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Large-scale production and evaluation of marker-free indica rice IR64 expressing phytoferritin genes.

ABSTRACT: Biofortification of rice (Oryza sativa L.) using a transgenic approach to increase the amount of iron in the grain is proposed as a low-cost, reliable, and sustainable solution to help developing countries combat anemia. In this study, we generated and evaluated a large number of rice or soybean ferritin over-accumulators in rice mega-variety IR64, including marker-free events, by introducing soybean or rice ferritin genes into the endosperm for product development. Accumulation of the protein was confirmed by ELISA, in situ immunological detection, and Western blotting. As much as a 37- and 19-fold increase in the expression of ferritin gene in single and co-transformed plants, respectively, and a 3.4-fold increase in Fe content in the grain over the IR64 wild type was achieved using this approach. Agronomic characteristics of a total of 1,860 progenies from 58 IR64 single independent transgenic events and 768 progenies from 27 marker-free transgenic events were evaluated and most trait characteristics did not show a penalty. Grain quality evaluation of high-Fe IR64 transgenic events showed quality similar to that of the wild-type IR64. To understand the effect of transgenes on iron homeostasis, transcript analysis was conducted on a subset of genes involved in iron uptake and loading. Gene expression of the exogenous ferritin gene in grain correlates with protein accumulation and iron concentration. The expression of NAS2 and NAS3 metal transporters increased during the grain milky stage.

SUBMITTER: Oliva N

PROVIDER: S-EPMC3890568 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCellular reprogramming in response to environmental stress involves alteration of gene expression, changes in the protein and metabolite profile for ensuring better stress management in plants. Similar to other plant species originating in tropical and sub-tropical areas, indica rice is highly sensitive to low temperature that adversely affects its growth and grain productivity. Substantial work has been done to understand cold induced changes in gene expression in rice plants. However, adequate information is not available for early gene expression, especially in indica variety. Therefore, a transcriptome profile was generated for cold shock treated seedlings of IR64 variety to identify early responsive genes.ResultsThe functional annotation of early DEGs shows enrichment of genes involved in altered membrane rigidity and electrolytic leakage, the onset of calcium signaling, ROS generation and activation of stress responsive transcription factors in IR64. Gene regulatory network suggests that cold shock induced Ca2+ signaling activates DREB/CBF pathway and other groups of transcription factors such as MYB, NAC and ZFP; for activating various cold-responsive genes. The analysis also indicates that cold induced signaling proteins like RLKs, RLCKs, CDPKs and MAPKK and ROS signaling proteins. Further, several late-embryogenesis-abundant (LEA), dehydrins and low temperature-induced-genes were upregulated under early cold shock condition, indicating the onset of water-deficit conditions. Expression profiling in different high yielding cultivars shows high expression of cold-responsive genes in Heera and CB1 indica varieties. These varieties show low levels of cold induced ROS production, electrolytic leakage and high germination rate post-cold stress, compared to IR36 and IR64. Collectively, these results suggest that these varieties may have improved adaptability to cold stress.ConclusionsThe results of this study provide insights about early responsive events in Oryza sativa l.ssp. indica cv IR64 in response to cold stress. Our data shows the onset of cold response is associated with upregulation of stress responsive TFs, hydrophilic proteins and signaling molecules, whereas, the genes coding for cellular biosynthetic enzymes, cell cycle control and growth-related TFs are downregulated. This study reports that the generation of ROS is integral to the early response to trigger the ROS mediated signaling events during later stages.

Project description:The presence of antibiotic resistance and other marker genes in genetically modified plants causes concern in society because of perceived risks for the environment and human health. The creation of transgenic plants that do not contain foreign genetic material, especially that of bacterial and viral origin, largely alleviates the tension and makes the plants potentially more attractive for consumers. To produce marker-free transgenic apple plants, we used the pMF1 vector, which combines Zygosaccharomyces rouxii recombinaseR and a CodA-nptII bifunctional selectable gene. The thaumatin II gene from the tropical plant Thaumatococcus daniellii, which is under the control of the plant E8 gene (a predominantly fruit-specific promoter) and rbsS3A terminator, was taken as the gene of interest for modification of the fruit taste and enhancing its sweetness. Exploitation of this gene in our laboratory has allowed enhancing the sweetness, as well as improving the taste characteristics, of fruits and vegetables of plants such as strawberry, carrot, tomato and pear. We have obtained three independent transgenic apple lines that have been analyzed by PCR and Southern blot analyses for the presence of T-DNA sequences. Two of them contained a partial sequence of the T-DNA. With one line containing the full insert we then used a delayed strategy for the selection of marker-free plants. After induction of recombinase activity in leaf explants on selective media with 5-fluorocytosine (5-FC) we obtained more than 30 sublines, most of which lost their resistance to kanamycin. Most of the apple sublines showed the expression of the supersweet protein gene in a wide range of levels as detected by RNA accumulation. The plants from the group with the highest transcript level were propagated and grafted onto dwarf rootstocks for early fruit production for future estimates of protein levels and organoleptic analyses. Thus, we developed a protocol that allowed the production of marker-free apple plants expressing the supersweet protein.

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Large-scale production and evaluation of marker-free indica rice IR64 expressing phytoferritin genes.

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