Genetic Transformation of Amylase Gene to Ruminal Bacteroides Species Using Conjugation Consequence for Improvement of Rumen Enzyme
محورهای موضوعی : Camel
ح. عقبی طلب
1
(Department of Animal Science, Sari Agricultural Science and Natural Resources University, Sari, Iran)
ف. مرادیان
2
(Department of Basic Science, Sari Agricultural Science and Natural Resources University, Sari, Iran)
ق. رحیمی
3
(Department of Animal Science, Sari Agricultural Science and Natural Resources University, Sari, Iran)
ح. رحیمیان
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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