الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
This part of this experiment was carried out in the laboratory of genetic and genetic engineering department, Faculty of Agriculture, Moshtohor, Benha University, and Molecular Genetics and Genome Mapping Laboratory (MGGM) at the Agriculture Genetic Engineering Research Institute (AGERI), Giza, Egypt.
Branched DNA (bDNA)is a novel technique used in genetic diagnosis of the important crop diseases. We used Ralstonia solanacearum isolates to synthesize branched DNA. Bacterial samples were collected from infected tubers, DNA was isolated from two isolates (virulence and avirulence). Two selected genes, Hrp and RS600, were amplified by PCR technique. The amplified PCR fragments for the two studied genes from the DNA template of both bacterial isolates were sequenced and registered in the NCBI database under accession numbers (PP195597, PP195598 for Hrp gene and PP214957, PP214958 for RS600 gene from virulence and avirulence bacterial isolates, respectively. Hrp gene encoding for type III secretion machinery, have been shown to be key determinants for pathogenicity in the vascular phytopathogenic bacterium ralstonia solanacearum and RS600 gene is a sequence within Hrp genes of R. solanacearum. These gene sequences were planned to be used to form branched DNA as a template for the diagnosis of different crop diseases. Sequencing results confirmed that all obtained DNA sequences were not appropriate for designing branched DNA (bDNA) which led us to search for an alternative sequence deposited in the NCBI database suitable for forming bDNA to achieve the aims of this work. Therefore, by using in-silico approach, branches Y and X of DNA in 3D mode using bioinformatics retainer databases were formed by software comprising Maya, bio-blender, and discovery studio (DS) software that can produce 3D models. The selected sequences were (Botrytis cinerea strain Sl3 chromosome 01, CP080979, consisting of 4 million nucleotides). These models, bDNA, were used in molecular diagnostic assays for many pathogens through the detection of gene expressions which are sensitive, specific, and reliable tools in the detection of virulence genes. Each branch binds to a specific primer which is designed and used in the detection of diseases such as fusarium oxysporum, phytphthora fragariae, and botrytis cinerea. This platform not only offers a means for screening the potential activity of molecular diagnostics but also presents opportunities for time and cost savings.
1-Collection of bacterial samples.
The samples of bacteria were isolated (two isolates of bacteria one virulent and avirulent) from infected potato tubers and then transplanted on Petri dishes containing nutrient agar and Tween B medium as a selective medium triphenyl tetra-zolium chloride (TTC). For 48–72 hours, all infected plates were incubated at 28°C. Re-streaking on the same medium allowed for the purification of some of the dominating colonies. Then bacteria were activated in liquid media called Luria Bertani broth (LB).
2-DNA Extraction.
DNA was isolated by using CTAB Bacterial DNA isolation protocol and determine the concentration of DNA, the DNA fluorescence level sample is compared to the different bands of DNA size markers.
3- Polymerase Chain Reaction PCR Protocol
PCR amplification was carried out using a Perkinelmer/Gene Amp® PCR system 9700 (PE Applied Biosystems) for two samples one virulent bacteria and avirulant by using three primers. Detection of PCR products was accomplished by using electrophoresis to separate PCR products on 1.5% agarose gels in 1X TBE buffer at 92 volts along with ethidium bromide (0.5 μg/ml). PCR results were photographed using a gel documentation system (BIO-RAD 2000) and seen under UV light.0
4-Cloning and Sequencing
The resulting DNA amplicons were eluted from agarose gel and purified using the QIAquick Gel Extraction Kit. The purified PCR fragments were ligated into pGEMR-T Easy Vector Systems according to its manufacturer. The competent cells of E. coli top 10 strain were prepared and transformed as described by (Inoue et al., 1990). from LB/Amp/Xgal plates, white colonies were selected and inoculated on LB/Amp broth media.
Then it was incubated overnight at 33 oC with shaking to stabilize the plasmid inside the transformed cells. The alkaline method of Birnboim and Doly (Bimboim and Doly, 1979) was used to isolate the plasmid. To confirm the recombinant plasmids the purified plasmids were examined by electrophoresis on 1.5% agarose gel. Macrogen Company (South Korea) sequenced the obtained recombinant plasmids. The obtained sequence for Hrp and RS600 from both virulence and avirulence bacterial isolates were examined for vector contamination using the VecScreen tool (http:// www.ncbi.nlm.nih.gov/tools/vecscreen). The obtained sequences were deposited at the NCBI database (http://www.ncbi.nlm.nih.gov) under accession numbers PP195597 and PP195598 virulence and avirulence Hrp, and PP214957 and PP214958 virulence and avirulence RS600 genes respectively.
5- Bioinformatics analysis of Hrp and RS600 sequences.
BLAST, SNPs, Gaps, Phylogenetic tree, protein SNPs and DNA comparison analysis were used for studying the genetic fidelity of Hrp and RS600 genes with other sequences of the same genes on database.
6- In-silico method.
6.1- Genetics and Network-based methods in-silico.
Branched DNA is a structure which consists of multiple DNA strands emanating from the point of branching; we designed by using in-silico method. (In-silico refers to computational models that investigate pharmacological hypotheses using methods such as databases, data analysis tools, data mining, homology models, machine learning, pharmacophores, quantitative structure-activity relationships, and network analysis tools)
6.2- DNA sequences and Databases.
First, search for the gene used in the formation of branched DNA and its shapes done. The most desirable sequence was found in NCBI database. Botrytis cinerea has been selected as a source of DNA. By using bioinformatics tools, we select Botrytis cinerea strain Sl3 chromosome 01 (CP080979) sequence which consists of 4 million nucleotide.
Sequences chosen for the Y-shaped branched DNA structures were selected based on their complementarity in a predetermined manner. Specifically, three sequences, each comprising 38 nucleotides, were identified, and retrieved from the NCBI database.
For the synthesis of X-shaped branched DNA, four sequences possessing specific characteristics were required. Palindromic sequences, essential for X-shaped branched DNA formation, were identified using the Palindromic Sequences Finder tool available at novoprolabs.com.
6.3- 3D Software for bDNA modelling
We used programs to create 3d bDNA shapes (Y and X shape) these programs are Blender and bio blender, Maya group software, Discovery Studio.
7- Diagnosis.
7.1- selection of crop.
We select strawberry crop due to its nutritional properties and suggested health advantages Strawberries are a highly sought-after fruit whose production has expanded over the past several decades.
7.2- Common disease.
There are many common diseases, but we focused on these three diseases. These diseases are wilting diseases (fusarium oxysporum); grey mold (botrytis cinerea); red stele or red core (phytphthora fragaria).
from each disease we select virulence gene, SIX8 (HQ260604.1) from fusarium oxysporum, BchX (XM024694372.1) from botrytis cinerea, and RxLR (MK530529.1) from phytphthora fragaria. (Home - Gene - NCBI (nih.gov))
7.3- Primer design.
Primers (forward and reverse) have been designed by using the NCBI database (primer blast/ Primer designing tool (nih.gov). primers will bind to the 5’ end of each branch of Y-shape bDNA. By using Single-stranded overhang sequences for ligation.
8- Diagnosis process.
The thermostable branching DNA nanostructures bind with the target genes of the diseases. Resulting in a “dumbbell-shaped” product