جداسازی، همسانه‌سازی، بررسی بیوانفورماتیکی ویژگی‌های پروتئینی و تحلیل بیان ژن دی هیدروبنزوفنانتریدین اکسیداز (DBOX) گیاه دارویی شقایق (Papaveer somniferum L.)

نوع مقاله: علمی - پژوهشی

نویسندگان

1 دانشجوی دکتری اصلاح نباتات، دانشکده کشاورزی، دانشگاه لرستان، لرستان، خرم‌آباد، ایران

2 استاد، گروه زراعت و اصلاح ‌نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

3 استاد، گروه زراعت و اصلاح‌نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

4 دانشجوی دکتری بیوتکنولوژی گیاهی، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

چکیده

چکیده
گیاه شقایق P. somniferum از مهم‌ترین گیاهان داروئی به شمار رفته و تنها منبع بسیاری از آلکالوئیدهای بنزیل ایزوکوینولینی (BIAs) محسوب می‌گردد. آلکالوئیدهای بنزیل ایزوکوینولینی گروه بزرگ و پیچیده‌ای از متابولیت‌های ثانویه گیاهی بوده که از خواص داروئی و درمانی قابل توجهی بر خوردار هستند. در مطالعه حاضر (در آزمایشگاه بیوتکنولوژی دانشگاه لرستان در سال 1393-1395) به منظور جداسازی و همسانه‌سازی ژن DBOXژنوتیپ ایرانی گیاه شقایق، ابتدا توالی مورد‌نظر با استفاده از آغازگرهای طراحی‌شده، تکثیر و سپس قطعه تولید‌شده به پلاسمید pTZ57R/T منتقل گردید. نتایج توالی‌یابی قطعه 1614 جفت‌بازی را معین نمود که با 100 درصد همسانی با ژن DBOX گیاه شقایق شباهت داشت. توالی پروتئینی کد‌شده توسط این ژن مورد تجزیه و تحلیل بیوانفورماتیکی قرار گرفت و سه دامنه عملکردی FAD binding، Berberine و FAD/FMN-containing dehydrogenase در توالی پروتئینی و وجود یک سیگنال پپتید در انتهای آمینی آن مشخص گردید. همچنین نتایج نشان داد که بیشترین تجمع پروتئین کد‌شده توسط ژن DBOX در ناحیه غشای سلولی و پلاسمایی بوده و بیشترین عملکرد این پروتئین در ناحیه خارج سلولی می‌باشد. جهت تجزیه و تحلیل بیان ژن DBOX و تعیین الگوی بیانی آن در بافت‌های مختلف گیاهی، تعداد 5 کتابخانه ترنسکریپتومی حاصل از داده‌های RNA-seq گیاه شقایق با شماره‌های دسترسی ERX651037، ERX651023، ERX651056، ERX651082 و ERX651062 از پایگاه SRA سایت NCBI دریافت شد. تعداد توالی‌های حاصل از همردیفی بین بافت‌های مختلف با استفاده از آزمون‌های آماری Fisher exact test و Chi-squared 2X2 مقایسه گردید. نتایج تجزیه و تحلیل بیان ژن با استفاده از تکنیک real time RT-PCR و آزمون‌های In silico نشان داد که بیشترین سطح بیان این ژن در ریشه و کمترین آن در ساقه می‌باشد.
 
کلیدواژه‌ها: الگوی بیان ژن، ترنسکریپتوم، توالی‌یابی، دامنه عملکردی، شقایق

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

Isolation, Cloning, Bioinformatics Analysis of Protein Properties and Gene Expression Analysis of Dihydrosanguinarine Oxidase (DBOX) from Opium Poppy (Papaveer somniferum L.)

نویسندگان [English]

  • Kamran Samiey 1
  • Ahmad Ismaili 2
  • Farhad Nazarian Firoz-abadi 3
  • Seyed Mohsen Sohrabi 4
1 Ph.D. Student of Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
2 Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
3 Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
4 Ph.D. Student of Plant Biotechnology, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
چکیده [English]

Abstract
 
Background and Objectives
Opium poppy (Paoaver somniferum L.) is one of the important medicinal plants considered the only source for several high-value pharmaceutically BIAs. Benzylisoquinoline alkaloieds (BIAs) are a very large and complex group of plant alkaloids. Today’s various methods of genetic engineering are applied to improve the biosynthetic pathways in medicinal plants. For successful implementation of techniques such as gene silencing and over-expression, it is important to get accurate and complete information about genetic characteristics of related genes in the biosynthesis of secondary metabolites. DBOX is one of the important genes in biosynthesis of sanguinarin and papaverin alkaloids of poppy. According to the cause, the present study was performed on DBOX to explain genetic characteristic and gene expression pattern.
 
Materials and Methods
Specific primers were designed based on databases, and complete sequences of DBOX gene were amplified using DNA and cDNA as a template, and then were cloned in pTZ57R/T plasmid. After sequencing of cloned fragments, CDD, CELLO and ProtParam tools were used to determine ORFs, domains and the location of protein accumulation. Phylogenetic relationship between the gene in this plant with other plants was determined. Finally, the pattern of gene expression in different tissues was assessed using real time RT-PCR technique and RNA-seq data derived from SRA data bank.
 
Results
Sequencing results showed the 1614-bp fragment that has 100% identity with the DBOX gene of P. somniferum. The protein encoded by this gene was analyzed by bioinformatics methods. Three domains including FAD binding, Berberine and FAD/FMN-containing dehydrogenase in the protein sequence were identified and the existence of an N-terminal signal peptide was also determined in this protein. The results also showed that the highest concentration of protein was present in the plasma membrane and highest function of this protein was in the extracellular. In order to analyze and determine the expression pattern of DBOX gene in different tissues, 5-gene libraries of RNA-seq data of opium poppy (with ERX651037, ERX651023, ERX651056, ERX651082 and ERX651062 access numbers) derived from NCBI-SRA were analyzed. The numbers of sequences from the alignment between different tissues were compared using the Fisher exact test and Chi-squared 2X2 tests. Results of the gene expression analysis showed that the highest level of gene expression of this gene performed in roots and the lowest level of gene expression occurred in the stems. In addition, results of the real-time RT-PCR experiment were similar to in silico tests and the present results demonstrated that DBOX gene transcript levels in root were significantly more than other plant tissues.
 
Discussion
Results showed that DBOX gene is an intron less gene. According to existing domains, this gene is a member of a FADOX gene family. Due to the presence of FAD and berberin like domains in protein sequence of this gene, its key function in biosynthesis of BIA alkaloids was confirmed. Different expression patterns of DBOX gene in root confirmed accumulation of high value sanguinarin alkaloid in this tissue. 

کلیدواژه‌ها [English]

  • Functional domain
  • Gene expression pattern
  • Opium poppy
  • Sequencing
  • Transcriptome
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