TaBASS2 isolation, sequence characterization and transformation

The fragment of the TaBASS2 sequence identified in the previous microarray was used to search wheat expression sequence tags (http://www.ncbi.nlm.nih.gov/nucest/?term=wheat) [23]. The resulting hits were assembled using CAP3 software [33], and the entire TaBASS2 coding region was cloned using a pair of primers (5′-ATG GCG CCT TCC GCG ACC TGC C-3′/5′-TCA TTC CTT GAA ATC GTC CTT G-3′) designed from the reconstructed sequence. The predicted protein sequence was aligned with other BASS2 sequences using the CLUSTALW2 algorithm (www.ebi.ac.uk/Tools/msa/clustalw2/), and a phylogenetic analysis was conducted using the neighbor-joining method implemented in the MEGA4 software [34]. To generate transgenic wheat plants, the TaBASS2 coding sequence was ligated into pGA3626 driven by a maize ubiquitin promoter and introduced into wheat cv. YM20 using the shoot apical meristem method [35]. To generate transgenic Arabidopsis, the TaBASS2 coding sequence was ligated into pROK2, and ABI4 into pJIM19 (transgene driven by a 35S promoter). The cassette containing TaBASS2 was introduced into either A. thaliana ecotype Col-0 or the knockout mutant bass2-1 (SALK_101808C), and the cassette containing the ABI4 coding sequence was introduced into TaBASS2 constitutive expressors using the floral dip method [36]. Transformants were selected as previously [32], and homozygous T₃ progeny was used in the subsequent experiments.