A candidate gene OsAPC6 of anaphase-promoting complex of rice identified through T-DNA insertion

• DOI: 10.1007/s10142-009-0155-6
• Publication Date: 2010-01-20T07:52:31Z
• Authors: Mankesh Kumar; P. Osman Basha; Anju Puri; Deepak Rajpurohit; Gursharn Singh Ra...; Tilak Raj Sharma; Harcharan Singh D...
• Keywords: DNA, Bacterial; 0303 health sciences; Reverse Transcriptase Polymerase Chain Reaction; Reproducibility of Results; Ubiquitin-Protein Ligase Complexes; Oryza; Genes, Plant; Anaphase-Promoting Complex-Cyclosome; Gibberellins; Blotting, Southern; Mutagenesis, Insertional; 03 medical and health sciences; Gene Expression Regulation, Plant; Mutation; Autoradiography; Plant Proteins

A dwarf mutant (Oryza sativa anaphase-promoting complex 6 (OsAPC6)) of rice cultivar Basmati 370 with 50% reduced plant height as compared to the wild type was isolated by Agrobacterium tumefaciens-mediated transformation using Hm(R) Ds cassette. This mutant was found to be insensitive to exogenous gibberellic acid (GA(3)) application. Homozygous mutant plants showed incomplete penetrance and variable expressivity for plant height and pleiotropic effects including gibberellic acid insensitivity, reduced seed size, panicle length, and female fertility. Single copy insertion of T-DNA and its association with OsAPC6 was confirmed by Southern hybridization, germination on hygromycin, and 3:1 segregation of HPT gene in F(2) from OsAPC6 x Basmati 370 cross. The T-DNA flanking region sequenced through thermal asymmetric interlaced polymerase chain reaction showed a single hit on chromosome 3 of japonica rice cultivar Nipponbare in the second exonic region of a gene which encodes for sixth subunit of anaphase-promoting complex/cyclosome. The candidate gene of 8.6-kb length encodes a 728-amino acid protein containing a conserved tetratricopeptide repeat (TPR) domain and has only a paralog, isopenicillin N-synthase family protein on the same chromosome without the TPR domain. There was no expression of the gene in the mutant while in Basmati 370, it was equal in both roots and shoots. The knockout mutant OsAPC6 interferes with the gibberellic acid signaling pathway leading to reduced height and cell size probably through ubiquitin-mediated proteolysis. Further functional validation of the gene through RNAi is in progress.