الفهرس 
REVIEW OF LITERATUREOnly 14 pages are availabe for public view
Abstract
Okra is an edible horticulture plant farmed for pods, leaves, and stems. Famous for its ability to tolerate long desiccation periods. In addition to its susceptibility to eight Begomovirus species, among them are Okra leaf curl virus (OLCV; monopartite), Okra yellow vein mosaic virus (OYVMV; monopartite), and Okra enation leaf curl virus (OELCuV; monopartite), which contribute between 30% and 100% yield losses in okra, particularly under field conditions. Okra belongs to the Malvaceae family and is a sister species to hibiscus, cotton, and cacao plants. However, it is considered an orphan crop in Africa, and little is known about its genetic makeup. The current study evaluated six elite okra lines for drought tolerance and viral infection sensitivity, in order to characterize and define tolerant and sensitive okra lines for further production and to form a core for future breeding programs in okra. furthermore, okra plants were used as a model to sequence, assemble, and analyze the evolutionary and functional characteristics of the infamous Dicer-like protein gene family. To achieve these goals, two main experiments were conducted:
- Part one: the drought tolerance experiment, was as follows:
Plants were grown in a growth chamber in replicates. Seedlings were subjected to drought after 20 days of vegetative growth in pots. After that, a water deficit was made, and irrigation stopped completely. Samples were collected from leaves before (Ctrl) and after 7 and 15 days of water stress. Then, they were isolated from the total RNA, then the NGS technique was applied to make a huge reading for them, and using bioinformatics analysis, the data was analyzed through the DESeq2 plug-in in the Geneious prime program. Which identifies the genes that differentiate between the control and treatment conditions.
Results could be concluded as follow:
1. RNA-Seq-based transcriptome has been successfully used to study differential drought stress gene expression in okra. They consider this the first study on okra that expands the basic understanding of its response to drought tolerance at the transcriptional level.
2. The overall results revealed a series of responses in okra under drought stress. The gene families found to be DEGs were transporter/channel proteins, methyltransferases, genes associated with protein modifications, and genes associated with gene silencing. Hence, it appears that okra uses multiple defense responses both for resistance and sensitivity to drought.
3. Our results showed that lines OEL01, OEL03, and OEL06 were sensitive to desiccation and lack of water, in contrast to lines OEL02, OEL04, and OEL05, which were drought-tolerant. In addition to the genes Sweet17, shspC2, DCL1, 2, and 3 in okra, we can use them as molecular markers for drought resistance.
- Part two: the virus resistance experiment, was as follows:
I: All the lines under study were evaluated for viral infection under the conditions of both the open field (as a natural infection) and the greenhouse (by mechanical inoculation using injection). Samples were collected from both infected and uninfected leaves in order to isolate the DNA, and then NGS technology was applied, and bioinformatics programs were used. The extracted results were analyzed, and through the single contigs approach (which is a collection of long contigs that were formed from short reads) the results were analyzed, search all circular contigs using the BLA ST collection of the local plant virus database, to identify virus-related circular sequences. Accordingly, assembled and reported a new complete OLCV DNA-A genome sequence identified in okra cultivated in the open experimental field, comprising 2764 bp and encoding 6 open reading frames (ORFs) with a GC content of 44.6% and 88.3% similarity to a reported virus previously, reported from Cameroon.
II: It is known that the leaf curl virus in okra is transmitted by the whitefly insect, but according to what was observed of the rugged appearance of viral symptoms on the leaves in the open field (as a natural infection), it was necessary to ascertain the way in which the virus is transmitted other than the insect. Accordingly, the goal was whether the virus was transmitted through seeds or not. Therefore, the qPCR technique was used to detect the presence of the virus in the seeds, and the results confirmed the presence of the virus in large quantities in the internal embryos of the seeds, which confirms that the virus is actually transmitted through the seeds. In addition, as a final result of the experiment, lines OEL02, OEL05, and OEL06 recorded sensitivity to viral infection, while lines OEL01, OEL03, and OEL04 were tolerant to viral infection. As a summary of the two experiments that were presented to evaluate the studied lines for viral infection and drought tolerance, OEL04 was evaluated as a resistant line both to viral infection and drought tolerance. In contrast, OEL06 was evaluated as a sensitive line to viral infection and drought tolerance.
- Part three: DICER-like gene family analysis in Okra.
The study represents a comprehensive genome-wide identification and analysis of DCL gene family in naturally drought-tolerant okra plants, an orphan crop that can be used as a model for further genomic and transcriptomic studies on drought and viral infection tolerance mechanisms in plants.
Four okra DCL single-copied genes were successfully assembled using the DNAseq technique. There were four copies of okra DCL (AeDCL) with 8494, 5214, 4731, and 9329 bp in length, for AeDCL1 to AeDCL4, respectively. Detected exons were one, 18, 29, and 24, for AeDCL3, AeDCL2, AeDCL1, and AeDCL4, respectively. The molecular weight ranged from 225.34 (AeDCL1) to 177.16 kDa (AeDCL3), while AeDCL2 was 137.04 and AeDCL4 was 178.75. The length of amino acids in the protein ranged from 1207, 1576, 1589, and 2008 for AeDCL1, AeDCL2, AeDCL3, and AeDCL4, respectively. A proximate isoelectric point was recorded around 6, where AeDCL1 and AeDCL4 recorded 6.07 and 6.2, respectively, while AeDCL2 was the highest at 6.73 in contrast to AeDCL3 which was the lowest at 5.87.
The results showed that AeDCL proteins have a high hydrophilicity property, and the Okra DCLs genes translate stable proteins, a factor ranging from 40.26 up to 50.44 for AeDCL1 to AeDCL4, ascendingly. All the AeDCLs proteins are subcellular-localized in the cytoplasm, showing a non-secretory nature and that all DCLs carry metabolic activities within the cell.
AeDCLs showed five functional domains of two DEAD-like helicases superfamilies, N and C, one Dicer domain, one Ribonuclease III domain (a-b), and one double-strand RNA-binding domain. Additionally, the PAZ domain was annotated only for AeDCL1 and 3.
Based on the DCLs protein alignment, Okra’s DCLs have followed Cacao in terms of gene copy number and distance compared to three diverse cotton species, apart from AeDCL3, which was part of the DCL3a subcluster.
All AeDCLs were up-regulated under drought conditions, with leaves showing a higher fold change than roots. In leaves, the AeDCL4 was the highest expressed DCL copy after seven days of dehydration and the lowest after 15 days. In okra roots, the AeDCL1 was the highest expressed DCL copy after seven days of dehydration, and the AeDCL3 was the lowest. While under viral infection conditions, our results showed that AeDCL3 leads to higher levels of expression in viral infection compared to AeDCL2 and AeDCL4 which are known for their traditional antiviral functions.