NANO ENCAPSULATION OF dsRNA OF METAMORPHOSIS RELATED GENE AND ITS INSECTICIDAL POTENTIAL AGAINST DIAMOND BACK MOTH.
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Date
2022-03-31
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Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola, Maharashtra.
Abstract
The present investigation entitled “Nano encapsulation of dsRNA of metamorphosis related gene and its insecticidal potential against diamond back moth” was carried out at Biotechnology Centre, Department of Agricultural Botany, Post Graduate Institute, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola during the academic year 2019-2021.
RNA interference (RNAi) technology post transcriptional mechanism triggerd by dsRNA to silence specific genes by down regulating their expression. It has become a potential strategy for functional and regulatory studies of insect genes and has potential to control insect pest. Though it has been challenging to produce effective RNAi in lepidopteran Diamond back moth (P. xylostella). Some important factors significantly influencing the silencing potential effects are concentration and length of dsRNA, nucleotide sequence, and determination of the silencing effect and life stage of the target organism. The P.xylostella shows RNAi effects after feeding of nanoencapsulated dsRNA. Considering the potential impact on the normal physiological functions of healthy adults, Juvenile Hormone Esterase gene select as targets for dsRNA mediated gene silencing. Juvenile Hormone Esterase gene sequence obtained from NCBI database. Off targets were predicted to avoid non target effects. Possible RNAi sites were depicted to identify the region having maximum RNAi targets.
The aim of studies was to prepared PLGA nanoparticles, because of their possibility to target specific cells with high biodegradability and biocompatibility. It is approved for human use by the Food and Drug Administration. Here, we describes in great details about the formation and characterization of PLGA nanoparticles. Poly (lactic-co-glycolic acid) nanoparticles synthesized using double emulsification method. PVA (poly vinyl alcohol) used as stabilizer during the synthesis of PLGA nanoparticles. This protocol can be readily adapted to use alternative emulsifiers PVA (e.g. Poly vinyl alcohol) or solvent (e.g. methanol).
A typical peak at 380nm having 0.56 absorbance on UV- visible spectroscopy indicates synthesis of PLGA nanoparticles. Particle size and zeta potential are determined with Nanoparticle size analyzer and Zeta potential analyzer. The PLGA nanoparticles characterized during present study showed 244.9 nm particle sizes and -4.59 mV zeta potential. FTIR analysis, carried out in wave range 1000- 4000 cm-1, showed different functional groups like 3884 cm-1 and 3410 cm-1 (Alcohol), 1780 cm-1 (Carbonyl group), 1494 cm-1 and 1666 cm-1 (Glycolic acid) 1382 cm-1, and 1187 cm-1 (Esters), 1472 cm-1 and 1096 cm-1(Vinyl) in PLGA NPs synthesis.
The process of encapsulation of dsRNA with PLGA NPs done to increase the insecticidal potential of dsRNA-JHE(A) and dsRNA-JHE(B). The encapsulated nanoparticles were also characterized by UV spectrophotometry, particle size was determined with Nanoparticle size analyzer and Zeta potential analyzer. We provide representative images for nanoparticles produced.
Effect of dsRNA and encapsulated dsRNA with PLGA NPs was checked out by performing insect bioassay studies on P. xylostella larvae. The dsRNAs and encapsulated dsRNA with PLGANPs were individually spread on to the cabbage leaf discs and twelve larvae were released into 3 replication. We detected that encapsulated dsRNA-JHE(A) and encapsulated dsRNA-JHE(B) which had the highest mortality 60% and 72% as compared to control 0% (only water) and dsRNA 36% JHE(A) and 48% JHE(B).
These findings largely broaden the target selection for RNAi-based technology and deliver dsRNA into the insect and potential against insect damage and insect control management.
Description
RNA interference (RNAi) is a mechanism through which small interfering RNA (siRNA) induces sequence-specific posttranscriptional gene silencing. RNAi is commonly recognized as a powerful tool not only for functional genomics but also for therapeutic applications. Juvenile hormone (JH) plays a crucial role in preventing precocious metamorphosis and stimulating reproduction. Thus, its hemolymph titer should be under a tight control. As a negative controller, juvenile hormone esterase (JHE) performs a rapid breakdown of residual JH in the hemolymph during last instar to induce a larval-to-pupal metamorphosis.
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Citation
PIMPALZARE, PRANALI SUBHASHRAO. (2021). Nano encapsulation of dsRNA of metamorphosis related gene and its insecticidal potential against diamond back moth. Biotechnology Centre, Department of Agricultural Botany. Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola. M. Sc. 2021. Print. xiii, 88p. (Unpublished)