Fasulye Genotiplerinde Tuz ve Kuraklık Stresleri Altında VPE Gen Ailesinin Genom Çapında Analizi ve Karakterizasyonu

Öz

Vakuolar işleme enzimleri (Vacoular Processing Enzymes; VPE’ler) önemli sistein proteazlarıdır ve protein öncüllerinin işlenmesi ve olgunlaşmasında, bitki gelişiminde, yaşlanmasında ve bağışıklığında, programlanmış hücre ölümünün yanı sıra şeker birikiminde işlev görür. Ayrıca biyotik ve abiyotik streslere karşı yanıt vermede önemli roller oynarlar. Çalışmanın amacı Phaseolus vulgaris bitkisinde in siliko olarak gerçekleştirilen VPE gen ailesi üyelerinin genom çaplı analizini ve karakterizasyonu sağlamak ve belirlenen gen ailesi üyelerinin abiyotik stresler (tuz ve kuraklık) altında vermiş olduğu yanıtların değerlendirilmesidir. Çeşitli in siliko yöntemler kullanılarak P. vulgaris genomunda 5 adet Pvul-VPE gen ailesi üyesi tespit edildi. Bu gen ailesi üyeleri 13,97 kDa ile 55,11 kDa arasında değişen moleküler ağırlığa sahipken, 123 ile 493 arasında değişen aminoasit sayısına sahip olduğu tespit edildi. İzoelektirik noktaları 5,48 (Pvul-VPE-4) ile 9,16 (Pvul-VPE-3) arasındadır. Ayrıca Pvul-VPE genleri fasulye genomunun 1, 3, 9 ve 11. kromozomları üzerinde konumlandığı tespit edilmiştir. Pvul-VPE gen ailesi üyeleri arasında yapılan ekzon ve intron analizleri sonucunda, toplam 40 ekzon ve 35 intron içerdikleri tespit edildi. Yapılan filogenetik analizler sonucunda Pvul-VPE proteinleri Arabidopsis thaliana ve Glycine max türleri ile 4 ana grupta kümelenmiştir. Pvul-VPE-1 ve Pvul-VPE-3 genleri arasında segmental duplikasyon olduğu tespit edildi. Pvul-VPE genlerinin ifade profilleri ile yapılan analizlerde farklı ifade seviylerine sahip oldukları tespit edildi ve bitkinin büyümesinde, gelişiminde ve olgunlaşmasında, protein öncüllerinin işlenmesinde ve bitkide şeker birikimi gibi görevlerin yanı sıra abiyotik ve biyotik streslere karşı yanıt oluşturmada önemli görevler üstlendiği tespit edildi. Yapılan çalışmanın sonuçları, VPE gen ailesinin P. vulgaris türünde kuraklık ve tuz streslerine karşı yanıtta ilk defa gerçekleştirilmiş olup, bitki ve tarımsal biyoteknoloji ve moleküler biyoloji alanlarında literatür bakımından kaynak ve ilave bilgiler sağlayacaktır.

Anahtar Kelimeler

• VPE
• Genom Çaplı Analiz
• Fasulye
• Biyoinformatik
• Abiyotik Stres

Genome-Wide Analysis and Characterization of the VPE Gene Family under Salt and Drought Stress in Common Bean Genotypes

Abstract

Vacuolar processing enzymes (VPEs) are cysteine proteases which are involved in the processing and maturation of protein precursors, as well as plant growth, senescence, and immunity, programmed cell death, and sugar accumulation. They also play a key role in biotic and abiotic stress responses. The goal of this study is to perform genome-wide analysis and characterization of VPE gene family members in Phaseolus vulgaris in silico, as well as to assess the reactions of the determined gene family members to abiotic stimuli (salt and drought). 5 members of the Pvul-VPE gene family was discovered in the P. vulgaris genome using a variety of in silico approaches. Members of this gene family have molecular weights ranging from 13.97 kDa to 55.11 kDa, and amino acid lengths ranging from 123 to 493 have been discovered. Its isoelectric points range from 5.48 (Pvul-VPE-4) to 9.16 (Pvul-VPE-3). Additionally, the Pvul-VPE genes were discovered on chromosomes 1, 3, 9, and 11 of the common bean genome. Exon and intron analysis of members of the Pvul-VPE gene family revealed that they have a total of 40 exons and 35 introns. Pvul-VPE proteins have been found to be clustered in four main groups with Arabidopsis thaliana and Glycine max species as a result of the phylogenetic analysis. Between the Pvul-VPE-1 and Pvul-VPE-3 genes, segmental duplication was discovered. The results of this study, which was performed for the first time in the response to drought and salt stresses in the VPE gene family's P. vulgaris species, will be a valuable source of information in the disciplines of plant breeding, agricultural biotechnology and molecular biology.

