2009 Annual Report
1a.Objectives (from AD-416)
1)Develop new promoter elements from potato that will allow refined expression profiles (tissue and/or developmental specificity) of transgenes to improve agronomic and quality properties of dicotyledonous crop species.
2)Discover and develop new molecular tools (promoters, terminators) from fruit trees. In particular, to isolate transcriptional control elements and polyadenylation signals from plum and apple.
3)Refine down-regulation technologies to improve general applications to metabolic regulation as well as improve design characteristics of pathogen-resistance transgenes.
1b.Approach (from AD-416)
Available EST, microarray and genomic DNA databases will serve as bioinformatics data sources to identify potato gene families, and specific family members, with requisite expression profiles to serve as sources of valuable transcriptional control elements. Putative elements will be isolated from a BAC library, assembled into marker-gene fusions, and function characterized in transgenic potatoes.
Polyubiquitin genes from apple and plum will serve as sources of transcriptional control elements for direction of commercial levels of transgene expression in these fruit trees. Appropriate polyubiquitin genes for these control sequences will be identified using EST databases to identify specific, highly constitutively transcribed family members. The molecular source of elements will include both BAC libraries and PCR-amplification. Putative promoter elements will be fused to standard marker and delivered to collaborators at the USDA/ARS Appalachian Fruit Research Station for introduction and characterization in apple.
Glycoalkaloids will be reduced in potatoes by suppression of both branches of the SGA pathway using small inverted hairpin structures of double stranded RNA-generating constructs. Small interfering RNAs (siRNAs) will be produced by these constructs specific for both the Sgt1 and Sgt2 gene family members responsible for the initial steps in each of the two SGA biosynthetic branches, resulting in gene inactivation. Suppression constructs will be tested for efficacy via standard genetic transformation, but will ultimately be adapted for intragenic transformation and eventual commercial application. Transgenic potato tubers producing siRNAs will be evaluated for transgene expression and SGA accumulation. Replacing 5325-21420-003-00D (08/2008).
The major problem areas being addressed are development of new molecular tools for potato and fruit tree improvement and construction of transgenic potatoes with improved postharvest and disease susceptibility properties. The project has three specific goals: .
1)to discover and develop new molecular tools (promoters, terminators) for improvement of dicotyledonous crop species including intragenic modifications,.
2)to reduce levels of glycoalkaloid toxicants in potatoes, and.
3)to reduce susceptibility of commercial potato cultivars to potato viruses and Late Blight.
Transcriptional control elements developed as part of the first goal (molecular tools) are expected to have specific application in construction of intragenic potatoes and fruit trees, as well as broader applications in transgene construction for dicotyledonous crops. Progress in this goal in FY09 includes completion of computational identification of citrus tree gene families to act as sources of novel transcriptional control elements for transgenes to confer resistance to Huanglongbing (HLB), also known as citrus greening disease. In addition, the first citrus promoter/terminator pair for HLB-resistance has been cloned and sequenced and marker gene constructs are currently being prepared to characterize expression profiles in citrus trees. Antibacterial transgenes previously developed in our laboratory, transcribed from potato control sequences, have already been entered into preliminary transformation by collaborators at the ARS U. S. Horticultural Research Laboratory (Fort Pierce, FL). Finally, the availability of significant citrus genomic sequence allowed the efficient isolation of these control sequences by direct PCR amplification from genomic DNA. A similar strategy is being employed for isolation of other fruit tree control sequences (apple).
Progress in the second goal (reduction of glycoalkaloids) in FY09 includes completion of characterization of second generation transgenes (employing intron-spliced hairpin RNA) in transgenic potatoes. In contrast to first generation (antisense) constructs, these assemblies did not result in significant alterations in glycoalkaloid accumulation. Transgenic potato lines are currently being evaluated to identify the sequence/design parameters responsible for this unexpected absence of phenotype.
Progress on the third goal (to reduce susceptibility of commercial potato cultivars to potato viruses and Late Blight) includes demonstration of efficacy of second generation virus-resistance genes (Potato Virus Y) (employing intron-spliced hairpin RNA constructs transcribed from improved promoter elements)in transgenic potato varieties. In addition, intragenic, marker-free, potato transformation for introduction of disease-resistance genes was successfully introduced into the laboratory.
New citrus gene components for resistance to citrus greening disease. Huanglongbing (HLB), also known as citrus greening or yellow dragon disease, is the most serious citrus disease in the world and is currently a threat to the US citrus industry. The causal agent of HLB is the phloem-limited bacterium Candidatus liberibacter, transmitted by the Asian citrus psyllid, and resistance to the disease does not exist in the available citrus germplasm. The immediacy of the HLB threat to the U.S. industry combined with the time frame required for development of new varieties makes the molecular genetic (transgenic) approach a logical choice for introducing resistance. ARS scientists at Albany, CA, used computational analysis of the available citrus DNA database to identify four specific genes with potential to act as sources of transcriptional control elements to direct expression of HLB-resistance genes. The first citrus control sequences to direct efficient gene expression in phloem were cloned and sequenced, and marker gene fusions for characterization in transgenic citrus are being constructed. These control sequences will be evaluated in transgenic citrus by ARS cooperators in Fort Pierce, FL and will be available for construction of HLB-resistance genes in public and private sector laboratories.
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Mc Cue, K.F. 2009. Potato Glycoalkaloids, Past Present and Future. Fruit, Vegetable, and Cereal Science and Biotechnology. 3:65-71.
Mc Cue, K.F., Allen, P.V., Shepherd, L., Blake, A., Whitworth, J.L., Maccree, M., Rockhold, D.R., Stewart, D., Davies, H., Belknap, W.R. 2006. The primary in vitro steroidal alkaloid glucosyltransferase from potato (solanum tuberosum). Phytochemistry. 67 (15):1590-1597