الفهرس 
REVIEW OF LITERATUREOnly 14 pages are availabe for public view
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Abstract
Banana (Musa spp.) is considered one of the most economic important fruit plants in Egypt as well as the tropic regions. Banana bunchy top virus (BBTV) the causal agent of Banana bunchy top disease (BBTD) was recorded to be the most economic impact on banana yield productivity worldwide. This work was designed to determine the complete nucleotide sequence of the circular ss-DNA genome of an Egyptian strain of BBTV. Bioinformatics analyses of the BBTV-DNA components were carried out compared to BBTV overseas strains and other Nanoviruses. The genetic variations of some banana cultivars (BBTV-infected or healthy) was estimated by using different molecular tools.
The experiments that achieve the aim of this study could be summarized as following:
1. Collection of banana plant cultivars exhibited BBTV-like symptoms from different regions in Egypt.
2. Biological characterization of BBTV-diseased banana plants.
3. Extraction and purification of DNA from BBTV-diseased plants.
4. Molecular confirmation of the presence of BBTV via polymerase chain reaction (PCR) using specific primer pair of BBTV-DNA-1 (DNA-R) component.
5. Design of primer pairs specific to the six BBTV components (DNA-R, DNA-U3, DNA-S, DNA-M, DNA-C and DNA-N) for their isolation via PCR.
6. Determination of nucleotide sequences of the six BBTV-DNA components followed by bioinformatics analyses at the level of DNA and deduced amino acids compared to those overseas BBTV strains as well as some Nanoviruses documented in GenBank.
7. Determining the genetic variations of some banana plant cultivars infected with BBTV compared to healthy ones by using different molecular tools were also aimed.
The experimental results of this study could be summarized as following:
1. A number of 15 banana plants belonging to three cultivars (Edward Cavendish, Williams, and Maghrabi, and exhibiting BBTD-like dark green streaks along the small veins on the underside of the leaves, midribs and stalks were collected from different regions of Qalyubia Governorate, Egypt. Banana black aphid (Pentalonia nigronervosa Coquerel), the vector of BBTV was hiding in the sheaths of banana leaves.
2. The total DNA was extracted and purified from collected banana cultivars samples with purity of 1.86 and concentration (87 ng µL-1). The DNA was successfully used as templates for PCR confirmation in the presence of one primer pair targeting BBTV-DNA-1 component (the master replication gene, BBTV-DNA-R, 1.1 Kb). No PCR product(s) was amplified from the healthy banana plant that used as a negative control.
3. The six BBTV-DNA components (DNA-R, DNA-U3, DNA-S, DNA-M, DNA-C and DNA-N) with sizes of about 1.1 Kb were amplified using six primer pairs with lengths ranged from 17 to 26 nucleotides.
4. The complete nucleotide sequences of the six BBTV DNA components were determined and documented in GenBank with accession numbers of LC468138.1 (1109 nts), LC468139.1 (1056 nts), LC468140 (1075 nts), LC468141 (1043 nts), LC468142 (1015 nts), and LC468143 (1043 nts), respectively.
5. Results showed that BBTV strain of this study had a genome that consists of six encapsidated single-stranded DNA components, each with one open reading frame (ORF) in the virion-sense.
6. A conserved stem-loop structure (from nucleotide 1 to 30) was located at the 5’ terminus of the large virion-sense ORF in all BBTV-DNA components.
7. Results also showed that ORF of BBTV-DNA-1 is 837 nts and encodes a rolling-circle replication initiation protein; ORF of BBTV-DNA-2 is 267 nts, and encodes a protein of unknown function; ORF of BBTV-DNA-3 is 528 nts, and encodes a coat protein peptide with estimated molecular mass of 20 kDa; ORF of BBTV-DNA-4 is 309 nts, and encodes the movement protein; ORF of BBTV-DNA-5 is 471 nts, and encodes cell cycle link protein and ORF of BBTV-DNA-6 is 390 nts, encodes a nuclear shuttle protein.
8. Two conserved common regions (CR) of the six BBTV-DNA components were scored. The 1st is called a common region-stem loop (CR-SL) starts from nucleotide number 1 to 44 and the 2nd is called common region-M (RC-M), and divided into three regions named CR-M1, CR-M2 and CR-M3 varied in their presence in the six components.
9. Results showed the presence of TATA box, i.e., nonanucleotide potential TATA and polyadenylation signals adjacent to GC rich regions was located upstream of the major ORFs of these components. It was noted that component-2 (DNA-U3) had a potential TATA box as well as polyadenylation signal location at the expected positions, and encoding a regulatory function of U3 protein.
10. Multiple sequence alignments of the six BBTV-DNA components at the levels of DNA and deduced amino acids sequences were compared to the most similar BBTV documented in GenBank.
