بررسی تنوع گونه ای دو عقرب Androctonus crassicauda و Hemiscorpius lepturus با استفاده از ژن های متابولیسمی متفاوت بیان شده در داده های RNA-seq

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه محیط زیست، واحد اصفهان (خوراسگان)، دانشگاه آزاد اسلامی، اصفهان، ایران

2 بخش جانوران سمی و تولید پادزهر، موسسه تحقیقات واکسن و سرم سازی رازی شعبه جنوب غرب کشور، سازمان تحقیقات، آموزش و ترویج کشاورزی، اهواز، ایران

3 گروه گیاه پزشکی، واحد اصفهان (خوراسگان)، دانشگاه آزاد اسلامی، اصفهان، ایران

10.22034/aej.2022.363899.2894

چکیده

راسته عقرب ‏ها جزء رده عنکبوتیان و شاخه بندپایان دسته بندی می شوند. با توجه به این که مطالعات گسترده ای در سراسر جهان در حوزه فیلوژنتیکی و مورفولوژیکی در حال انجام است، دسته بندی عقرب ها نیز در حال به روز رسانی می باشد. این مطالعه در سال 1400 انجام گردیدد. هدف از پژوهش حاضر، بازسازی شبکه متابولیسمی عقرب‌های Androctonus crassicauda و Hemiscorpius lepturus با استفاده از مطالعات RNA-seq و معرفی ژن های متابولیسمی کلیدی متفاوت بیان شده بین دو گونه عقرب به عنوان نشانگر ملکولی برای ارزیابی تنوع ژنتیکی بین گونه های مختلف عقرب بود. صید عقرب ها با استفاده از نور فرابنفش و در نواحی مختلف استان خوزستان انجام شد (10 نمونه از هر گونه). سم‌گیری از عقرب‌ها به روش شوک الکتریکی انجام شد و پس از گذشت 72 ساعت از سم‌گیری، غده زهر جداسازی و با استفاده از هاون چینی و ازت مایع پودر گردید. استخراج RNA از غده زهر با استفاده از کیت تجاریRNeasy Animal Mini Kit (Qiagen, Valencia, CA, USA) طبق دستور العمل شرکت انجام گردید. به این منظور توالی یابی ترانسکریپتوم دو گونه عقرب انجام شد و تفاوت بیان ژن ها توسط نرم افزار RSEM آنالیز گردید. نتایج آنالیز ها نشان داد که در مجموع 688060 یونی ژن‌ در بین دو گونه شناسایی شدند که از این تعداد ۴۲۶۷۶ ژن دارای تفاوت بیان معنی‌داری بودند (0/05>p). هستی شناسی ژن‌های متفاوت بیان شده با استفاده از پایگاه های KEEG و GO انجام شد و نتایج نشان داد که ژن های دخیل در مسیر متابولیسمی بیش ترین فراوانی را دارا می باشند. بازسازی شبکه متابولیسمی عقرب و تجزیه و تحلیل آن با استفاده از پایگاه داده استرینگ و نرم افزار سایتواسکیپ منجر به شناسایی 5 ژن هاب متابولیسمی گردید. در نهایت با استفاده از توالی اسید آمینه ای ژن هاب، (تریوز فسفات ایزومراز، سیترات سنتاز، گلوگز 6- فسفات ایزومراز، کاتالاز و انولاز) درخت فیلوژنتیکی بین 7 گونه عقرب ترسیم شد. این تحقیق استفاده از این 5 ژن متابولیسمی را بر ای بررسی روابط فیلوژنتیکی در عقرب ها کارآمد ارزیابی کرد.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Species diversity of Androctonus crassicauda and Hemiscorpius lepturus using different metabolic genes expressed in RNA-seq data

نویسندگان [English]

  • Elham Pondehnezhadan 1
  • Atefeh Chamani 1
  • Fatemeh Salabi 2
  • Reihaneh Soleimani 3
1 Department of Environment, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran
2 Department of Venomous Animals and Antidote Production, Razi Vaccine and Serum Research Institute, Southwest Branch, Agricultural Research, Education and Extension Organization, Ahvaz, Iran
3 Department of Plant Protection, Isfahan (Khorasgan) Branch, Islamic Azad University, Isfahan, Iran
چکیده [English]

