Characterization of x-type high-molecular-weight glutenin promoters (x-HGP) from different genomes in Triticeae

Abstract

The sequences of x-type high-molecular-weight glutenin promoter (x-HGP) from 21 diploid Triticeae species were cloned and sequenced. The lengths of x-HGP varied from 897 to 955 bp, and there are 329 variable sites including 105 singleton sites and 224 polymorphic sites. Genetic distances of pairwise X-HGP sequences ranged from 0.30 to 16.40% within 21 species and four outgroup species of Hordeum. All five recognized regulatory elements emerged and showed higher conservation in the x-HGP of 21 Triticeae species. Most variations were distributed in the regions among or between regulatory elements. A 22 bp and 50 bp insertions which were the copy of adjacent region with minor change, were found in the x-HGP of Ae. speltoides and Ps. Huashanica, and could be regarded as genome specific indels. The phylogeny of media-joining network and neighbour-joining tree both supported the topology were composed of three sperate clusters. Especially, the cluster I comprising the x-HGP sequences of Aegilops, Triticum, Henrardia, Agropyron and Taeniatherum was highly supporting by both network and NJ tree. As conferring to higher level and temporal and spatial expression, x-HGP can used as the source of promoter for constructing transgenic plants which allow endosperm-specific expression of exogenous gene on higher level. In addition, the x-HGP has enough conservation and variation; so it should be valuable in phylogenetic analyses of Triticeae family members.

Keywords: Evolution analysis; Regulatory element; Triticeae; x-type high-molecular-weight glutenin promoter (x-HGP).

Figure 1

Schematic structure of HMW glutenin gene promoter and the strategy of cloning. The regulatory elements were indicated by boxes, E: E motif, N: N motif, PE: partial HMW enhancer, EN: complete HMW enhancer, TA: TATA box. The specific primers (HGPF and HGPxR) for amplifying x-type HMW glutenin gene promoter and their target region are marked. The deletion of regulatory element partial enhancer in y-type HMW glutenin gene promoter of some species is also indicated by broken lines.

Figure 2

PCR amplification of x-HGP from partial of 21Triticeaespecies. Line 1–12: T. urartu, T. monococcum L. subsp.aegilopoides, Ae. bicornis, Ae. longissima, Ae. sharonensis, Ae. speltoides, Ae. tauschii, Ps. huashanica, Se. cereale, Ta. caput-medusae, Th. Elongatum and Th. Elongatum; M is DNA marker.

Figure 3

The nucleotide variations in the sequences of x-HGP from 21 Tritceae species and y-HGP ofHordeum. The number at the top of figure indicated the variation position. See Table 1 for species abbreviations.

Figure 4

Multiple sequence alignment of x-HGP of 21Triticeaediploid species and four species ofHordeumas outgroup. The species-specific indels were indicated by boxes, a, partial HMW enhancer appears in all x-HGP of all 21 species, but deleted in those of four Hordeum; b and c, d represent unique indels in Psathyrostachys and Aegilops. The inserted fragments in Psathyrostachys and Aegilops are duplication of adjacent region.

Figure 5

The media-joining network derived from the x-HGP sequences from 21 diploid species ofTriticeaeand four y-HPG sequences ofHordeum. The x-HGP of all Aegilops formed the biggest subcluster around which two minor clade comprising Triticum, and He. persica, Ag. cristatum and Ta. caput-medusae emerged at the top of network. The topology was cluster into three main separate groups with placing PSHU aside the group II.

Figure 6

The neighbor-joining (NJ) tree derived from x-HGP sequences from 21 diploid species ofTriticeae. The NJ tree was constructed by using the substitute model of Maximum Composite Likelihood. The bootstrap values were calculated based on 1000 replications to estimate the topological robustness.