Identification and expression analysis of PHT5 genes to P dynamics in B. napus
To identify the PHT5 genes in B. napus , we performed a BLAST search in B. napus cultivar ’Darmor-bzh ’ genome (Chalhoub et al., 2014) using the AtPHT5;1 , AtPHT5;2 andAtPHT5;3 gene sequences. A total of eight close homologs ofAtPHT5 were identified, namely BnA09PHT5;1a ,BnC09PHT5;1a , BnA09PHT5;1b , BnCnPHT5;1b ,BnA09PHT5;2 , BnC09PHT5;2 , BnA01PHT5;3 andBnC01PHT5;3 (Figure 1a). Gene structure analysis showed that these BnPHT5 genes had 9 or 10 exons, encoding comparable length proteins (Figure S1, 695-705 amino acid residues). Based on protein sequence alignment, eight members of BnPHT5 family showed more than 90% protein identities between the homologs of Arabidopsis andB. napus (Table 1).
To estimate the response of theBnPHT5genes to P supply, quantitative RT-PCR (qRT-PCR) was performed on 3-week-old B. napus leaves grown with different external Pi supply (0-100 μM). Both BnPHT5;1a and BnPHT5;3genes had the highest expression at 0 μM Pi supply and significantly declined at the 5-100 μM Pi supply (Figure 1a, d, e, g). In contrast,BnA09PHT5;1b and BnCnPHT5;1b were gradually induced as Pi supply increased (Figure 1c, f). These results suggest thatBnPHT5;1b genes might play a different role from theBnPHT5;1a and BnPHT5;3 genes in the B. napus Pi response. The expression of BnA09PHT5;2 and BnC09PHT5;2could not be detected in our experimental conditions (data not shown).