Keywords

• VPE
• Genome Wide Analysis
• Common Bean
• Bioinformatics
• Abiotic Stress

References

1. Adams, K. L., & Wendel, J. F. (2005). Polyploidy and genome evolution in plants. Current opinion in plant biology, 8(2), 135-141.
2. Bailey, T. L., Williams, N., Misleh, C., & Li, W. W. (2006). MEME: discovering and analyzing DNA and protein sequence motifs. Nucleic acids research, 34(suppl_2), W369-W373.
3. Broughton, W. J., Hernandez, G., Blair, M., Beebe, S., Gepts, P., & Vanderleyden, J. (2003). Beans (Phaseolus spp.)–model food legumes. Plant and soil, 252(1), 55-128.
4. Chen, C., Chen, H., Zhang, Y., Thomas, H. R., Frank, M. H., He, Y., & Xia, R. (2020). TBtools: an integrative toolkit developed for interactive analyses of big biological data. Molecular plant, 13(8), 1194-1202.
5. Chung, H.-J., Liu, Q., Pauls, K. P., Fan, M. Z., & Yada, R. (2008). In vitro starch digestibility, expected glycemic index and some physicochemical properties of starch and flour from common bean (Phaseolus vulgaris L.) varieties grown in Canada. Food Research International, 41(9), 869-875.
6. Crooks, G. E., Hon, G., Chandonia, J.-M., & Brenner, S. E. (2004). WebLogo: a sequence logo generator. Genome research, 14(6), 1188-1190.
7. De Pinto, M. C., Locato, V., & De Gara, L. (2012). Redox regulation in plant programmed cell death. Plant, cell & environment, 35(2), 234-244.
8. De Ron, A. M., Papa, R., Bitocchi, E., González, A. M., Debouck, D. G., Brick, M. A., . . . Geffroy, V. (2015). Common bean. In Grain legumes (pp. 1-36). Springer.

