Genetic Transformation of Amylase Gene to Ruminal Bacteroides Species Using Conjugation Consequence for Improvement of Rumen Enzyme
محورهای موضوعی : Camelح. عقبی طلب 1 , ف. مرادیان 2 , ق. رحیمی 3 , ح. رحیمیان 4
1 - Department of Animal Science, Sari Agricultural Science and Natural Resources University, Sari, Iran
2 - Department of Basic Science, Sari Agricultural Science and Natural Resources University, Sari, Iran
3 - Department of Animal Science, Sari Agricultural Science and Natural Resources University, Sari, Iran
4 - Department of Plant Protection, Sari Agricultural Science and Natural Resources University, Sari, Iran
کلید واژه: 16S rRNA gene, Manipulation, <, i>, Bacteroides<, /i>, spp, conjugation, shuttle vector,
چکیده مقاله :
Rumen bacterial strains can potentially be manipulated to perform functions different from wild type species. The most numerous species of bacteria in the rumen and gut are species of the familyBacteroidetes, whichcan have the potential for genetic modification for enzyme production. One of the genetic manipulation of rumen bacteria can perform for production of starch digestive enzyme for the enhancement of nutrient flow to the rumen. In this study, Bacteroides species were isolated from rumen of cows. The 16S rRNA gene analysis was used to confirm the classification of the Bacteroides species. The amylase gene from Bacillus spp. was cloned into expression vector pET28A and then subcloned in Escherichia coli-Bacteroides pGFK114.1 shuttle vector. Conjugation between Escherichia coli and a Bacteroides strainwas accomplished with two Escherichia coli donors containing pGFK114.1 and pRK231 and one Bacteroides strain as a recipient. Transfer of amylase gene by vector was confirmed using a marker cefoxcitin antibiotic resistant gene. The amylase activity assay showed that successful expression of enzyme occurred in ruminal Bacteroides species.
سویههای باکتریهای شکمبهای قابلیت دستکاری برای انجام عملکرد متفاوت از نوع وحشی را دارند. بیشترین تعداد سویههای باکتری شکمبه و روده از خانواده باکتروئیدها هستند که توانایی تغییر ژنتیکی جهت تولید آنزیم را دارند. یکی از دستورزی ژنتیکی باکتری شکمبه که میتواند انجام شود، تولید آنزیم هضم کننده نشاسته جهت افزایش جریان مواد غذایی به شکمبه است. در مطالعه حاضر، سویههای باکتروئید از شکمبه گاو جدا شدند. ازآنالیز ژن 16S rRNA جهت تأیید طبقهبندی گونه باکتروئیدی استفاده شد. ژن آمیلاز در وکتور بیانی pET28A همسانهسازی شد و سپس در وکتور انتقالی pGFK114.1 باکتروئید-اشرشیاکولی زیر همسانهسازی گردید. کانژوگاسیون بین اشرشیاکولی و سویه باکتروئیدی با دو دهنده اشرشیاکولی شامل وکتورهایpGFK114.1 و pRK231 و یک سویه دریافت کننده باکتروئیدی انجام شد. انتقال ژن آمیلاز توسط مارکر ژن مقاومت به آنتی بیوتیک سفوکسیتین در وکتور تأییدگردید. سنجش فعالیت آمیلاز نشان داد که بیان آنزیم در سویه باکتروئید شکمبهای به طور موفقیت آمیزی انجام گرفته است.
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