همسانه‌سازی و بررسی بیوانفورماتیکی ژن‌های CCD4a و CCD4b در زعفران (Crocus sativus L. ) ایران

نوع مقاله: مقاله علمی پژوهشی

نویسندگان

1 دانشجوی دکتری رشته بیوتکنولوژی کشاورزی، گروه به‌نژادی و بیوتکنولوژی گیاهی، دانشکده کشاورزی دانشگاه تبریز، تبریز، ایران.

2 دانشیار، گروه به‌نژادی و بیوتکنولوژی گیاهی، دانشکده کشاورزی دانشگاه تبریز،تبریز، ایران

3 استادیار، گروه زیست فناوری مواد غذایی، مؤسسه پژوهشی علوم و صنایع غذایی، مشهد، ایران.

4 استاد، گروه به‌نژادی و بیوتکنولوژی گیاهی، دانشکده کشاورزی دانشگاه تبریز. تبریز، ایران.

10.22048/jsat.2019.182490.1343

چکیده

زعفران به عنوان یکی از گران­بهاترین ادویه­ها و رنگ­های طبیعی است که در صنایع مختلف از جمله غذایی، دارویی و آرایشی- بهداشتی مورد استفاده قرار می­گیرد. در سال‌های اخیر، خانواده‌‌ای از آنزیم‌ها که پیش ماده­های کاروتنوئیدی را در جایگاه پیوندهای دوگانه برش می‌دهند، در گیاهان شناسایی و معرفی شده‌اند. به این خانواده از آنزیم‌های برش­دهنده کارتنویید دی اکسیژناز CCD)) گفته می­شوند. در این تحقیق به دلیل اهمیت ژن‌های CCD در بیوسنتز آپوکاروتنوئیدهای زعفران، دو ایزوفرم از این ژن با استفاده از روش نسخه­برداری معکوس همسانه­سازی، تعیین توالی و نتایج با موارد مشابه خارجی مورد مقایسه قرار گرفتند. بررسی­های بیوانفورماتیکی شامل بررسی ارتباطات خویشاوندی و نیز ساختار­های پروتتینی مورد ارزیابی قرار گرفت. مدل‌سازی سه ‌بعدی این پروتئین‌ها به روش همولوژی مدلینگ و با استفاده از پایگاه Swiss Model پس از انتخاب الگوی مناسب انجام گرفت. همچنین جهت اعتبار­سنجی ساختاری مدل ترسیم شده سه‌بعدی، پلات راماچاندران ترسیم گردید. نتایج حاصله نشان داد دو ایزوفرم CCD4a و CCD4b هر دو دارای دو اگزون (641 و 1099 جفت باز برای CCD4a  و 614 و 1099 جفت باز برای CCD4b) و یک اینترون (670 جفت باز در CCD4a و 668 جفت باز در CCD4b) هستند. بررسی In silico  خصوصیات فیزیکوشیمیایی پروتئین‌های CsCCD4a و CsCCD4b بیانگر این حقیقت بود که پروتئین­های بدست آمده از این دو ایزوفرم از نظر وزن مولکولی، تعداد اسیدهای آمینه، نقطه ایزوالکتریک، شاخص آلیفاتیک، شاخص ناپایداری و حلالیت مشابه می­باشند. نتایج بررسی ساختارهای سه بعدی بدست آمده نشان داد این دو ایزوفرم دارای ساختارهای سه بعدی مشابهی می­باشند. یافته­های این تحقیق می­تواند اطلاعات با ارزشی در رابطه با رفتار و واکنش آنزیم CCD4 در مسیر سنتز آپوکاروتنوئیدهای زعفران فراهم کند و همچنین این نتایج می­تواند در  برنامه­های آتی اصلاح ژنتیکی زعفران ایران مفید واقع گردد.

کلیدواژه‌ها


عنوان مقاله [English]

Cloning and Bioinformatics Investigation on CCD4a and CCD4b Genes from Iranian Saffron (Crocus sativus L.)

نویسندگان [English]

  • Mohammad Javad Habibzadeh 1
  • Ebrahim Dorani 2
  • Seyed Mahdi Ziaratnia 3
  • Mostafa Valizadeh 4
1 PhD. Student, Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran.
2 Associate Professor, Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Tabriz, Tabriz
3 Assistant Professor, Department of Food Biotechnology, Research Institute of Food Science and Technology (RIFST), Mashhad, Iran
4 Professor, Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Tabriz, Tabriz, Iran.
چکیده [English]

