Cloning The Plastid Lipid-Associated Protein Gene From 'Hong Anliu' Sweet Orange And Its Primary Functional Analysis

'Hong anliu'sweet orange (Citrus sinensis [L.] Osbeck) is a red-fleshed mutant dominantly accumulating lycopene. In the comparative proteomics study of'Hong anliu' (MT, mutant) and'Anliu'(WT, wild type), a plastid lipid-associated protein (PAP) was detected to increase in parallel to the accumulation of pigments during the development of fruits and its expression in MT was over two-fold of that in WT. PAP is a large group of protein family involving in the formation of plastoglobules and fibrils, plastid differentiation and carotenoid sequestration. The research aimed at cloning the gene encoding CsPAP and primarily analyzing its function by genetic transformation.The isolated cDNA was 972 bp in length and had high identity rate with PAP genes reported in other species. The prokaryotic expression vector carrying cDNA fragment was constructed and transformed into E. coli to induce protein with a predicted molecular weight of 29 kD.The eukaryotic constitutive expression vector was also constructed to over-express CsPAP gene in callus (Citrus unshiu), epicotyls (Fortunella hindsii) and tomato cotyledon (Lycopersicon esculentum) by Agrobacterium-mediated transformation. Kanamycin-resistant citrus callus and regenerated tomato plants were obtained by selective culture and verified to be transgenic by PCR.Real-time PCR analysis revealed that CsPAP gene expressed over 50-fold in one line of transgenic citrus callus than in CK. Meanwhile, ABA treatment and certain stress conditions (NaCl, H2O2, and dehydration treatment) could bring out effects on the expression of CsPAP in Valencia callus (Citrus sinensis).