Isolation And Functional Analysis Of Seed-specific Promoters Of Zpu1 And Brittle2 From Zea Mays

Maize (Zea mays L.) is one of the most important food crops in the world, and the seed of maize is an unusual organ due to high level of starch and storage protein produced. Although the understanding of these processes has significant agronomic value, relatively few studies of regulation of these processes have been performed. With the development of the plant bioreactor the seed of maize become an important organ as a bioreactor. But efficient genetic engineering lies on being able to produce a specific gene product at the desired expression level in the appropriate tissues. This can be accomplished easily by driving the gene with specific promoters or cis-regulatory elements. It is important that isolation and comparison of seed-specific promoters may help elucidate required promoter sequences, the tissue specificity, relative strengths, or developmental program spatially and temporally as seen with the endogenous genes.In this work, we have studied three promoters from the maize Zpul encoding maize pullulanase-type starch debranching enzyme, brittlε2 encoding the small subunit of maize ADP-glucose pyrophosphorylase(AGPase) and zE19 encoding a maize 19kDa zein, respectively.By screening a genomic library of maize, a genomic DNA fragment consisting of the 5' flanking sequence and part of the coding sequence corresponding to the maize pullulanase-type starch debranching enzyme (ZPU1) cDNA were isolated. Promoter fragments of various lengths, a full isolated 5' flanking sequence (-2267 to -1) (Z1), a 3' deletion (-2267 to -513) (Z5) and three 5' deletions extending -1943 (Z2), -1153(Z3) and -516(Z4) upstream of the translational initiation ATG codon were fused to a gus reporter gene encoding β -glucuronidase and introduced into tobacco via an Agrobacterium tumefuciens-mediated leaf disc transformation. No GUS activity was detectable in the roots, stems, young leaves, stigma and ovaries of the transgenic tobacco plants, which had integrated the full isolated sequence or its deletions. Analysis of the 5' promoter deletions indicated that the region between -2267 and -1943 contains positive cis-regulatory elements necessary for seed-specific, high level expression and that the region (-1153 to -516) negatively regulated gus gene expression. A comparison of promoter activity of the Z4 fragment with Z5 indicated that the proximal region (-516 to -1) of the Zpu1 promoter was essential for directing the expression of gus reporter gene in transgenic tobacco plants. These results suggest that the expression pattern of the Zpu1 promoter is beneficial in understanding the function of ZPU1 in starch synthesis and hydrolysis and it also offers great potential in plant genetic engineering.Two fragments from the 5' sequence of the zE19 and the brittle2 were PCR amplified with two pairs of primers based on the sequences in GenBank (Accession no.AF334959, X63667). The characterization of the 0.9kb 5' flanking sequences of brittle2 gene, encoding the small subunit of AGPase, was studied via expression of gus reporter gene in transgenic tobacco. Analysis of GUSactivities showed that the 0.9kb 5' flanking fragment of brittle! was sufficient to initiate transcription and the expression pattern of brittld promoter is seed-specific when comparing the expression in the target tissues and organs to that shown in non-target tissues and organs. To further analyze its activity, a comparison of the 5' flanking fragment activity with promoter of zE19, encoding a maize 19KD zein, indicated that both promoters were seed-specific. At the same time, the result indicated that the activity of zE19 promoter was three to four fold higher than that of the 0.9kb 5' flanking fragment of brittle2 in seeds. In next generation of transgenes we also studied the temporal and spatial expression pattern of brittle2 and zE19 promoter. At the 20 days after flowering GUS activity was detected in transgenic lines integrated with zE19 promoter, but no GUS activity was found in brittle2 transgenic lines. These results indicated that the temporal expression of zE19 promoter is earlier than that of brittle2 promter.