9. Díaz-Batalla, L., Widholm, J. M., Fahey, G. C., Castaño-Tostado, E., & Paredes-López, O. (2006). Chemical components with health implications in wild and cultivated Mexican common bean seeds (Phaseolus vulgaris L.). Journal of Agricultural and Food Chemistry, 54(6), 2045-2052.
10. Fagundes, D., Bohn, B., Cabreira, C., Leipelt, F., Dias, N., Bodanese-Zanettini, M. H., & Cagliari, A. (2015). Caspases in plants: metacaspase gene family in plant stress responses. Functional & integrative genomics, 15(6), 639-649.
11. Hara-Nishimura, I., Kinoshita, T., Hiraiwa, N., & Nishimura, M. (1998). Vacuolar processing enzymes in protein-storage vacuoles and lytic vacuoles. Journal of plant physiology, 152(6), 668-674.
12. Hatsugai, N., Yamada, K., Goto-Yamada, S., & Hara-Nishimura, I. (2015). Vacuolar processing enzyme in plant programmed cell death. Frontiers in plant science, 6, 234.
13. Hiz, M. C., Canher, B., Niron, H., & Turet, M. (2014). Transcriptome analysis of salt tolerant common bean (Phaseolus vulgaris L.) under saline conditions. PloS one, 9(3), e92598.
14. Horton, P., Park, K.-J., Obayashi, T., Fujita, N., Harada, H., Adams-Collier, C. J., & Nakai, K. (2007). WoLF PSORT: protein localization predictor. Nucleic acids research, 35(suppl_2), W585-W587.
15. Hu, B., Jin, J., Guo, A.-Y., Zhang, H., Luo, J., & Gao, G. (2015). GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics, 31(8), 1296-1297.
16. Jenkins, A. L. (2007). The glycemic index: Looking back 25 years. Cereal foods world, 52(2), 50.
17. Juretic, N., Hoen, D. R., Huynh, M. L., Harrison, P. M., & Bureau, T. E. (2005). The evolutionary fate of MULE-mediated duplications of host gene fragments in rice. Genome research, 15(9), 1292-1297.
18. Kalefetoğlu, T., & Ekmekçi, Y. (2005). The effects of drought on plants and tolerance mechanisms. Gazi University Journal of Science, 18(4), 723-740.
19. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., & Sternberg, M. J. E. (2015). The Phyre2 web portal for protein modeling, prediction and analysis. Nature protocols, 10(6), 845-858.
20. Kinoshita, T., Yamada, K., Hiraiwa, N., Kondo, M., Nishimura, M., & Hara‐Nishimura, I. (1999). Vacuolar processing enzyme is up‐regulated in the lytic vacuoles of vegetative tissues during senescence and under various stressed conditions. The Plant Journal, 19(1), 43-53.
21. Lamesch, P., Berardini, T. Z., Li, D., Swarbreck, D., Wilks, C., Sasidharan, R., . . . Garcia-Hernandez, M. (2012). The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic acids research, 40(D1), D1202-D1210.
22. Lamkanfi, M., Declercq, W., Kalai, M., Saelens, X., & Vandenabeele, P. (2002). Alice in caspase land. A phylogenetic analysis of caspases from worm to man. Cell death and differentiation, 9(4), 358-361.
23. Lescot, M., Déhais, P., Thijs, G., Marchal, K., Moreau, Y., Van de Peer, Y., . . . Rombauts, S. (2002). PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic acids research, 30(1), 325-327.
24. Letunic, I., & Bork, P. (2011). Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucleic acids research, 39(suppl_2), W475-W478.
25. Lynch M, Conery JS (2003) The evolutionary demography of duplicate genes. Genome Evolution:35-44.
26. Nagashima, Y., von Schaewen, A., & Koiwa, H. (2018). Function of N-glycosylation in plants. Plant Science, 274, 70-79.
27. Nakaune, S., Yamada, K., Kondo, M., Kato, T., Tabata, S., Nishimura, M., & Hara-Nishimura, I. (2005). A vacuolar processing enzyme, δVPE, is involved in seed coat formation at the early stage of seed development. The Plant Cell, 17(3), 876-887.
28. Mortazavi A, Williams BA, Mccue K, Schaeffer L, Wold B (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods 5(7):621-628. https://doi.org/10.1038/nmeth.1226
29. Quevillon, E., Silventoinen, V., Pillai, S., Harte, N., Mulder, N., Apweiler, R., & Lopez, R. (2005). InterProScan: protein domains identifier. Nucleic acids research, 33(suppl_2), W116-W120.
30. Schmutz, J., McClean, P. E., Mamidi, S., Wu, G. A., Cannon, S. B., Grimwood, J., . . . Chavarro, C. (2014). A reference genome for common bean and genome-wide analysis of dual domestications. Nature genetics, 46(7), 707-713.
31. Selda, Ö., & EkİNcİ, M. (2015). Kuraklık stresi ve bitki fizyolojisi. Derim, 32(2), 237-250.
32. Song, J., Yang, F., Xun, M., Xu, L., Tian, X., Zhang, W., & Yang, H. (2020). Genome-wide identification and characterization of vacuolar processing enzyme gene family and diverse expression under stress in apple (Malus× domestic). Frontiers in plant science, 11, 626.
33. Suyama, M., Torrents, D., & Bork, P. (2006). PAL2NAL: robust conversion of protein sequence alignments into the corresponding codon alignments. Nucleic acids research, 34(suppl_2), W609-W612.
34. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular biology and evolution, 28(10), 2731-2739.
35. Tang, Y., Wang, R., Gong, P., Li, S., Wang, Y., & Zhang, C. (2016). Gene cloning, expression and enzyme activity of Vitis vinifera vacuolar processing enzymes (VvVPEs). PloS one, 11(8), e0160945.
36. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F., & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic acids research, 25(24), 4876-4882.
37. Valliyodan, B., Cannon, S. B., Bayer, P. E., Shu, S., Brown, A. V., Ren, L., . . . Daum, C. G. (2019). Construction and comparison of three reference‐quality genome assemblies for soybean. The Plant Journal, 100(5), 1066-1082.
38. Voorrips, R. E. (2002). MapChart: software for the graphical presentation of linkage maps and QTLs. Journal of heredity, 93(1), 77-78.
39. Wang, Y., Tang, H., DeBarry, J. D., Tan, X., Li, J., Wang, X., . . . Guo, H. (2012). MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic acids research, 40(7), e49-e49.
40. Yang, Z. (2007). PAML 4: phylogenetic analysis by maximum likelihood. Molecular biology and evolution, 24(8), 1586-1591.
41. Zhang, H., Tao, X., & Zhang, F. (2021). Genome-wide identification and expression analysis of the vacuolar processing enzyme (VPE) family genes in pear. The Journal of Horticultural Science and Biotechnology, 96(4), 469-478.
42. Zhu, L., Wang, X., Tian, J., Zhang, X., Yu, T., Li, Y., & Li, D. (2022). Genome-wide analysis of VPE family in four Gossypium species and transcriptional expression of VPEs in the upland cotton seedlings under abiotic stresses. Functional & Integrative Genomics, 1-14.