The order of scorpions belongs to the category of arachnids and the branch of arthropods, and according to the extensive studies that are being carried out today in the field of phylogenetic and morphological around the world, the classification of scorpions is also being updated. The aim of this studyis reconstruct the metabolic network of Androctonus crassicauda and Hemiscorpius lepturus using RNA-seq studies for suggestion of key metabolic genes differentially expressed between two scorpion species as a molecular marker to evaluate the genetic diversity of different scorpion species. For this purpose, Scorpions were caught using ultraviolet light in different areas of Khuzestan province (10 samples of each species). Scorpions were poisoned by electric shock method, and after 72 hours of poisoning, the venom gland was isolated and powdered using a porcelain mortar and liquid nitrogen. RNA extraction from the venom gland was performed using the commercial RNeasy Animal Mini Kit (Qiagen, Valencia, CA, USA) according to the company's instructions. Transcriptome sequencing and differential gene expression analysis of two scorpion species were performed by RSEM software. The results of analyzes showed that a total of 688060 genes were identified in two species, of which 42676 genes had significant differences in expression (p<0.05). The gene ontology of the differentially expressed genes was performed using Kegg and GO databases and the results showed that the genes involved in the metabolic pathway are the most abundant. Reconstruction of scorpion metabolic network and its analysis using string database and cytoscope software led to the identification of 5 metabolic hub genes. Finally, using the amino acid sequences of Hub genes (triose phosphate isomerase, citrate synthase, glucose 6-phosphate isomerase, catalase and enolase), a phylogenetic tree was constructed using 7 scorpion species. This study evaluated the use of these 5 metabolic genes to study phylogenetic relationships in scorpions.

کلیدواژه‌ها [English]

  • Genetic diversity
  • Gene ontology
  • Metabolic network
  • Phylogenetic tree
  • Phylogenetic relationships

