جداسازي، همسانه‌سازي، بررسي بيوانفورماتيكي ويژگي‌هاي پروتئيني و تحليل بيان ژن دي هيدروبنزوفنانتريدين اكسيداز (DBOX) گياه دارويي شقايق (Papaveer somniferum L.)

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

گياه شقايق P. somniferum از مهم‌ترين گياهان داروئي به شمار رفته و تنها منبع بسياري از آلكالوئيدهاي بنزيل ايزوكوينوليني (BIAs) محسوب مي‌گردد. آلكالوئيدهاي بنزيل ايزوكوينوليني گروه بزرگ و پيچيده‌اي از متابوليت‌هاي ثانويه گياهي بوده كه از خواص داروئي و درماني قابل توجهي بر خوردار هستند. در مطالعه حاضر (در آزمايشگاه بيوتكنولوژي دانشگاه لرستان در سال 1393-1395) به منظور جداسازي و همسانه‌سازي ژن DBOXژنوتيپ ايراني گياه شقايق، ابتدا توالي مورد‌نظر با استفاده از آغازگرهاي طراحي‌شده، تكثير و سپس قطعه توليد‌شده به پلاسميد pTZ57R/T منتقل گرديد. نتايج توالي‌يابي قطعه 1614 جفت‌بازي را معين نمود كه با 100 درصد همساني با ژن DBOX گياه شقايق شباهت داشت. توالي پروتئيني كد‌شده توسط اين ژن مورد تجزيه و تحليل بيوانفورماتيكي قرار گرفت و سه دامنه عملكردي FAD binding، Berberine و FAD/FMN-containing dehydrogenase در توالي پروتئيني و وجود يك سيگنال پپتيد در انتهاي آميني آن مشخص گرديد. همچنين نتايج نشان داد كه بيشترين تجمع پروتئين كد‌شده توسط ژن DBOX در ناحيه غشاي سلولي و پلاسمايي بوده و بيشترين عملكرد اين پروتئين در ناحيه خارج سلولي مي‌باشد. جهت تجزيه و تحليل بيان ژن DBOX و تعيين الگوي بياني آن در بافت‌هاي مختلف گياهي، تعداد 5 كتابخانه ترنسكريپتومي حاصل از داده‌هاي RNA-seq گياه شقايق با شماره‌هاي دسترسي ERX651037، ERX651023، ERX651056، ERX651082 و ERX651062 از پايگاه SRA سايت NCBI دريافت شد. تعداد توالي‌هاي حاصل از همرديفي بين بافت‌هاي مختلف با استفاده از آزمون‌هاي آماري Fisher exact test و Chi-squared 2X2 مقايسه گرديد. نتايج تجزيه و تحليل بيان ژن با استفاده از تكنيك real time RT-PCR و آزمون‌هاي In silico نشان داد كه بيشترين سطح بيان اين ژن در ريشه و كمترين آن در ساقه مي‌باشد.

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.