The Effect Of Fusion Expression Of β-subunit And α-subunit In Rhodobacter Sphaeroides On Formation Of Light-harvesting Ⅱ Complexes

Rb. sphaeroides is a purple non-sulfur photosynthetic bacteria, its photosynthetic apparatus consists of light-harvesting II complex (LHII), light-harvesting I complex (LHI) and the light reaction center (RC). LHII of Rb. sphaeroides is encoded by pucBA genes. Firstly the pucB gene and pucA gene were respectively expressed to form β-subunits and a-subunits which were all transmembrane structure, and then they assembled into a heterogeneous dimerization structure, finally formed a pigment-protein complexes. Due to the combination of the photosynthetic pigment molecules, the maximum characteristic spectral absorption peaks of LHII are at800nm and850nm.In this study, flexible sequence (FS) was inserted into a paragraph betweenpuclB gene and puc1A gene to increase protein flexibility and swinging, to verify the ability of forming a normal structure and function of LHII. Then the expression vector puclB-FS-1A was constructed and transfered to the dual-deletion mutant Rb. sphaeroides CQU68(genomic deletion of puc1BA, puc2BA and pufBALMX) strain using the combination of the transfer method. Analyzing near infrared700nm-900nm spectral absorption of live bacteria showed that two typical absorption peaks were at800nm and850nm, but the absorption peaks were slightly lower and blue shift. So it is infered that the fusion protein can form LHII and enter the membrane system. Using affinity chromatography to purify fusion protein and the SDS-PAGE electrophoresis, the results showed that the fusion protein was successfully expressed in Rb. Sphaeroides.This topic not only can help us to understand the basic structure and molecular mechanism of LHII, but also can provide us the theoretical support and the experimental evidence of photosynthetic apparatus which will be efficient and correct expression of membrane proteins.