title: The relationship between PMI (manA) gene expression and optimal selection pressure in Indica rice transformation.
abstract: An efficient mannose selection system was established for transformation of Indica cultivar IR58025B . Different selection pressures were required to achieve optimum transformation frequency for different PMI selectable marker cassettes. This study was conducted to establish an efficient transformation system for Indica rice, cultivar IR58025B. Four combinations of two promoters, rice Actin 1 and maize Ubiquitin 1, and two manA genes, native gene from E. coli (PMI-01) and synthetic maize codon-optimized gene (PMI-09) were compared under various concentrations of mannose. Different selection pressures were required for different gene cassettes to achieve corresponding optimum transformation frequency (TF). Higher TFs as 54 and 53% were obtained when 5 g/L mannose was used for selection of prActin-PMI-01 cassette and 7.5 g/L mannose used for selection of prActin-PMI-09, respectively. TFs as 67 and 56% were obtained when 7.5 and 15 g/L mannose were used for selection of prUbi-PMI-01 and prUbi-PMI-09, respectively. We conclude that higher TFs can be achieved for different gene cassettes when an optimum selection pressure is applied. By investigating the PMI expression level in transgenic calli and leaves, we found there was a significant positive correlation between the protein expression level and the optimal selection pressure. Higher optimal selection pressure is required for those constructs which confer higher expression of PMI protein. The single copy rate of those transgenic events for prActin-PMI-01 cassette is lower than that for other three cassettes. We speculate some of low copy events with low protein expression levels might not have been able to survive in the mannose selection.
authors.affiliation: Syngenta Biotechnology China Co., Ltd, 25 Life Science Park Road, Changping District, Beijing, 102206, People's Republic of China.
authors.forename: Huaping
authors.initials: H
authors.lastname: Gui
authors.forename: Xia
authors.initials: X
authors.lastname: Li
authors.forename: Yubo
authors.initials: Y
authors.lastname: Liu
authors.forename: Kai
authors.initials: K
authors.lastname: Han
authors.forename: Xianggan
authors.initials: X
authors.lastname: Li
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D018506:Gene Expression Regulation, Plant; D005818:Genetic Engineering / Q000379:methods; D008358:Mannose / Q000494:pharmacology; D008359:Mannose-6-Phosphate Isomerase / Q000235:genetics*; D012275:Oryza / Q000187:drug effects / Q000235:genetics*; D018515:Plant Leaves / Q000235:genetics; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D012641:Selection, Genetic; D014170:Transformation, Genetic / Q000187:drug effects; D003313:Zea mays / Q000235:genetics
chemical_list: D008359:Mannose-6-Phosphate Isomerase; D008358:Mannose