11. The six BBTV-DNA components showed identities ranged from 97.39-98.56 % (DNA-R, encoding replication associated protein gene (rep), 92.5-98.85% (DNA-U3, encoding U3 protein gene), 97.18-98.98% (DNA-S, encoding coat protein gene (cp)), 91.71-95.40 (DNA-M, encoding movement protein gene (mv), 96.16- 98.33% (DNA-C, encoding cell-cycle link protein gene (ccl) and 95.03-99.45% (DNA-N, encoding nuclear shuttle protein (ns) gene encoding ) when compared to the most similar oversea BBTV strains.
12. Results showed that numbers of 16, 12, 11, 48, 17 and 6 nucleotide differences, were recorded between the nucleotide sequences of DNA-R (LC468138.1), DNA-U3 (LC468139.1), DNA-S (LC468140.1), DNA-M (LC468141.1), DNA-C (LC468142.1) and DNA-N (LC468143.1) of the current study and the most similar oversea BBTV strains; AF416465.1, LC155097.1, NC_003473.1, NC_003474.1., NC_003477.1 and AY948438.1, respectively) that were fallen into the same clusters when the distance genetic tree was determined.
13. Results of the genetic relationships between the six components of BBTV-DNA of this study and the most similar oversea BBTV strains could be reflecting the strong genetic relationship between the Australian and Rwandan strains, and one of them may have evolved from the same origin.
14. The genetic relationship between the BBTV isolate under investigation and some BBTV isolates belonging to different locations of the some continents (Egypt, Burundi, Congo, Australia, Pakistan, India, Indonesia, Taiwan, Philippines and China) was determined by comparing the BBTV-DNA-1 (DNA-R) component. It was observed that two Egyptian strains were fallen in clusters with the strains of Australia and Taiwan, while strains of Asia continent were found together in subclusters indicating the extent genetic relationship between them.
15. By studying the genetic relationship between the six BBTV components and other virus components in the Nanoviridae family documented in the GenBank, it became clear from the experimental results showed that there is a very high genetic relatedness between BBTV and FBNYV.
16. When the nucleotide sequences of the six components of BBTV were subjected to analysis of their encoded amino acids and their function, results showed CDS translation in reading frames on the direct strand. These ORFs were characterized with; 837 nts/278 aa, extends from base 132 to base 968 of BBTV-DNA-1 (DNA-R) (BBJ34149.1); 267 nts/88 aa extends from base153 to base 419 of BBTV-DNA-2 (DNA-U3) (BBJ34150.1.); 528 nts/175 aa, extends from base 213 to base 740 of BBTV-DNA-3 (DNA-S) (BBJ34151.1); 309 nts/102 aa, extends from base 279 to base 587 of BBTV-DNA-4 (DNA-M) (BBJ34152.1); 471 nts/156 aa, extends from base 240 to base 710 of BBTV-DNA-5 (DNA-C) (BBJ34153.1) and 390 nts/129 aa, extends from base 281 to base 670 of BBTV-DNA-6 (DNA-N) (BBJ34154.1).
17. At the level of deduced amino acids and ORFs, the six BBTV-DNA components showed identities of 99.64-100% (DNA-R, encoding rep, 97.73% (DNA-U3, encoding U3 protein gene), 97.71-99.43% (DNA-S, encoding cp), 98-100% (DNA-M, encoding mv, 98% (DNA-C, encoding ccl and 99.15% (DNA-N, encoding ns gene) when compared to the most similar oversea BBTV strains.
18. Data show that NEBcutter was successfully used in generating the restriction enzymes map of the circular sequence of BBTV-DNA components appearing each of cleavage code and enzyme name code. Both of closest enzymes at 5’ end and 3’ end were occurred. At the level of closest enzymes at 5’ end the restriction sites of HphI, ApoI, BtsIMutI, Fnu4HI, Hpy99I and BccI were detected for the six BBTV-DNA components, receptively. While, the restriction sites of Cac8I, AluI, Hpy166II, DpnII, AccI and AseI were detected as closest enzymes at 3’ end for the same six BBTV-DNA components, respectively.
19. At the level of domains of the six BBTV-DNA components, results showed that the ORF of BBTV-DNA-1 (DNA-R) has two domains, BBTV-DNA-2 (DNA-U3) has a hypothetical protein belonging to un-characterized protein U3 domain, BBTV-DNA-3 (DNA-S) has one domain called nanovirus_coat the protein family membership is Nanovirus coat protein, BBTV-DNA-4 (DNA-M) has one domain Babovirus_MP beginning from 1-101 which is a membership of movement and RNA silencing protein, BBTV-DNA-5 (DNA-C) has cell cycle protein domain belonging to cell cycle link protein Clink and BBTV-DNA-6 (DNA-N) has a domain belonging to nuclear shuttle protein domain.
20. The genetic changes in the genome of banana plants BBTV-infected compared to healthy ones of three banana cultivars (Maghrabi, Edward Cavedish and Williams) grown in the same governorate (Qalyubia) as well as one cultivar (Maghrabi) grown in different locations (Qalyubia, Qena and Aswan) were determined by using some molecular tools.
