Ribozymes Targeted to Stearoyl-ACP D9 Desaturase mRNA Produce Heritable Increases of Stearic Acid in Transgenic Maize Leaves

Merlo, Ann Owens; Cowen, N. M.; Delate, Tom; Edington, Brent V.; Folkerts, Otto; Hopkins, Nicole; Lemeiux, Christine; Skokut, Tom; Smith, Kelley; Woosley, Aaron; Yang, Yajing; Young, Scott D.; Zwick, Michael E.

doi:10.2307/3870760

Cited by 4 publications

(4 citation statements)

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“…The down-regulation of the expression of genes encoding stearoyl-ACP desaturases has been repeatedly reported and is associated with changes in fatty acid composition and alterations in various physiological traits and growth of mutants or transgenic plants (Knutzon et al 1992;Lightner et al 1994a, b;Merlo et al 1998;Kachroo et al 2001;Nandi et al 2003). However, there are relatively few data concerning up-regulation of the same genes.…”

Section: Discussionmentioning

confidence: 99%

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

Craig¹,

Lenzi²,

Scotti³

et al. 2008

Transgenic Res

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The possibility of altering the unsaturation level of fatty acids in plant lipids by genetic transformation has implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. While the integration and expression of transgenes in the plastome has several potential advantages over nuclear transformation, very few attempts have been made to manipulate fatty acid biosynthesis using plastid transformation. We produced transplastomic tobacco plants that express a D 9 desaturase gene from either the wild potato species Solanum commersonii or the cyanobacterium Anacystis nidulans, using PEG-mediated DNA uptake by protoplasts. Incorporation of chloroplast antibioticinsensitive point mutations in the transforming DNA was used to select transformants. The presence of the transcript and the D 9 desaturase protein in transplastomic plants was confirmed by northern and western blot analyses. In comparison with control plants, transplastomic plants showed altered fatty acid profiles and an increase in their unsaturation level both in leaves and seeds. The two transgenes produced comparable results. The results obtained demonstrate the feasibility of using plastid transformation to engineer lipid metabolic pathways in both vegetative and reproductive tissues and suggest an increase of cold tolerance in transplastomic plants showing altered leaf fatty acid profiles. This is the first example of transplastomic plants expressing an agronomically relevant gene produced with the ''binding-type'' vectors, which do not contain a heterologous marker gene. In fact, the transplastomic plants expressing the S. commersonii gene contain only plant-derived sequences, a clear attraction from a public acceptability perspective.

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Section: Discussionmentioning

confidence: 99%

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

Craig¹,

Lenzi²,

Scotti³

et al. 2008

Transgenic Res

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“…[9][10][11][12] Hammerhead ribozymes, for instance, have been used to cleave RNA in transgenic plants and animals. [13][14][15][16][17][18] Although it is clear that hammerhead ribozymes can cleave specific viral RNA or mRNA in cell-free systems effectively, the efficiency of cleavage in vivo remains to be improved. 19 Part of the explanation for the low efficiency of ribozymes in an intracellular environment is the misfolding and the degradation of ribozymes by exonucleases present in cells.…”

Section: Introductionmentioning

confidence: 99%

Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus

et al. 2003

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The DNA-packaging pRNA of bacterial virus phi29, which forms dimers and then hexamers, contains two independent tightly self-folded domains. Circularly permuted pRNAs were constructed without impacting pRNA folding. Connecting the pRNA 5 0 /3 0 ends with variable sequences did not disturb its folding and function. These unique features, which help prevent two common problems -exonuclease degradation and misfolding in the cell, make pRNA an ideal vector to carry therapeutic RNAs. A pRNA-based vector was designed to carry hammerhead ribozymes that cleave the hepatitis B virus (HBV) polyA signal. The chimeric HBV-targeting ribozyme was connected to the pRNA 5 0 /3 0 ends as circularly permuted pRNA. Two cis-cleaving ribozymes were used to flank and process the chimeric ribozyme. The hammerhead ribozyme including its two arms for HBV targeting was able to fold correctly while escorted by the pRNA. The chimeric ribozyme cleaved the polyA signal of HBV mRNA in vitro almost completely. Cell culture studies showed that the chimeric ribozyme was able to enhance the inhibition of HBV replication when compared with the ribozyme not escorted by pRNA, as demonstrated by Northern blot and e-antigen assays. pRNA could also carry another hammerhead ribozyme to cleave other RNA substrate. These findings suggest that pRNA can be used as a vector for imparting stability to ribozymes, antisense, and other therapeutic RNA molecules in vivo.

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“…As a means to raise stearate levels in seed oil researchers have implemented two approaches, a biotechnology avenue (Hawkins and Kridl, ; Merlo et al ., ) and a mutational breeding strategy (Bubeck et al ., ; Pérez‐Vich et al ., ; Salas et al ., ; Zhang et al ., ). In soybean, high stearate mutants have been identified (Rahman et al ., ; Spencer et al ., ).…”

Section: Discussionmentioning

confidence: 99%

Stacking of a stearoyl‐ACP thioesterase with a dual‐silenced palmitoyl‐ACP thioesterase and ∆12 fatty acid desaturase in transgenic soybean

Park

Graef

et al. 2014

Plant Biotechnology Journal

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Soybean (Glycine max (L.) Merr) is valued for both its protein and oil, whose seed is composed of 40% and 20% of each component, respectively. Given its high percentage of polyunsaturated fatty acids, linoleic acid and linolenic acid, soybean oil oxidative stability is relatively poor. Historically food processors have employed a partial hydrogenation process to soybean oil as a means to improve both the oxidative stability and functionality in end-use applications. However, the hydrogenation process leads to the formation of trans-fats, which are associated with negative cardiovascular health. As a means to circumvent the need for the hydrogenation process, genetic approaches are being pursued to improve oil quality in oilseeds. In this regard, we report here on the introduction of the mangosteen (Garcinia mangostana) stearoyl-ACP thioesterase into soybean and the subsequent stacking with an event that is dual-silenced in palmitoyl-ACP thioesterase and ∆12 fatty acid desaturase expression in a seed-specific fashion. Phenotypic analyses on transgenic soybean expressing the mangosteen stearoyl-ACP thioesterase revealed increases in seed stearic acid levels up to 17%. The subsequent stacked with a soybean event silenced in both palmitoyl-ACP thioesterase and ∆12 fatty acid desaturase activity, resulted in a seed lipid phenotype of approximately 11%-19% stearate and approximately 70% oleate. The oil profile created by the stack was maintained for four generations under greenhouse conditions and a fifth generation under a field environment. However, in generation six and seven under field conditions, the oleate levels decreased to 30%-40%, while the stearic level remained elevated.

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Ribozymes Targeted to Stearoyl-ACP D9 Desaturase mRNA Produce Heritable Increases of Stearic Acid in Transgenic Maize Leaves

Cited by 4 publications

References 0 publications

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

Transplastomic tobacco plants expressing a fatty acid desaturase gene exhibit altered fatty acid profiles and improved cold tolerance

Bacterial virus phi29 pRNA as a hammerhead ribozyme escort to destroy hepatitis B virus

Stacking of a stearoyl‐ACP thioesterase with a dual‐silenced palmitoyl‐ACP thioesterase and ∆12 fatty acid desaturase in transgenic soybean

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