بررسی امکان انتقال cDNA ژن منگنز‌پراکسیداز (mnp) از قارچ صدفی به قارچ دکمه‌ای سفید با کمک آگروباکتریوم

نوع مقاله : مقالات پژوهشی

نویسندگان

1 دانشگاه فردوسی مشهد

2 دانشگاه زنجان

چکیده

قارچ دکمه‌ای سفید یکی از مهم‌ترین محصولات باغبانی در دنیا به شمار می‌رود که در برداشت سوم محصول قابل قبولی تولید نمی‌کند. دلیل این امر کاهش مواد غذایی و ناتوانی این قارچ در استفاده بهینه از کمپوست ذکر شده است. تغییر بیان یا نوع آنزیم‌های موثر در تجزیه ترکیبات لیگنینی نظیر منگنزپراکسیداز راه‌حل‌های احتمالی حل این مشکل می‌باشند، که به نظر می رسد بتوان با بهره‌گیری از روش انتقال ژن از طریق آگروباکتریوم به این هدف دست یافت. در این پژوهش از قارچ خوراکی صدفی واریته فلوریدا به‌عنوان منبع ژن منگنزپراکسیداز و بافت‌های تیغه و کلاهک قارچ دکمه‌ای سفید نژاد 737 به‌عنوان گیرنده ژن استفاده شدند. باکتری آگروباکتریوم سویه‌ی LBA4404 دارای پلاسمید p13H88-FM نیز به‌عنوان ناقل به کار رفت. محیط کشت گزینشگر حاوی 30 میکروگرم بر میلی‌لیتر آنتی‌بیوتیک هیگرومایسین برای انتخاب ریزنمونه‌های تراریخت مورد استفاده قرار گرفت. ریزنمونه‌های تیغه که نرخ تراریزش آن‌ها پنج درصد بود، بهتر از ریزنمونه‌های کلاهک که نرخ تراریزش آن‌ها صفر درصد بود، به روش تراریزش مورد استفاده پاسخ دادند. علاوه بر توانایی رشد بر روی محیط کشت انتخابی، واکنش زنجیره‌ای پلیمراز با آغازگرهای اختصاصی ژن‌های hph و mnp به‌عنوان یکی از روش‌های تأیید تراریختگی، سبب تکثیر قطعات به ترتیب 1049 و 1086 نوکلئوتیدی شد و تراریختگی کلنی‌های قارچی را تأیید کرد.

کلیدواژه‌ها


عنوان مقاله [English]

Investigation on Possibility of Transferring OysterMushroom (Pleurotusostreatus) Manganese Peroxidase Gene (mnp) to the White Button Mushroom (Agaricusbisporus)

نویسندگان [English]

  • Mojgan Parvandi 1
  • Mohammad Farsi 1
  • Mohsen Ashrafi 2
1 Ferdowsi University of Mashhad
2 University of Zanjan
چکیده [English]

Introduction: The white button mushroom does not produce remarkable yield in the third flash. Nutritional deficiency and the inability of this mushroom to efficient use of compost are mentioned as its reasons. Basically, compost includes two major food components, lignocellulose and microbial biomass. But this microbial biomass provides just 10% of button mushroom food needs. According to research studies, differentenzymes in both white button mushroom and oyster mushroom are responsible for decomposition of lignin compounds in compost media, from begin of mycelium grows to the end of fruiting. Lacasse, manganese peroxidase, lignin peroxidase, glyoxal oxidase enzymes contribute to degradation of lignin compounds in degradation mushroom has proven by researchers however itis dependent on mushroom types. Manganese peroxidase enzyme (EC. 1.11.1.13) is an extracellular parser lignin enzyme that has a central peroxidase core. Manganese peroxidase enzyme oxidizesMn2+ to Mn3+ and then Mn3+ oxidizes phenolic structure to fonoxile radical. Produced Mn3+ is very active and makes complex by chelating organic acids that is produced by mushrooms such as oxalate or malate. Mn3+ ions become stable by helping of these chelates and it can penetrate through materials such as wood. On the other hand, in recent years, plant biotechnology provides new solutions for old problems such as use of microorganisms, particularly using bacteria for gene transfer and improvement of superlatives. For a sample of this method, Agrobacterium-mediated transformation system can be noted. In addition, the use of suitable promoters for heterologous genes expression in suitable hosts is an important strategy in functional biotechnology that has been raised in edible mushroom genetic engineering. The lack of efficient and sufficient use of compost, low power of white button mushroom in competition with other rivals, lack of yield per area unit due to production costs, pests and diseases, low flexibility and adaptability with environmental conditions changes are some of the problems that the mushroom reformers are faced. Unlike the great efforts made by researchers, conventional breeding techniques to produce the A. bisporus mushroom only have been led to produce a few new races. Therefore, todays some problems associated with traditional methods of breeding of edible mushrooms, including the need to provide races that have desired characteristics, the traditional method performance tests and low chances of success in the transfer of important agronomic characteristics such as functionality and disease resistance. So, they almost have been replaced with new biotechnology methods. Anexample of this method is to manipulateproperties transformation for the particular purpose. Modification of both expression or type of lignin degrading enzyme are possible solutions to deal with this problem, but these are not applicable or are difficult to be done with traditional breeding programs. In recent years, gene transformation mediated with Agrobacterium routinely is used for gene transformation to mushrooms and is proposed as a method for removing limitations of white button mushroom breeding.
Materials and Methods: In this research, the oyster mushroom strain Florida was used as the source of manganese peroxidase (mnp) gene and white button mushroom strain 737 gill and cap tissue were used as transformation host. Agrobacterium strain LBA4404 harbors p133H88-FM plasmid thatcontainsmnp gene of oyster mushroom and also hph gene under control of gpdII promoter of the button white mushroom strain IM008 was used as a transformer. Selection medium containing 30 mg/ml Hygromycin B and was used for selecting transformed explants. To confirm transformation, PCR with specific primers of mnp and hph genes was performed on genomic DNA of selected colonies.
Results and Discussion: Results showed the gill tissue explants, with transformation rate 5%, have a better response to applied transformation method than cap tissue explants, with transformation rate zero percent. As expected, polymerase chain reaction with specific primers ofhph and mnp genes amplified 1049 and 1086 bp fragments and verified the transformation of mycelium's grown on selection medium. It seems that Bacterial strain and also used plasmid were one of the responses for observed low rate transformation which is in accordance with leach and co-workers study. Finally, we could propose that cap tissue is more suitable for further gene transformation of this mushroombecause of high transformation rate of cap tissue.

کلیدواژه‌ها [English]

  • Agrobacterium
  • Compost
  • Gene transformation
  • Lignin degradation
  • white button mushroom
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