Monica A. Schmidt

Suppression of Soybean Oleosin Produces Micro-Oil Bodies that Aggregate into Oil Body/ER Complexes

Using RNAi, the seed oil body protein 24-kDa oleosin has been suppressed in transgenic soybeans. The endoplasmic reticulum (ER) forms micro-oil bodies about 50 nm in diameter that coalesce with adjacent oil bodies forming a hierarchy of oil body sizes. The oil bodies in the oleosin knockdown form large oil body-ER complexes with the interior dominated by micro-oil bodies and intermediate-sized oil bodies, while the peripheral areas of the complex are dominated by large oil bodies. The complex merges to form giant oil bodies with onset of seed dormancy that disrupts cell structure. The transcriptome of the oleosin knockdown shows few changes compared to wild-type. Proteomic analysis of the isolated oil bodies of the 24-kDa oleosin knockdown shows the absence of the 24-kDa oleosin and the presence of abundant caleosin and lipoxygenase. The formation of the micro-oil bodies in the oleosin knockdown is interpreted to indicate a function of the oleosin as a surfactant.

Silencing of Soybean Seed Storage Proteins Results in a Rebalanced Protein Composition Preserving Seed Protein Content without Major Collateral Changes in the Metabolome and Transcriptome

The ontogeny of seed structure and the accumulation of seed storage substances is the result of a determinant genetic program. Using RNA interference, the synthesis of soybean (Glycine max) glycinin and conglycinin storage proteins has been suppressed. The storage protein knockdown (SP−) seeds are overtly identical to the wild type, maturing to similar size and weight, and in developmental ontogeny. The SP− seeds rebalance the proteome, maintaining wild-type levels of protein and storage triglycerides. The SP− soybeans were evaluated with systems biology techniques of proteomics, metabolomics, and transcriptomics using both microarray and next-generation sequencing transcript sequencing (RNA-Seq). Proteomic analysis shows that rebalancing of protein content largely results from the selective increase in the accumulation of only a few proteins. The rebalancing of protein composition occurs with small alterations to the seed’s transcriptome and metabolome. The selectivity of the rebalancing was further tested by introgressing into the SP− line a green fluorescent protein (GFP) glycinin allele mimic and quantifying the resulting accumulation of GFP. The GFP accumulation was similar to the parental GFP-expressing line, showing that the GFP glycinin gene mimic does not participate in proteome rebalancing. The results show that soybeans make large adjustments to the proteome during seed filling and compensate for the shortage of major proteins with the increased selective accumulation of other proteins that maintains a normal protein content.

Endoplasmic reticulum to vacuole trafficking of endoplasmic reticulum bodies provides an alternate pathway for protein transfer to the vacuole.

One of the emerging differences that appears to separate plants from the other eukaryotes is the plasticity of the endoplasmic reticulum (ER) to form protein, oil, or rubber containing subcellular structures best termed ER bodies, as proposed by Hara-Nishimura and colleagues ([Matsushima et al.,

Quantitative detection of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) by real-time polymerase chain reaction

Abstract. Quantitative real-time polymerase chain reaction (PCR) assays were designed that enabled the zygosity of transgenes in soybean [Glycine max (L.) Merrill] and peanut (Arachis hypogaea L.) to be determined. The two zygosity assays, based on TaqMan technology that uses a fluorogenic probe which hybridizes to a PCR target sequence flanked by primers, were both accurate and reproducible in the determination of the number of transgenes present in a cell line. In the first assay, in which TaqMan assays were performed on increasing amounts of a plasmid containing the transgene of interest, a linear relationship between the level of fluorescence and the template amount was produced. Using the resultant linear relationships as standard curves, we were able to determine the zygosity of both soybeans segregating for the cry1Ac transgene and that of a T1 peanut segregating for the hph transgene. In the second assay, a relative determination of copy number (referred to as comparative Ct) was performed on transgenic soybeans by comparing the amplification efficiency of the transgene of interest to that of an endogenous gene in a multiplexed PCR reaction. Both methods proved to be sufficiently sensitive to differentiate between homozygotes and hemizygotes. These assays have numerous potential applications in plant genetic engineering and tissue culture, including the hastening of the identification of transgenic tissue, selecting transformation events with a low number of transgenes and the monitoring of the transmission of transgenes in subsequent crosses.