21. Results showed that the total number of DNA fragments obtained was 70 distributed as follows: 13, 15, 18, 15 and 14 by the five SCo-T primers used. Results showed that when DNA extracts of Maghrabi, Williams and Edward Cavendish banana cultivars were used as templates, numbers of 64, 51 and 52 of DNA fragments were amplified from healthy banana plants, respectively, while the average number of DNA fragments amplified for the same BBTV-infected cultivars was 58.6, 50 and 52, respectively.
22. Regarding the type of the 70 amplified DNA fragments, results showed that DNA fragments were distributed as follows: 33 polymorphic and 37 monomorphic and only two out of the 33 polymorphic fragments were considered as unique DNA markers and were amplified by SCoT-03 and SCoT-22 primers, one fragment for each.
23. Results showed that the percentage of identities between healthy banana plants and those infected with BBTV of the same variety ranged between 91-94, 94-97, and 96-97% in Maghrabi, Williams and Edward Cavendish cultivars, respectively. In other words, the amount of genetic variation in the genome of the same variety whatever was infected with BBTV or healthy was the lowest possible in the Edward Cavendish cultivar (3-4%), followed by Williams cultivar (3-6%) and Maghrabi cultivar (6-9%), and as a conclusion, Edward Cavendish cultivar is closer in its genetic origin to the Williams cultivar compared to the Maghrabi cultivar.
24. When the genetic variation of Magharbi banana cultivar grown in different climate governorates was estimated by using SCoT technique, results showed that a number of 72 DNA fragments were amplified and distributed as follows: 11, 8, 5, 8, 14, 9, 10 and 7 belonging to the eight SCoT primers; 1, 2, 3, 4, 7 and 9 and 12, respectively. Also, the number of DNA fragments of Qalyubia Governorate samples was 29 and 34 in BBTV-infected plants, and 30 and 44 fragments in the healthy ones. The number of BBTV-infected samples compared to uninfected ones of Qena and Aswan Governorates was as follows: 48 and 50 in Qena and 38 and 45 in Aswan. As overall view, the number of DNA fragments in the DNA polymorphism resulting from BBTV-infected samples was less than in healthy samples in all Governorates.
25.It was found that 63 out of 72 pieces were polymorphic fragments, while 9 fragments were of the monomorphic type. Among the polymorphic fragments, it was found that there were five unique DNA markers were amplified by SCoT-03 (one fragment), SCoT-07 (one fragment) and SCoT-11 (three fragments).
26. Results paid an attention to that the BBTV-infected samples as well as the healthy ones for each of the region were present together in a separate cluster. This is in addition to the fact that the samples from Qena and Aswan were genetically closer to each other.
27. Based on the obtained findings, it was found that the BBTV has affected the genome of the Maghrabi banana plant, and this is evident from the low percentage of identities between BBTV-infected plants when compared to healthy plants in all four regions.
28. Finally, the different climatic conditions had an effect on the plant genome of the same variety, whether it was virus-infected or healthy, and SCoT technique was successfully used to determine the genetic variation of tested samples.
29. Results of SDS-PAGE (12%) analysis of the Maghrabi banana plant, whether healthy or BBTV-infected, grown in three Qalyubia, Qena and Aswan Governorates showed that, a number of twelve protein bands were obtained from all banana samples, and were distributed as follows: 10, 11, 10, 10, 11, 12, 7 and 8 from samples of Qa01-I, Qa01-H, Qa02-I, Qa02-H and Qe-I and Qe-H, As-I, and As-H, respectively.
30. A similarity was observed in the number of protein bands obtained in healthy and BBTV-infected plants. The number of protein bands for healthy plant samples was found to be higher than for BBTV-infected plant samples in the majority of tested samples.
31. At the level of the type of bands, five bands out of twelve were of the monomorphic type, while seven bands were considered as polymorphic type, and no protein bands were obtained that could be considered as a unique marker protein.
32. Results showed the percentage of similarity between the protein patterns obtained from banana samples of Maghrabi cultivar, whether were BBTV-infected or healthy and grown in three different climatic regions belonging to Delta (Qalyubia Governorate) and two regions belonging to Upper Egypt (Qena and Aswan Governorates).
33. Results showed that the genetic relationship based on percentage similarity results of protein patterns were somewhat different from those that were recorded between the same samples at the DNA level. This was due to; it was not necessarily the presence of samples of the same region whether they were BBTV-infected or healthy together in the same cluster as was reported in the case of DNA. Results also showed that the healthy samples from Qena (Qe-H) and Aswan (As-H) were fell in the same cluster contains BBTV-infected sample of Qalyubia (Qa01-I), while the Qa02-H sample was fell with the BBTV-infected samples (Qe-I & As-I) of Qena and Aswan Governorates.