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Gene silencing of Diatraea saccharalis using the interference RNA technique (RNAi)

Grant number: 17/17882-7
Support Opportunities:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): December 01, 2017
Effective date (End): December 31, 2021
Field of knowledge:Biological Sciences - Genetics - Molecular Genetics and Genetics of Microorganisms
Principal Investigator:Antonio Vargas de Oliveira Figueira
Grantee:Bruna Garbatti Factor
Host Institution: Escola Superior de Agricultura Luiz de Queiroz (ESALQ). Universidade de São Paulo (USP). Piracicaba , SP, Brazil

Abstract

The caterpillar Diatraea saccharalis (Lepidoptera: Crambidae) is the most important insect-pest of sugarcane in Brazil. The larval stage of the insect causes high damages, since it develops inside the stems, hindering its control. In this sense, a promising control strategy has been the use of gene silencing of the target insect through the interfering RNA technique (RNAi). The efficiency of RNAi for insect-pest control varies depending on the target species, the mode of delivery of the dsRNA and the target genes to be silenced. The potential delivery mode of double stranded RNA (dsRNA) to agricultural pests includes the production of transgenic plants expressing inverted versions (hairpin) of target insect genes and spraying of synthetic dsRNA molecules. Another alternative to consider would be the use of transformed bacteria expressing dsRNA because they present lower costs, they allow large-scale production, and still, protect the dsRNA molecules from degradation, thus contributing to greater efficiency of the technique. In this context, genetically modified endophytic bactéria can also offer potential to colonize the host and produce dsRNA to control insect pests. Therefore, the objective of this project will be to evaluate the use of bacteria, Escherichia coli HT115 (DE3) and the endophytic Pantoea agglomerans 33.1 as a form of delivery of dsRNA corresponding to insect target genes, aiming to validate the use of these transformed bacteria expressing dsRNA by the technique of RNAi for the control of Diatraea saccharalis. dsRNA expression vectors of the target genes of D. saccharalis will be constructed using plasmid L4440, which will be used to transform the two bacterial species. The transformed bacteria expressing dsRNA will be supplied via artificial diet to D. saccharalis caterpillars in bioassays whose parameters will be optimized. In order to increase the efficiency of the technique, the knockout of the gene encoding the RNaseIII enzyme of P. agglomerans 33.1 will be sought, using the genomic-editing technique by CRISPR-Cas9 with the purpose of accumulating a higher concentration of dsRNA to be supplied to the insect. With the results obtained, it is expected to observe the potential of the use of the genetically modified bacteria, E. coli HT115 (DE3) and the endophytic P. agglomerans 33.1, for gene silencing and control of D. saccharalis via RNAi. Finally, the genes that present the best results in the bacterial assays will be selected for use in transgenic sugarcane "RNAi" constructs. (AU)

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Academic Publications
(References retrieved automatically from State of São Paulo Research Institutions)
FACTOR, Bruna Garbatti. Gene silencing of Diatraea saccharalis using an RNA interference (RNAi) technique. 2022. Doctoral Thesis - Universidade de São Paulo (USP). Escola Superior de Agricultura Luiz de Queiroz (ESALA/BC) Piracicaba.

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