با همکاری انجمن علمی گیاهان دارویی 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.

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
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