬菌体DNA聚合酶的截短N端以及ptac-cI噬菌体λ阻遏基因的截短N端读框内克隆到载体pEX34和pEA305中。两种融合蛋白均表达，并且利用来自pMTBEX1的融合蛋白制备抗体。这些抗体能特异性识别pL-MS2-rolB和ptac-cI-rolB融合蛋白中的RolB部分。通过对rolB进行体外转录获得的转录本在兔网织红细胞系统中进行体外翻译，从而获得未融合的完整RolB蛋白。针对杂交蛋白MS2-RolB产生的抗体能特异性免疫沉淀RolB蛋白。