A comparison of strategies for transformation with multiple genes via microprojectile-mediated bombardment

The stable insertion and expression of multiple transgenes in crops is highly desirable, as the manipulation of complex agronomic traits and the introduction of novel biosynthetic pathways are dependent upon it. This study was performed to explore the frequency and efficiency of introducing multiple genes in soybean by using somatic embryogenesis and microprojectile bombardment transformation. The co-transformation frequency of six selectable marker or reporter genes (GusA, bleomycin resistance, glufosinate resistance, hygromycin resistance, green fluorescent protein, and kanamycin resistance) were followed throughout the T0, T1, and T2 generations. Three bombardment strategies were compared to determine the best method to generate transgenic plants that express the introduced transgenes and have a simple insertion pattern that would facilitate any downstream breeding. The plasmid bombardment treatments were (1) a six-gene-containing plasmid, (2) an equimolar treatment of five individual plasmids that collectively contained the six transgenes of interest (genes of glufosinate and hygromycin resistance were on the same plasmid), and (3) a 1:9 ratio mixture of the five plasmids, in which the plasmid containing the selectable marker used in the regeneration process, hygromycin resistance, was used in ninefold excess to all the other plasmids. Of the six bombardments performed per plasmid treatment, the results of seven independent events for the six-gene plasmid, four events for the 1:9 treatment, and a single regenerated event for the equimolar treatment indicate that containing all the transgenes on one plasmid just had an advantage in terms of frequency of a successful transformation events. Based on Southern analysis, the only events that contained all six transgenes was the one obtained by the equimolar treatment. No event was obtained that expressed all six transgenes, and certain transgenes seem to be non-randomly lost, namely gusA, bleomycin resistance, and glufosinate resistance, regardless of treatment. The addition of elements to optimize the expression of each gene cassette when multiple genes are in close proximity needs to be further investigated.

Aflatoxin-free transgenic maize using host-induced gene silencing

Suppression of an Aspergillus biosynthetic enzyme effectively alleviates aflatoxin production in transgenic maize kernels. Aflatoxins, toxic secondary metabolites produced by some Aspergillus species, are a universal agricultural economic problem and a critical health issue. Despite decades of control efforts, aflatoxin contamination is responsible for a global loss of millions of tons of crops each year. We show that host-induced gene silencing is an effective method for eliminating this toxin in transgenic maize. We transformed maize plants with a kernel-specific RNA interference (RNAi) gene cassette targeting the aflC gene, which encodes an enzyme in the Aspergillus aflatoxin biosynthetic pathway. After pathogen infection, aflatoxin could not be detected in kernels from these RNAi transgenic maize plants, while toxin loads reached thousands of parts per billion in nontransgenic control kernels. A comparison of transcripts in developing aflatoxin-free transgenic kernels with those from nontransgenic kernels showed no significant differences between these two groups. These results demonstrate that small interfering RNA molecules can be used to silence aflatoxin biosynthesis in maize, providing an attractive and precise engineering strategy that could also be extended to other crops to improve food security.

Reduction of protease inhibitor activity by expression of a mutant Bowman-Birk gene in soybean seed

A mutant Bowman-Birk gene was created that encoded an inactive high-sulfur product. It was used to transform soybean line Asgrow 3237. Transformants bearing the mutant gene were identified by GUS expression, PCR analysis, and Southern analysis. The amount of steady state mRNA from the mutant gene in the transformed plants showed that the gene was highly expressed, but the amount of message from the unmodified Bowman-Birk gene did not change detectably. Proteins synthesized at the direction of the mutant Bowman-Birk gene accumulated in seeds of the transformed plants, and there was a marked decrease in the ability of extracts prepared from these seeds to inhibit trypsin and chymotrypsin despite the presence of Kunitz trypsin inhibitor. The more prevalent mRNA from the mutant gene was considered to out-compete message from the native genes to decrease the amount of active Bowman-Birk inhibitor.