مراجع

  1. Akbari, A., Tabatabai S.M., Hedayat, A., Moder Rosta, H., Alizadeh, M.H. and Zare Muslim, K., 1997. Study of geographical distribution of scorpions in southern Iran. Journal of Research and Construction. 34: 115-112.
  2. Alfonso Prieto, M., Vidossich, P. and Rovira, C., 2012. The reaction mechanisms of heme catalases: an atomistic view by ab initio molecular dynamics. Archives of Biochemistry and Biophysics. 525(2): 121-130.
  3. Ashokan, K.V., Mundaganur, D.S. and Mundaganur, Y.D., 2010. Catalase: Phylogenetic characterization to explore protein cluster. International Journal of Bioinformatics Research and Application. 1: 1-8.
  4. ‏Bader, G.D. and Hogue, C.W., 2003. An automated method for finding molecular complexes in large protein interaction networks. BMC bioinformatics. 4(1): 1-27.‏
  5. Bolger,M., Lohse, M. and Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics Bioinformatics. 30(15): 2114-2120.
  6. Bruggeman,J. and Westerhoff, H.V., 2007. The nature of systems biology. Trends in Microbiology. 15(1): 45-50.
  7. Chahari, K., Jelodar, A. and, Jafari, H., 2020. Molecular phylogeny of Scorpion Hemiscorpius lepturus based on Sequences Ribosomal DNA in the Internal Transcribed Spacers (ITS2) in Khuzestan province. Veterinary Research and Biological Products. 33(4): 112-122.
  8. Dehghan, R. and Kassiri, H., 2018. 2003. A Checklist of Scorpions in Iran (2017) Asian Journal of Pharmaceutics (AJP): Free Full Text Articles from Asian Journal of Pharmcolgy. 12(3): 25-29.
  9. Dennis, G., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C. and Lempicki, R.A., 2003. DAVID: database for annotation, visualization, and integrated discovery. Genome biology. 4(9): 1-11.‏
  10. Fet, V., Gantenbei,, Gromov, A.V., Lowe, G. and Lourenco, W.R., 2003. The first molecular phylogeny of Buthidae (Scorpiones). Euscorpius. 4: 1-10.
  11. Grabherr, G., Haas, B.J., Yassour, M., Levin, J.Z., Thompson, D.A., Amit, I., Adiconis, X., Fan, L., Raychowdhury, R. and Zeng, Q., 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnology.
  12. Grashof, D.G.B., Kerkkamp, H., Archer, S.A., Harris, J., Richardson, M.K., Vonk, F.J. and Meijden, A., 2019. Transcriptome annotation and characterization of novel toxins in six Scorpion species. BMC Genomics. 20: 645- 655.
  13. Grauvogel, C., Brinkmann, H. and Petersen, J., 2007. Evolution of the glucose-6-phosphate isomerase: the plasticity of primary metabolism in photosynthetic eukaryotes. Molecular biology and evolution. 24(8): 1611-1621.‏
  14. Haas, B.J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P.D., Bowden, J., Couger, M.B., Eccles, D., Li, B., Lieber, M., MacManes, M.D, Ott, M., Orvis, J., Pochet, N., Strozzi, F., Weeks, N., Westerman, R., William, T., Dewey, C.N., Henschel, R., Leduc, R.D., Friedman, N. and Regev, A., 2013. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nature Protocols. 8: 1494-1512. DOI 10.1038/nprot.2013.084.
  15. Harms, D., Robrts, J.D. and Harvey, M., 2019. Climate variability impacts on diversification processes in a biodiversity hotspot: a phylogeography of ancient pseudoscorpions in south-western Australia. Zoological Journal of the Linnean Society. 186: 934-952.
  16. Hernández Lucas, I., Rogel-Hernández, M. A., Segovia, L., Rojas-Jiménez, K. and Martínez Romero, E., 2004. Phylogenetic relationships of Rhizobia based on citrate synthase gene sequences. Systematic and applied microbiology. 27(6): 703-706.‏
  17. Jafari, H., Salabi, F., Navidpour, S.H. and Forouzan, S.H., 2020. The phylogenetic and morphological analyses of Androctonus crassicuda from Khuzestan province (Scorpiones: Buthidae). Razi Vaccine and Serum Research Institute. 10.22092/ari.2020.342071.1451.
  18. Jeyaprakash, A. and Hoy, M.A., 2009. First divergence time estimate of spiders, scorpions, mites and ticks (Sub phylum: Chelicerata) inferred from mitochondrial phylogeny. Experimental and Applied Acarology. 47: 1-18.
  19. Kazemi Lomedasht, F., Khalaj, V., Bagheri, K.P., Behdani, M. and Shahbazzadeh, D., 2017. The first report on transcriptome analysis of the venom gland of Iranian scorpion, Hemiscorpius lepturus. Toxicon. 125: 123-130.‏
  20. Keeling, P.J. and Doolittle, W.F., 1997. Evidence that eukaryotic triosephosphate isomerase is of alpha proteobacterial origin. Proceedings of the National Academy of Sciences. 94(4): 1270-1275.‏
  21. Kovařík, F. and Ahmed, Z., 2013. A Review of Androctonus Finitimus Fet, V., Gantenbein, B., Gromov, A., Lowe, G. and Lourenço, W.R., 2003. The First Molecular Phylogeny of Buthidae (Scorpiones). Euscorpius. 4: 1-10.
  22. Kumar, S., Stecher, G., Li, M., Knyaz, C. and Tamura, K., 2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular biology and evolution. 35: 1547-1549.
  23. Li, B. and Dewey, C.N., 2011. RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC bioinformatics. 12(1): 1-16.‏
  24. Liu, K.C., Liu, C., Fan, M.X., Zhang, C.Y., Cai, F.C., Li, Q.Y. and Lin, F., 2019. Phylogenetic analysis of enolase gene family in different species. Applied Ecology and Environmental Research. 17(5): 11537-11550.‏
  25. Michels, P.A.M., Opperdoes, F.R., Hannaert, V., Wiemer, E.A.C., Allert, S. and Chevalier, N., 1992. Phylogenetic analysis based on glycolytic enzymes. Belgian journal of botany. 164-173.‏
  26. Monod, L. and Lourenco, W.R., 2005. Hemiscorpiidae (Scorpiones) from Iran, with descriptions of two new species and notes on biogeography and phylogenetic relationships. Revue suisse de Zoologie. 112(4): 869.
  27. Morgenstern, D., Rohde, B.H., King, G.F., Tal, T. and Sher, D., 2011. The tale of a resting gland: transcriptome of a replete venom gland from the scorpion Hottentotta judaicus. Toxicon. 57: 695-703.
  28. Mozduri, Z. and Qaderzadeh, M., 2017. Study gene regulatory network in Iactogenesis in dairy cow mammary tissue using RNA-seq data. Animal Sciences (Research and Construction). 30(115): 193-205.
  29. Patwardhan, A., Ray, S. and Roy, A., 2014. Molecular markers in phylogenetic studies-a review. Journal of Phylogenetics & Evolutionary Biology.
  30. Piast, M., Kustrzeba Wójcicka, I., Matusiewicz, M. and Banaś, T., 2005. Molecular evolution of enolase. Acta Biochimica Polonica. 52(2): 507-513.‏
  31. Pondehnezhadan, E., Chamani, A., Salabi, F. and Soleimani, R., 2022. Identification, characterization and molecular phylogeny of scorpion enolase (Androctonus crassicauda and Hemiscorpius lepturus). Toxin review.
  32. Rendo, N., Anaya, M., Delaye, L., Possani, L.D. and Herrera Estrella, A., 2012. Global Transcriptome Analysis of the Scorpion Centruroides noxius: New Toxin Families and Evolutionary Insights from an Ancestral Scorpion Species. PLoS ONE. 7(8): e43331. doi:101371/ journal.pone.0043331.
  33. Rohipoor, M. and Nazari, M., 2019. Genetic and Phylogenetic Analysis of Adani Goat Population Based on Cytochrome B Gene. Research On Animal Production. 10(26): 84-89.
  34. Rohipoor, M. and Nazari, M., 2021. Population structure, Genetic diversity and phylogenetic analysis of control region of mtDNA in Adani goat breed. Journal of Modern Genetics. 15(4): 297-304.
  35. Santibáñez López,, Ojanguren Affilastro, A. and Prashant, P., 2020. Another one bites the dust: taxonomic sampling of a key genus in phylogenomic datasets reveals more non-monophyletic groups in traditional scorpion classification. Invertebrate Systematics. 34: 133-143.

