الفهرس 
REVIEW OF LITERATUREOnly 14 pages are availabe for public view
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Abstract
The main aim of the present study is the construction and production of long dsRNA invitro via using bacteria, to trigger plant defense mechanism against viral diseases and increase dsRNA stability via binding with chitosan nanoparticles.
RNAi is a powerful tool for investigating gene function and regulation, with potential applications in molecular biology, genetics, medicine, and agriculture. Synthetic dsRNA molecules can be introduced into plants through exogenous application methods such as spraying, infiltration, injection, spreading, mechanical inoculation, and root/seed soaking. Optimization of the concentration and delivery method is critical to achieving efficient RNAi induction while avoiding non-specific effects.
Plasmids T777T and pYL192 (TRV1) were used to choose restriction enzymes for experiment. To construct expressed plasmid. Followed by transformation in E. coli expression strain HT115. Blue- white screening was used to select colonies followed by colony PCR reaction to obtain target cells, optimizing production condition using two types of media (LB and 2xYT) different concentration of IPTG (0, 0.25, 0.5, 0.75, 1) mM, and incubation time (3, 6, 9, 12, 15, 18). using LB media with 0.25 mM IPTG, and incubation time 18 h was the best condition of production.
Total dsRNA was extracted using the phenol/chloroform method. The dsRNA concentration was measured using a spectrophotometer. The highest concentration was achieved using LB media with 0.25 mM IPTG, and incubation time 18 h using LB media with 0.25 mM IPTG, and incubation time 18 h.
To confirm its stability, it was treated with DNAase and RNAase to degrade any contaminating DNA and confirm that only double-stranded RNA was present. This is an important step in the extraction and confirmation of dsRNA stability, as it ensures only double-stranded RNA is present in the sample.
Chitosan has been explored as a potential antiviral agent in plant virology, which can induce systemic resistance against viral infections. RNAi induction is a valuable tool for plant genetic engineering.
Chitosan samples molecular weight were determined using viscometry method. Sample was 935 KDa, which located in high molecular weight. Titration method was used to determine the degree of deacetylation (DDA) 58.64%.
Chitosan/dsRNA complex retention was studied on agarose gel electrophoresis. It was found that changing chitosan concentrations and keeping the dsRNA concentration constant varied the +/ratio particle formulations. The dsRNA binding is dependent on both the molecular weight and the degree of deacetylation of the chitosan used.
Chitosan has been studied for its antimicrobial effect on Agrobacterium. A lethal effect of CNP on Agrobacterium tumefaciens GV3101 cells was studied. Results showed that CNP reduced the bacterial population as the concentration increased, with a lethal effect of 44.34%.
Agroinfiltration and dsRNA delivery are promising plant genetic engineering techniques due to their potential efficiency in modifying gene expression. Agroinfiltration involves the delivery of foreign DNA into plant cells by infiltrating leaf tissue with an Agrobacterium intermediate host containing expression vectors. Agrobacterium tumefaciens mediated gene delivery is the preferred method in agroinfiltration and can be introduced into the plant leaf through direct injection using system using siring. Treatments with agroinfiltration and dsRNA were injected to study the effect on plant morphological level, pigments analysis, and qPCR for the PDS gene.
Pigments analysis is in the same manner to morphological effects, with the lowest amount of pigments in T1, and the highest amount in T8. Treatments before viral infection within 15 days with dsRNA naked or complex had a significant effect on plant growth and stability of dsRNA.