2012 Annual Report
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.
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.
Progress in the first objective included development of a citrus genomic DNA database as well as several requisite constructs for intragenic modification of citrus and potatoes. Over the past year a 15X genome of the citrus rootstock variety Carrizo was completed. This genome contains the only known source of resistance to Huanglongbing disease in citrus that is currently threatening citrus production worldwide. This database is being employed to identify potential huanglongbing-resistance genes.
This year we constructed additional components for potato and citrus “intragenic” toolboxes to allow direct genetic modification of these crops without introduction of non-native DNA. We identified and demonstrated functionality of potato sequences to allow introduction of novel sequences into the genome without introducing any sequences from bacteria. We also completed construction of a novel citrus intragenic vector, currently being evaluated by ARS cooperators in Fort Pierce, Florida. The expression of novel potato and citrus promoters was evaluated using marker gene fusions in transgenic plants.
Progress in the second goal (reduction of glycoalkaloids) in FY12 includes construction and evaluation of novel siRNA constructs to down-regulate component enzymes of the glycoalkaloid biosynthetic pathway. Five constructs designed to down-regulate genes that carry out addition of galactose or glucose to the aglycone (solanidine galactosyltransferase SGT1 and solanidine glucosyltransferase SGT2) were evaluated. The suppression of both branches of the pathway was not observed in any of the dual constructs.
Progress on the third goal (to reduce crop disease susceptibility included completion of first year field evaluation of transgenic potatoes expressing a multiple virus-resistance transgene (PVY, PVA, PVX and PLRV) showed successful conference of resistance to two of target pathogens (PVY and PLRV). Over the past year we, in cooperation with ARS scientists in Wapato, WA, have continued development of a bacterial potato disease (Zebra Chip) for evaluating potential transgenic resistance strategies.
Mc Cue, K.F., Rockhold, D.R., Chhan, A., Belknap, W.R. 2011. Structure of two solanum tuberosum steroidal glycoalkaloid glycosyltransferase genes and expression of their promoters in transgenic potatoes. American Journal of Potato Research. 88(6):485-492 doi:10.1007/s12230-011-9215-2.
Belknap, W.R., Wang, Y., Wu, J., Huo, N., Rockhold, D.R., Gu, Y.Q., Stover, E.W. 2011. Characterizing the citrus variety Carrizo genome through 454 shotgun sequencing. Genome. 54:1005-1015. doi:10.1139/G11-070.