Genome-wide Screening, Cloning And Functional Characterization Of Promoters Specifically And Strongly Expressed In Maize Embryos

The use of maize seeds as bioreactors has several advantages for the production of recombinant proteinsin plant biotechnology, which include eukaryotic posttranslational modifications, low risk of human andanimal pathogen contamination, excellent scalability, a relatively high protein yield, and low-costproduction potential, but available embryo-specific and strong promoters are limited. Here, we describea genome-scale microarray-based approach to identify embryo-specifically and strongly expressedgenes and their promoters in maize. We identified28embryo-preferred and abundantly expressed genesbased on our microarray data. These embryo-preferred genes were further analyzed using the UniGenedatabase and by quantitative reverse transcriptase-PCR leading to the identification of seven genes(Zm.2098, Zm.13387, Zm.66589, Zm.85502, Zm.68129, Zm.3896, and Zm.2941) as embryo-specificgenes with higher expression levels relative to maize globulin-1. The putative promoters of fiveembryo-specific genes (Zm.13387, Zm.66589, Zm.85502, Zm.3896, and Zm.2941) were isolated and allexhibited strong promoter activities when transiently expressed in maize embryos of20DAP. Theembryo specificity and expression levels of the promoters of four genes (Zm.13387, Zm.85502,Zm.3896, and Zm.2941) were further examined in transgenic maize plants, revealing that they arestrong promoters in embryos of all four developmental stages tested compared to reference globulin-1promoter. Moreover, Zm.2941and Zm.3896promoters are stringently embryo-specific promoters whileZm.85502promoter is basically embryo-specific yet wounding-inducible in non-seed tissues, andZm.13387promoter is developmentally expressed in both embryo and aleurone with wounding-inducedactivity in non-seed tissues. Our study provides novel embryo-specific and strong promoters that aresuitable for production of high-level recombinant proteins in maize embryos and for functional andregulatary analysis of genes involved in the development of maize kernal.Bidirectional gene pairs exist as a specific form of gene organization in microorganisms and mammalsas well as in model plant species, such as Arabidopsis and rice. Little is known about bidirectional genepairs in maize, which has a large genome and is one of the most important grain crops. We conducted agenome-wide search in maize using genome sequencing results from the inbred maize line B73. In total,1696bidirectional transcript pairs were identified using a modified search model. The Blast2GO wasemployed to make a functional classification of these transcript pairs, and we found that plastid,membrane, mitochondria and nucleus were the major subcellular localizations, and nucleotide andnucleic acid binding, transferase activity, and hydrolase activity were the most relevant molecularfunctions, and biosynthetic processes, nucleobase-containing compound metabolic processes, cellularprotein metabolic processes, and stress responses were the most relevant biological processes. Wefunctionally characterized the promoter activity of the intergenic regions of most of theembryo-expressing bidirectional transcript pairs in a maize embryo transient expression system usingthe GFP and GUS as reporter genes, revealing that34out of156promoters were able to drivebidiectional transcription in maize embryos. An expanded study about the bidirectional transcript pairsin two monocot (Sorghum bicolor, and Oryza sativa) and two dicot (Arabidopsis thaliana and Glycine max) species showed that bidirectional transcript pairs were603,2809,654and4386,acounting for4.1%,8.46%,2.3%and24.78%of the total transcripts in the four plants, respectively, and the functionclassification patterns were consistent with that in maize. Moreover the orthologous bidirectional genespairs were clearly distinguishable between the monocot and dicot species although the total numbers oforthologous bidirectional genes were similar. Another comparative analysis of orthologous bidirectionalgenes among maize, Brachypodium distachyon, Selaginella moellendorfii, and Ostreococcus tauriconfirmed that the functions of the bidirectional genes and the structure of the bidirectional genes pairswere conserved but the gene components of the bidirectional genes pairs was dynamic. Theevolutionary analysis of the function and structure of orthologous bidirectional genes pairs in variousplant species revealed a potential pathway of their origin, which may be required for the evolution of anew species.