Studying Of The Thylakoid Membrane Protein Interactions Through The Split-ubiquitin Yeast Two Hybrid System

The major photosynthetic complexes PSI, PSII, the cytochrome b₆f complex and the ATP synthase complex are located in the thylakoid membrane. To study the mechanism of photosynthesis in the postgenomic era, the split-ubiquitin yeast two hybrid were used as a strategy through which a cDNA library would be screened, if this could be reached, some novel factors involved in the PSII biogenesis would be found and their functions will be subsequently investigated . Here, some initial trials for this idea have been performed.1. The newly developed split-ubiquitin two hybrid system was used to investigate protein-protein interactions of thylakoid membrane proteins. The reaction center D1 protein of PSII, which is encoded by plastid gene psbA, was used as the bait protein, while the D2 encoded by plastid gene psbD , another PSII reaction center protein, and Cyt_b6 encoded by plastid gene petB, a component of Cytb₆f complex, were used as prey proteins. The yeast two hybrid analysis showed that the D1 protein interacts with D2, but not with the Cyrtb₆. This result confirms the protein interaction model of thylakoid membrane protein complex, and also means the availability of this system in detecting thylakoid membrane protein interactions. Thus, the split-ubiquitin two hybrid systems could provide an efficient tool to reveal the mechanism of chloroplast proteins biogenesis.2. An Arabidopsis leaf cDNA entry library were constructed by using the Gateway cloning technology. The first strand cDNA was synthesized with the reverse transcript primer attB2-Oligod(T). After the synthesis of the second strand of cDNA by virtue of Ecoli DNA polymerase I and RNase H, the attB1 adapter were ligated to 5' end of the cDNA for the BP recombination reaction. The resulting donor vector cloned cDNA fragment were subjected to electronical transformation into DH10B. The generating transformants were termed entry library. Subsequent quality analysis indicated that its representation is 0.97×10⁷, with the average insert of 648bp and insert length ranging from 500bp to 950bp.