36.                Shahabi, A., Por Mojtaba, T. and Kazemipour, A., 2019. Reconstruction, analysis and comparison of    Gene Networks Topology Based on Rna-Seq Data Involved in Reproductive and Fertility Complex Traits. Journal of Agricultural Biotechnology. 11(2): 57-78.

  1. Soleglad, E., Fet, V. and Kovarík, F., 2005. The systematic position of the scorpion genera Heteroscorpion Birula, 1903 and Urodacus Peters, 1861 (Scorpiones: Scorpionoidea). Euscorpius. 20: 1-38.
  2. Temate Tiagueu, Y., Al Seesi, S., Mathew, M., Mandric, I., Rodriguez, A., Bean, K., Cheng, Q., Glebova, O., andoiu, I.B., Lopanik, N. and Zelikovsky, A., 2016. Inferring metabolic pathway activity levels from RNA-Seq data. BMC Genomics. 17(5): 542.
  3. Thompson, J.D., Higgins, D.G. and Gibson, T.J., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic acids research. 22: 4673-4680.
  4. Wiegmann, B.M., Mitter, C., Regier, J.C., Fried lander, T.P., Wagner, D.M. and Nielsen, E.S., 2000. Nuclear genes resolve mesozoic-aged divergences in the insect order Lepidoptera. Molecolar Phylogentics and Evolution. 15: 242-259.
  5. Zámocký, M., Phylogenetic relationships in class I of the superfamily of bacterial, fungal & plant peroxidases. European Journal of biochemistry. 271(16): 3297-3309.
فصلنامه محیط زیست جانوری
دوره 15، شماره 3
آبان 1402
صفحه 329-340

فایل ها

هم رسانی

ارجاع به این مقاله

آمار