Sakakibara, 2,4-dichlorophenoxyacetic acid. The chemicals used in

Sakakibara, Kasahara, Ueda, Kojima, Takei, Hishiyama, Asami, Okada, Kamiya, Yamaya, and Yamaguchi (2005) conducted an experiment in order to discover how Tmr modifies CK biosynthesis in host plants by investigating the substrate preference and subcellular localization of Tmr in plant cells. The method in which Agrobacterium Tmr modifies CK biosynthesis in plant cells, the localization of Tmr to plastids in tumors, and its existence as substrate was examined. It was found that Agrobacterium tumefaciens modifies CK biosynthesis by sending a key enzyme into plastids of the host plant, which infects the plants and brings about the formation of tumors, or “crown galls.”The plant materials used in this experiment include Arabidopsis thaliana ecotype Columbia, a crown gall cell line of periwinkle V208 (Catha-ranthus roseus G. Don), which was cultured in hormone-free Murashige-Skoog (MS) liquid medium and supplemented with 3% sucrose, in addition to a wild-type cell line of periwinkle CRA, which was cultured in MS liquid medium and supplemented with 3% sucrose and 0.5gml 2,4-dichlorophenoxyacetic acid. The chemicals used in this experiment include HMBDP, which was synthesized by Wako Pure Chemical, and1-13C1-deoxy-D-xylulose (DX). 3,4-18O2DX was synthesized using H218O and 5-Ketoclomazone (KC) was synthesized using 2-chlorophenylmethylhydroxylam. Recombinant enzymes were ligated into pQE60. The coding region of Tmr and AtIPTs were ligated into pTA7001 and tagged.  V208 and CRA cells were cultured with KC. Crown Galls formed and were harvested, then extracted, purified, and analyzed. Coding regions of Tmr were fused and controlled by the cauliflower mosaic virus 35S (35S–sGFP S65T). Seedlings were homogenized and extracted. Polyclonal antibodies against Tmr were prepared with rabbits, and was purified by the HiTrap Mab purification kit (Amersham Pharmacia Biosciences) using  epitope selection method.Using the recombinant protein produced in E-coli, the Km values of Tmr for dimethylallyl diphosphate (DMAPP) and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBDP) were found. It was determined the major substrate for Tmr is likely to be HMBDP because it was  overexpressed in Arabidopsis. Tmr’s uses, if any, of HMBDP in planta were examined and it was determined Tmr uses HMBDP as a substrate in Arabidopsis seedlings, though it cannot be detected without using the engineering method of Tmr in this experiment. Tmr was expected to exist in plastids and use HMBDP as a substrate. Therefore, using Immunoblot analysis, Tmr was detected in a chloroplast-enriched fraction with the absence of plastid- and cytosol-localized glutamine synthetase isoforms. By purifying enacted plastids from the cell line, the localization of Tmr in Crown Gall Cells were found to be in the stroma of plastids. Ultimately, it was found Agrobacterium tumefaciens infects plants and brings about the formation of tumors.

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