Saffron is one of the most expensive spices and natural colors used in various food, pharmaceutical and cosmetic industries. In recent years, a family of enzymes that digest carotenoid substrates into double bonds are identified and introduced in plants. This family is of enzymes Carotenoid Cleavage Dioxygenase (CCD) enzymes. In this study, two isoforms of this gene were cloned and sequenced due to the importance of CCD genes in biosynthesis of apocarotenoids. Bioinformatics analyses including phylogenetic relationships and protein structures were evaluated. 3D modeling of these proteins was done by homologous modeling and using the Swiss Model database after selecting the appropriate pattern. The Ramachandran plot was drawn in order to validate the structure of the 3D model. The results show that the two CCD4a and CCD4b isoforms have both exons and one intron. In silico analysis, the physicochemical properties of CsCCD4a and CsCCD4b proteins also show that the proteins derived from these two isoforms are similar in terms of molecular weight, amino acids, isoelectric points, aliphatic index, instability index and solubility. The results of study of 3D structures resulted in proposal of similar structures for two isoforms. The results of this study can provide valuable information on the behavior and response of CCD4 enzyme in the pathway for synthesis of apricotines in saffron, and these results can be useful in future protein engineering programs.

کلیدواژه‌ها [English]

  • Crocus sativus
  • CCD4
  • Phylogenetic
  • Bioanphormatic
  • Modeling
Ahrazem, O., Trapero, A., Gomez, M.D., Rubio-Moraga, A., and  Gomez-Gomez, L. 2010. Genomic analysis and gene structure of the plant carotenoid dioxygenase 4 family: A deeper study in Crocus sativus and its allies. Genomics 96: 239-250.
Ahrazem, O., Rubio-Moraga, A., Berman, J., Capell, T., Christou, P., Changfu, Z., and Gomez-Gomez, L. 2015. The carotenoid cleavage dioxygenase CCD2 catalysing the synthesis of crocetin in spring crocuses and saffron is a plastidial enzyme. New Phytologist 209 (2): 650-663.
Ahrazem, O., Rubio-Moraga, A., Argandola-Picazo, J., Castillo, R., and Gomez-Gomez, L. 2016. Intron retention and rhythmic diel pattern regulation of carotenoid cleavage dioxygenase 2 during crocetin biosynthesis in saffron. Plant Molecular Biology 91 (3): 355-374.
Almagro Armenteros, J.J., Salvatore, M., Emanuelsson, O., Winther, O., von Heijne, Arne Elofsson, G., and Nielsen, H. 2019. Detecting sequence signals in targeting peptides using deep learning. Life Science Alliance 2 (5): 1-14.  
Artimo, P., Jonnalagedda, M., Arnold, K., Baratin, D., Csardi, G., de Castro, E., Duvaud, S., Flegel, V., Fortier, A., Gasteiger, E., Grosdidier, A., Hernandez, C., Ioannindis, V., Kuznetsov, D., Liechti, R., Moretti, S., Mostaguir, K., Redaschi, N., Rossier, G., Xenarios, I., and Stockinger, H. 2012. ExPASy: SIB bioinformatics resource portal, Nucleic Acids Research 40 (W1): W597-W603.
Baba, Sh.A, Mohiuddin, T., Basu, S., Swarnkar, M.K., Malik, A.H., Wani1, Z.A., Abbas, N., Singh, A.K., and Ashraf, N. 2015. Comprehensive transcriptome analysis of Crocus sativus for discovery and expression of genes involved in apocarotenoid biosynthesis. BMC Genomics 16 (698): 1-14.
Bathaie, S.Z., Ashrafi, M., Bolhasani, A., Etemadi-kia, B., and Moosavi-movahedi, A.A. 2006 Purification of carotenoids and monoterpen aldehydes from Iranian saffron and investigation of their effect on the structure of DNA, histone H1 and H1-DNA complex. Iranian Journal of Medicinal and Aromatic Plants 22 (2): 85-97. (In Persian with English Summary).
Beiki,  A.H.,  Keify,  F.,  and  Mozafari,  J.  2011. Rapid  genomic  DNA  isolation  from  corm  of  Crocus species  for  genetic  diversity  analysis. Journal of Medicinal Plants Research 5 (18): 4596-4600.
Bendtsen, J.D., Nielsen, H., Von Heijne, G., and Brunak, S. 2004. Improved prediction of signal 3.0. Journal Moulecular Biology 340 (4): 783-795.
Bertoni, M., Kiefer, F., Biasini, M., Bordoli, L., and Schwede, T. 2017. Modeling protein quaternary structure of homo- and hetero-oligomers beyond binary interactions by homology. Scientific Reports 7 (1): 10480.
