A novel Gram-stain-negative strain, designated ZYY5T, was isolated from rice roots. Results of 16S rRNA gene analysis indicated that strain ZYY5T was a member of the genus Dickeya , with a highest similarity to Dickeya zeae DSM 18068T (98.5%). The major fatty acids were summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:0 and summed feature 8 (C18:1 ω7c and/or C18:1 ω6c). Multi-locus sequence analysis using five concatenated genes (16S rRNA, atpD, infB, recA and gyrB) and phylogenomic analysis based on 2940 core gene sequences showed that strain ZYY5T formed a robust cluster with strains EC1, ZJU1202, DZ2Q, NCPPB 3531 and CSL RW192, while separated from the other strains of D. zeae . The orthologous average nucleotide identity (ANI) and digital DNA–DNAhybridization (dDDH) values among these six strains ranged from 96.8–99.9% and 73.7–99.8%, which supported that they were belonged to the same species. However, strain ZYY5T shared 58.4 of dDDH and 94.5% of ANI values with type strain D. zeae DSM 18068T, which were lower than the proposed species boundary cut-off for dDDH and ANI. The genomic analysis revealed that strain ZYY5T contained virulence-associated genes, which is same as the phylogenetic-related strains of the genus Dickeya . Based on the results of the polyphasic approaches, we propose that strain ZYY5T represents a novel species in the genus Dickeya , for which the name Dickeya oryzae sp. nov. (=JCM 33020 T=ACCC 61554 T) is proposed. Strains EC1, ZJU1202, DZ2Q, NCPPB 3531 and CSL RW192 should also be classified in the same genomospecies of D. oryzae same as ZYY5T.
Genetic engineering plays a unique role in fundamental plant biology studies and in improving crop traits. These efforts often necessitate introduction and expression of multiple genes using promoters from a very limited repertoire. Current common practice of expressing multiple genes is the repeated use of the same or similar promoters. This practice causes more frequent transgene silencing due to a high degree of sequence homology and a greater chance of rearrangement among repeatedly used promoter sequences. Therefore, availability and use of natural bidirectional promoters to minimize gene silencing and achieve desirable expression pattern of transgenes is a critical issue in the field of plant genetic engineering. Here we describe the use of a single natural bidirectional promoter to drive the expression of two reporter genes in onion epidermal cells and in transgenic tobacco plants. We show that (1) the promoter drives the simultaneous expression of GUS and GFP reporter genes after transient expression and stable transformation, (2) the transcription is equally strong in both directions, (3) immediate upstream regions in each direction control transcription independently from each other, and (4) the reporter genes are expressed in leaves and stems but not in roots, as expected from the fact that the endogenous promoter controls the expression of two photosynthetic genes in Arabidopsis. Hence, use of bidirectional promoters in heterologous background provides a means to express multiple genes in transgenic plants and aids genetic engineering-based crop improvement.
BackgroundThe development of plant gene transfer systems has allowed for the introgression of alien genes into plant genomes for novel disease control strategies, thus providing a mechanism for broadening the genetic resources available to plant breeders. Using the tools of plant genetic engineering, a broad-spectrum antimicrobial gene was tested for resistance against head blight caused by Fusarium graminearum Schwabe, a devastating disease of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) that reduces both grain yield and quality.ResultsA construct containing a bovine lactoferrin cDNA was used to transform wheat using an Agrobacterium-mediated DNA transfer system to express this antimicrobial protein in transgenic wheat. Transformants were analyzed by Northern and Western blots to determine lactoferrin gene expression levels and were inoculated with the head blight disease fungus F. graminearum. Transgenic wheat showed a significant reduction of disease incidence caused by F. graminearum compared to control wheat plants. The level of resistance in the highly susceptible wheat cultivar Bobwhite was significantly higher in transgenic plants compared to control Bobwhite and two untransformed commercial wheat cultivars, susceptible Wheaton and tolerant ND 2710. Quantification of the expressed lactoferrin protein by ELISA in transgenic wheat indicated a positive correlation between the lactoferrin gene expression levels and the levels of disease resistance.ConclusionsIntrogression of the lactoferrin gene into elite commercial wheat, barley and other susceptible cereals may enhance resistance to F. graminearum.
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