الفهرس 
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Abstract
The major aim of this work is to employ some defense genes against CLCV disease in some Egyptian cotton (G. barbadense) cultivars in Lower Egypt (El Beheira and El Gharbeya), through manipulation of the chitinases and β-1,3-glucanases genes, which can be a defense line in the cotton plant against CLCV disease. Furthermore, to explore the genetic relationship between the different G. barbadense cultivars and the two ancestor species; G. arboreum and G. raimondaii.
Results:
1. Integrity of the genomic RNA was examined by amplifying the 18S rRNA gene. The detection of the expected amplicon for 18S rRNA indicate the integrity of cotton genomic RNA. In other words, the extracted and purified RNA and the reverse transcription reaction into cDNA is intact and suitable for further study.
2. The employment of differential diaplay PCR revealed that the Egyptian cotton cultivars showed different polymorphic and monomorphic genes along the expressed genes in the cotton genome as it is different from one gene to other. Furthermore, these genes were amplified and found in some Egyptian cotton cultivars but not in others.
3. The genetic diversity between the Egyptian cotton cultivars was done based on differential display PCR results using the cDNA it showed high similarity between the healthy cultivars; it was 87 % between (G.77 and G.80) and (G.70 and G.90) and 83 % between (G.80 and G.86). In the infected cultivars with the CLCV disease it gave 87 % between (G.80 and G.86) and 83 % between (G.67 and G.70), (G.70 and G.90) and (G.76 and G.80).
4. The phylogenetic relationship between the cotton cultivars was done based on the genetic diversity as it was 0.25 between the healthy cultivars. It also reached 0.225 between the infected cultivars. That means the different cotton cultivars came from the same ancestor which is the Gossypium spp.
5. Partial sequencing of the β-1,3-glucanase gene sequence was recorded in the GeneBank as defense_related_gene with accession number JQ307153.
6. Specific primers for the defense genes in plants were produced an amplicon for the chitinases gene it was at molecular weight 320 bp in the Egyptian cotton cultivars and their progenitors. While it was at molecular weight 350 bp with the β-1,3-glucanases gene.
7. Gene expression in the Egyptian cotton cultivars by RT-PCR was done based on the results of the specific primers. The chitinases gene showed different levels of expression, the highest level of the gene expression was (2.96) in cultivar G.86 and the least level of the gene expression was (0.78) in cultivar G.70 in the healthy samples. While the level of gene expression in the infected samples with CLCV was higher than the healthy, the highest level of the infected gene expression was (3.4) in cultivar G.86 and the least level of the gene expression was (1.7) in cultivar G.77.
8. The level of gene expression for the β-1,3-glucanases showed different levels of expression. The highest level of gene expression was (1.8) in cultivar G.67 and the least level of the gene expression was (0.19) in cultivar G.77 in the healthy
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samples. While the level of gene expression in the infected samples with CLCV was higher than the healthy, the highest level of the infected gene expression was (2.98) in cultivar G.86 and the least level of the gene expression was (0.38) in cultivar G.76.
9. Chitinase gene expression (gene copy number) in the infected samples showed the highest percent of gene expression was in cultivar G.70 it was (219.23 %) than in the uninfected and the lowest level of percent of gene expression (gene copy number) was in cultivar G.86 it was (14.86 %) than in the uninfected.
10. β-1,3-glucanase gene expression (gene copy number) in the infected samples showed the highest percent of gene expression (gene copy number) was in cultivar G.77 it was (905.26 %) than in the uninfected and the lowest level of gene expression was in cultivar G.67 it was (7.77 %) than in the uninfected.
In conclusion, the current study revealed that the Egyptian cotton cultivars and their progenitors contain chitinases and β-1,3-glucanases genes. As the mean of fold chain in chitinases gene expression in the healthy samples ranged from 0.78 till 2.96 and in the infected samples ranged from 1.54 till 3.4. While the mean of fold chain in β-1,3-glucanases gene expression was in the healthy cultivars ranged from 0.19 till 1.8 and in the infected cultivars ranged from 0.38 till 2.98. That means that the RT-PCR amplification for chitinases and β-1,3-glucanases gene proved that these genes were expressed in all the samples but with different levels of polymorphism. from the obtained result the gene expression was high in the G. barbadense infected cultivars and decreases in the healthy cultivars that means that the infection with the CLCV disease increases the expression of the gene level for the plant to defend itself against the disease.
These results suggested that chitinases and β-1,3-glucanases are standard component of the Gossypium genome. Furthermore, their wide distribution and detection of their encoded transcripts illustrate their activity role in the Gossypium genome and supporting the fact that these sequences represent a major of a plant genome. Further experimental data such as copy number determination and sequencing of large contiguous regions of the Gossypium genome will significantly add up fundamental knowledge about the role of the defense genes in shaping and evolution of the Gossypium genome.
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CHAPTER SIX
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