Bhat, A., Mishra, S., Kaul, S., and Dhar, M.K. 2018. Elucidation and functional characterization of CsPSY and CsUGT promoters in Crocus sativus L.. Plos One 13 (4): 1-15.
Bienert, S., Waterhouse, A., de Beer, T.A.P., Tauriello, G., Studer, G., Bordoli, L., and Schwede, T. 2017. The SWISS-MODEL Repository - new features and functionality. Nucleic Acids Research 45: D313-D319.
Bilas, R., Szafran, K., Hnatuszko-Konka, K., and Kononowicz, A.K. 2016. Cis-regulatory elements used to control gene expression in plants. Plant cell, Tissue and Organ Culture (PCTOC) 127 (2): 269-286.
Brown, AK., Sridharam, S., Kremer, L., Lindenberg, S., Dover, LG., Sacchettini, JC., and Besra, GS., 2005. Probing the mechanism of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III mtFabH: factors influencing catalysis and substrate specificity. The Journal of Biological Chemistry 280 (37): 32539-32547. 
Brown, T.A., 2007. Genome 3, 3rd ed, c2006. Khosravi Publication, Tehran. (In Persian).
Busconi, M., Colli, L., Sanchez, R. A., Santaella, M., Pascual, M., Santana, O., Roldan, M., Fernandez, A. 2015. AFLP and MS-AFLP analysis of the variation within Saffron Crocus (Crocus sativus L.) germplasm. PLoS ONE 10 (4): e0123434.
Combet, C., Blanchet, C., Geourjon, C., and Deleage, G. 2000. NPS@: Network protein sequence analysis. Trends in Biochemical Science 25 (3): 147-150.
Cunningham, F.X., and Gantt, E. 1998. Genes and enzymes of carotenoid biosynthesis in plants. Annual Review of Plant Physiology and Molecular Biology 49: 557-583.
El-Gebali, S., Mistry, J., Bateman, A., Eddy, S.R., Luciani, A., Potter, S.C., Qureshi, M., Richardson, L.J., Salazar, J.A., Smart, A., Sonnhammer, E.L.L., Hirsh, L.,  Paladin, L., Piovesan, D., Tosatto, S.C.E., and Finn, R.D. 2019. The Pfam protein families database in 2019. Nucleic Acids Research  48 (D1): D427- D432.
Emanuelsson, O., Nielsen, H., and von Heijne, G. 1999. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Science 8 (5): 978- 984.
Gasteiger, E., Hoogland, Ch.,  Gattiker, A., Duvaud, S.,  Wilkins, M.R., Appel, R.D.,  and Bairoch, A. 2005. Protein Identification and Analysis Tools on the ExPASy Server. The Proteomics Protocols Handbook. Humana Press, New York. pp. 571-607.
Fallah Ziarani, M., Tohidfar, M., and Aminfar, Z. 2017. Bioinformatic analysis of Acyl Carier Protein (ACP) in eukaryotes and prokaryotes. Crop Biotech 17: 15-29 (In Persian with English Summary).
Giuliano, G., Al-Babili, S., and Lintig, J. 2003. Carotenoid oxygenases: cleave it or leave it. Plant Science 8 (4): 145-148.
Goli, S.A.H., Mokhtari, F., and Rahimmalek, M. 2012. Phenolic Compounds and antioxidant activity from saffron (Crocus sativus L.) petal. Journal of Agricultural Science 4 (10): 175-181.
Golovnina, K.A., Glushkov., S.A., Blinov., A.G., Adkison, L.R., and Goncharov, N.P. 2007. Molecular phylogeny of the genus Triticum. Plant Systematics and Evolution 264 (3-4): 195-216.
Gomez-Gomez, L., Rubio-Moraga, A., and Ahrazem, O. 2010. Understanding carotenoid metabolism in saffron stigmas: unravelling aroma and colour formation. Functional Plant Science and Biotechnology 4 (2): 56-63.
Grilli, M.C., and Canini, A. 2004. Ultrastructure of chromoplasts and other plastids in Crocus sativus L. (Iridaceae). Plant Biosystems 138 (1): 43-52.
Guex, N., Peitsch, M.C., and Schwede, T. 2009. Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective. Electrophoresis 30 (S1): S162-S173. 
Hosseinpour azad, N., Nematzadeh, G.H., Gouliano, G., Ranjbar, G.A., and Yamch, A. 2016. Identification of Apo- Carotenoids' crocin and crocetin isomers in saffron crude extracts by HPLC coupled to atmospheric pressure chemical ionization and high resolution orbitrap mass spectrometry. Saffron Agronomy and Technology 4 (4): 291-299. (In Persian with English Summary).
Huang, FC., Horváth, G., Molnár, P., Turcsi, E., Deli, J., Schrader, J., Sandmann, G., Schmidt, H., and Schwab, W. 2009. Substrate promiscuity of RdCCD1, a carotenoid cleavage oxygenase from Rosa damascene. Phytochemistry  70 (4): 457-464.
Krogh, A., Larsson, B., von Heijne, G., and Sonnhammer, EL. 2001. Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. Journal of Molecular Biology 305 (3): 567-580.
