الفهرس 
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Abstract
This study was carried out in the laboratories of the Genetics Dept., Fac. of Agric., Ain Shams Univ. and Ain Shams Center for Genetic Engineering and Biotechnology (ASCGEB) during the period from 2015 to 2020.
This study aimed at testing the potential effect of salinity stress on retrotransposons movement; study the effect of this retrotransposition on coding regions and to study the effectiveness of two PCR-techniques such as IRAP and SCoT in detection retrotransposition with their effect on several coding regions. Also to study the effectiveness of semi-quantitative analysis to detect if the retrotransposition occurs due to salinity stress. This purpose was achieved by comparing between the results obtained from the TY1B and Actin primer
Transposons which are also known as ”transposable elements” are specific sequences found in the genomes. Retrotransposons; type I of transposons comprise the major part of eukaryotic genomes. They have the ability for replicating themselves through RNA intermediate via reverse transcription process. These elements are not always active but during normal development they become quiescent, and are stimulated by several stresses.
In this study, the possibility of PCR-based techniques; IRAP, SCoT and semi-quantitative to detect the variation in retrotransposition rate due to salinity stress was tested. IRAP, SCoT markers were applied in two salinity-tolerant eukaryotic genomes: Yeast (Saccharomyces cerevisiae L.); a single cell organism and Barley (Hordeum vulgare L.) as a higher organism. While semi-quantitative analysis for a partial sequence of reverse transcriptase gene (TY1B) was applied in only the two barley cultivars.
The yeast strain EMCC-49 was grown in pure culture of YPG medium; with 0.5 M, 1 M, 1.5 M NaCl and the control. The barley cultivars were grown in normal conditions for 14 days then treated with high concentration of NaCl salt for nine days where cultivars were irrigated with 0.25 M, 0.6 M NaCl or just distilled water (control). After that the DNA of the yeast strain and two barley cultivars Giza-123 and Giza-2000 were extracted. The results were as follows:-
1. Five IRAP primers (5’LTR, Sukkula and Wltr2105 for barley cultivars, ScM1 and ScM2 for yeast strain) with two combinations (5’LTR- Sukkula in barley and ScM1-ScM2 in yeast).
2. IRAP technique developed three markers in the yeast under the different levels of salinity. ScM1 IRAP primer showed a band with MS of 456 bp in yeast under 0.5 and 1.5 M only. Another band with molecular size of 409 bp appeared under the control and disappeared in all salinity treatments. The third IRAP marker was shown by the ScM2 primer with molecular size of 1952 bp under the 0.5 M treatment.
3. Two IRAP markers appeared in barley due to salinity stress. The 5’LTR IRAP primer showed an 886 bp band in the barley cultivar Giza-2000 under the control condition only. Sukkula IRAP primer displayed the second IRAP marker in the cultivar Giza-2000 of barley with molecular size of 330 bp under the 0.6 M only.
4. Nine SCoT primers (SCoT 5, SCoT 7, SCoT 18, SCoT 22, SCoT 26, SCoT 31, SCoT 32, SCoT 34 and SCoT35) were applied.5. SCoT results for yeast showed that eight from nine primers gave different bands between the control and treatments. The number of polymorphic bands ranged from five different marker bands with SCoT-26 (the highest number of markers per SCoT primer) to only one different band in SCoT-31, SCoT-32 and SCoT-34. While in barley, seven primers gave different bands between them. The number of polymorphic bands ranged from five bands with SCoT-32 to only one different band in SCoT-5, SCoT-31 and SCoT-35; SCoT-35 didn’t show any different bands in Giza-123 between the control and treatments but showed only one band in Giza 2000.
6. SCoT primer gave 17 markers in response of salinity stress in yeast with molecular sizes ranged from 1911 to 271 bp with SCoT 31 and SCoT 26 primers, respectively, for example SCoT 26 exhibited bands with MS of 814, 423, 350 bp were found in both the control and 1.5 M NaCl. While there is band with MS of 321 bp that found only in 1.5 M whereas, one band with MS of 271 bp appeared only in the control.
7. In barley, 18 SCoT markers were detected under salinity stress. They MS were between 1762 (SCoT-26) and 281 bp (SCoT-07), for example SCoT-26 primer exhibited two different bands with Giza 123 with MS of 1762 and 1594 bp, where the first band appeared in both 0.25 M and 0.6 M, the second band was found only at 0.25 M NaCl while it was absent in both the control and other treatment.
8. The results showed different patterns between the control and treatments and the high levels of salinity led to new retrotransposition. Also these results revealed the potential effect of retrotransposition on other coding regions.
9. Semi-quantitative analysis gave positive result where the banding patterns obtained with actin primer as housekeeping gene were the same in the control and all treatments in Giza 123 and Giza 2000.
10. The specific primer for TY1B gene gave difference in intensity of bands between the control and treatments. In Giza 123, the band in 0.25 M NaCl (T1) had low intensity compared with the control. Giza 2000 showed different result where the intensity of the band was the same in the control and 0.25 M NaCl (T1) while was high in 0.6 M NaCl (T2) treatment.
This study was applied to detect the activity of retrotransposition under salinity stress in yeast and barley. The obtained data indicated that not only real time PCR technique can detect the retrotransposition due to different stresses but other techniques can be used if it is easy, fast, cheap and have effectiveness for detect retrotransposition, like IRAP and SCoT-PCR techniques. The two techniques achieved these purposes and gave good positive results to be recommended for different purposes. Semi-quantitative analysis of DNA gave positive results for confirmation of the activation of retrotransposition due to the salinity stress in barley cultivars where semi-quantitative analysis based on the detection of DNA concentration of the PCR reaction.
Scientific addition for this study, the possibility of using the IRAP-PCR, SCoT-PCR and semi-quantitative molecular techniques to study the effect of environmental stresses on retrotransposons activation and determine the effect of this activation on the encoded regions of the genome in eukaryotic organisms (barley and yeast).
Salinity stress as an environmental stress played a central role in activation of retrotransposons, the sensitive plant exhibited retrotransposition rate more than the tolerant plant.