ARTIFICIAL GENE-CLUSTERS ENGINEERED INTO PLANTS USING A VECTOR SYSTEM BASED ON INTRON- AND INTEIN-ENCODED ENDONUCLEASES

SummaryThe ability to create artificial gene-clusters for genetic transformation could facilitate the development of crops with multiple engineered traist, or with traits which result from the expression of multiple genes. A simple method to assemble artificial gene-clusters was developed by designing a multiple cloning site consisting of an array of homing endonuclease cleavage sites into a single vector. These enzymes are also known as intron-or intein-encoded endonucleases, and have very long recognition sequences, which makes them very rare cutters. The resulting vectors are pUGA for microprojectile-mediated transformation, and pUGA2 for Agrobacterium-mediated transformation. In addition, a series of unidirectional shuttle vectors containing various combinations of homing endonuclease restriction sites was constructed. Gene cassettes can be cloned into individual shuttles, and then transferred to either pUGA or pUGA2 to construct artificial gene-clusters. To test the feasibility of this approach, a six-gene cluster was constructed and transformed into soybean via microprojectile bombardment and into tobacco via Agrobacterium. The genes were assayed for expression in both the T0 and T1 generations for three independent transgenics. Up to five of the six genes were expressed. Additional changes to the construction of individual gene cassettes may improve the frequency with which all genes in the cluster are expressed.

Adaptation of an ecdysone-based genetic switch for transgene expression in soybean seeds.

Soybean was used as a model for studies of chemical induction of gene expression in seeds. A chimeric transcriptional activator, VGE, driven by the soybean seed glycinin G1 promoter, was used to induce the expression of an ER-targeted GFPKDEL reporter protein upon addition of the chemical ligand, methoxyfenozide. The chemical gene switch activated gene expression under in vitro conditions in somatic cotyledonary embryos and zygotic seed embryos cultured from transgenic soybean plants, as well as in seeds in planta under greenhouse conditions. The efficiency of induction of GFP expression under different growth conditions was strongly influenced by the developmental stage of the seed and availability of the inducer. The formation of ER-derived GFP-containing protein bodies in seed storage parenchyma cells was correlated with the level of induced expression.

Transgenic Soybean Production of Bioactive Human Epidermal Growth Factor (EGF)

Necrotizing enterocolitis (NEC) is a devastating condition of premature infants that results from the gut microbiome invading immature intestinal tissues. This results in a life-threatening disease that is frequently treated with the surgical removal of diseased and dead tissues. Epidermal growth factor (EGF), typically found in bodily fluids, such as amniotic fluid, salvia and mother’s breast milk, is an intestinotrophic growth factor and may reduce the onset of NEC in premature infants. We have produced human EGF in soybean seeds to levels biologically relevant and demonstrated its comparable activity to commercially available EGF. Transgenic soybean seeds expressing a seed-specific codon optimized gene encoding of the human EGF protein with an added ER signal tag at the N’ terminal were produced. Seven independent lines were grown to homozygous and found to accumulate a range of 6.7 +/- 3.1 to 129.0 +/- 36.7 μg EGF/g of dry soybean seed. Proteomic and immunoblot analysis indicates that the inserted EGF is the same as the human EGF protein. Phosphorylation and immunohistochemical assays on the EGF receptor in HeLa cells indicate the EGF protein produced in soybean seed is bioactive and comparable to commercially available human EGF. This work demonstrates the feasibility of using soybean seeds as a biofactory to produce therapeutic agents in a soymilk delivery platform.