Kyte, J., and Doolitelle, R.F. 1982. A simple method for displaying the hydropathic character of a protein. Journal of Molecular biology 157 (1): 105-132.
Ma, G., Zhang, L., Matsura, A., Matsura, K., Yamawaki, K., Yahata, M., Wahyudi, A., Motohashi, R., and Kato, M. 2013. Enzymatic formation of beta-citraurin from beta-cyptoxanthin and zeaxanthin by carotenoid cleavage dioxygenase4 in the flavedo of citrus fruit. Plant Physiology 163: 682-695.
Maniatis, T., Fritsch, E., and Sambrook, F. 1995. Molecular Cloning. A laboratory Manual. Cold Spring Harbor Laboratory, New York .
Messing, S.A.,  Gabelli, S.B., Echeverria, I., Vogel,  T.J., Guan, J.C.,   Cai Tan, B., Klee, H.J., McCarty, R.D., and Amzel, L.M. 2010. Structural insights into maize viviparous14, a key enzyme in the biosynthesis of the phytohormone abscisic acid. Plant Cell 22 (9): 2970-2980.
Mirhoseini, S.Z., Pezeshkian, Z., and Ghovvati, Sh. 2016. Phylogenetic and In Silico Analysis of Interferon Beta-1b Protein. Journal Mazandaran University Medical Science 26 (145): 70-82. (In Persian).
Naghavi, M R., Malboobi, M.A., and Rashidi, S. 2009. Bioanformatics. University of Tehran Press. (In Persian).
Nassaj Hoseini, S.M., and Shamsbakhsh, M. 2010. Phylogenetic Analysis Methods. Haghshenass Publication, Rasht.(In Persian).
Nogueira, M., Mora, L., Enfissi, E.M., Bramley, P.M., and Fraser, P.D. 2013. Subchromoplast sequestration of carotenoids affects regulatory mechanisms in tomato lines expressing different carotenoid gene combinations. Plant Cell 25: 4560–4579.
Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. 2004. UCSF Chimera-avisualization system for exploratory research and analysis. Journal of Computational Chemistery 25 (13): 1605-1612.
Petersen, T.N., Brunak, S., Von Heijne, G., and Nielsen, H. 2011. SignalP 4.0: discriminating signal peptides from transmembrane region. Nat Metthod 8 (10): 785-786.
Rubio, A., Rambla, J.L., Santaella, M., Gomez, D., Orzaez, D., Granell, A., and Gomez-Gpmez, L. 2008. Cytosolic and plastoglobule-targeted carotenoid dioxygenases from Crocus sativus are both involved in β-Ionone release. The Journal of Biological Chemistry 283 (36): 24816–24825.
Rubio, A., Rambla, JL., Ahrazem, O., Granell, A., and Gomez-Gomez, L. 2009. Metabolic and target transcript analysis during Crocus sativus stigma development. Phytochemistry 70 (8): 1009-1016.
Schwartz, S.H., Tan, B.C., Gage, D., Zeevaart, J.A.D., and McCarty, D.R. 1997. Specific oxidative cleavage of carotenoids by VP14 of Maize.  Science 276 (5320): 1872- 1874.
Messing, S.A., Gabelli, S.B., Echeverria, I., Vogel, J.T., Guan, J.C., Tan, B.C., Klee, H.J., McCarty, D.R., and Amzel, L.M. 2010. Structural insights into maize viviparous14, a key enzyme in the biosynthesis of the phytohormone abscisic acid. Plant Cell 22 (9): 2970-2980.
Sivakumar, K. 2005.WWW.Databases tools and prediction server for protein sequence analysis and characterization. Advance Biotechnology 27-31.
Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. 2013. MEGA6: Moist lecular evolutionary genetics analyze version 6.0. Molecular  Biology and Evolution 30: 2725-2729.
Tarmontano, A., Leplae, R., and Morea, V. 2001. Analysis and assessment of comparative modeling predictions in proteins. Proteins 45 (S5): 22-38.
Varjonen, E., Vainio, E., Kalimo, K., and Juntunen-Backman, K. 2002. Clinical importance of non-specific lipid transfer proteins as food allergens. Biochemical Society Transactions 30 (6): 910-913.
Wang, Y., You, F.M., Lazo, G.R., Lou, M-C., Thilmony, R., Gordon, S., Kianian, Sh.F., and Gu, Y.Q. 2013. PIECE: a database for plant gene structure comparison and evolution. Nucleic Acids Research 41:1159–1166.
Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., de Beer, T.A.P., Rempfer, C., Bordoli, L., Lepore, R., and Schwede, T. 2018. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Research 46 (W1): W296-W303.
Yousefi Javan, I., and Gharari, F. 2017. The structure of the protein and gene expression of PIC2 affecting blooming flowers (Crocus sativus L.). Saffron Agronomy and Technology 5 (1): 73-90.