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Achillea Millefolium L. جداسازی ژنهای لینالول سنتاز و پینن سنتاز از گیاه دارویی بومادران ) (

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Achillea Millefolium L. جداسازی ژنهای لینالول سنتاز و پینن سنتاز از گیاه دارویی بومادران ) ( پژوهشهای ژنتیک گیاهی / جلد 2 / شماره 1 Achillea millefolium L. جداسازی ژنهای لینالول سنتاز و پینن سنتاز از گیاه دارویی بومادران ) ( مریم جاودان اصل1، حمید رجبی معماری2،*، داریوش نباتی احمدی2 و افراسیاب راهنما قهفرخی2 1- دانشآموخته کارشناسی ارشد، گروه زراعت و اصﻻحنباتات، دانشکده کشاورزی، دانشگاه شهید چمران، اهواز 3- استادیار، گروه زراعت و اصﻻحنباتات، دانشکده کشاورزی، دانشگاه شهید چمران، اهواز )تاریخ دریافت: 12/70/1232 – تاریخ پذیرش: 1232/13/30( چکیده بومادران ).Achillea millefolium L( گیاهی علفی و چندساله از خانوادهی گل ستتاره ایهتا )Asteraceae( متی باشتد استان بومادران دارای ترکیبهایی از جمله مونوترپن و سزکوئیترپنهای مختلف است که لینالول و پیتنن از اجتزای اصتلی تشتکیل دهنده آن میباشند این دو ترکیب دارای ارزش دارویی و اثرات ضدآفت و ضدمیکروبی هستند و در صنایع غذایی، عطرسازی و آرایشی و بهداشتی کاربرد دارند هدف از تحقیق حاضر، استفاده از راهکار آغازگرهای هرز برای جداسازی ژن های لینتالول سنتاز و پینن سنتاز از گیاه دارویی بومادران میباشد در این تحقیق RNA کل از گیاه بومادران استتخرا شتد، ستس ژنهتای موردنظر با استفاده از آغازگرهای هرز و واکتنش زنجیت رهای پلیمتراز )PCR( تکثیتر گردیدنتد نتتای حاصتل از PCR، تکثیتر باندهای مورد نظر به ترتیب حدود 037 و 357 جفت باز را نشان داد با آنالیزهای بیوانفورماتیکی، نتای توالییابی با دادههتای موجود در بانک ژن جهانی )NCBI( مقایسه گردید نتای بررسی تنوع بین گونهها و خت انوادههتای مختلتف گیتاهی و روابت فیلوژنتیکی بر اساس ژنهای Pis و Lis نشان داد بیشترین میزان مشابهت بین گیاه بومادران و درمنه )Artemisia annua( و نیز بین خانواده های Asteraceae و Lamiaceae وجود دارد، به نحوی که در یک گروه قرار گرفتند نتای این پتووهش، مشتابهت نسبتاً باﻻی توالی این ژنها را با ژنهای متناظر در سایر گیاهان نشان داد و صحت توالییابی را تأیید نمود واژگان کلیدی: آغازگر هرز، بومادران، پینن سنتاز، ترپنها، لینالول سنتاز Downloaded from pgr.lu.ac.ir at 4:08 IRST on Tuesday October 5th 2021 [ DOI: 10.29252/pgr.2.1.23 ] * نویسنده مسئول، آدرس پست الکترونیکی: [email protected] 32 …. جداسازی ژنهای لینالول سنتاز و پینن سنتاز از گیاه جاودان اصل و همکاران - Geranyl-diphosphate مقدمه ژرانیل دی فسفات ) ( بیوسنتز متی C H O Cseke et al., 1998 بومتتادران ).Achillea millefolium L( گیتتاهی علفتتی و شوند ) ( لینتالول ) 11 17 ( یتک چنتتدستتاله از ختتانواده گتتل ستتتارهایهتتا )Asteraceae یتتا مونوترپن طبیعتی و ترکیبتی بتا بتوی معطتر بتوده کته در Compositae( استتت )Omidbeygi, 2007( کتته در طتتب بسیاری از گلها وگیاهان معطر یافت میشتود و بته دلیتل سنتی برای درمان بیماریهتا بته طتور عتا و زخت هتا و بوی معطر، در ساخت 17-67 % از مواد بهداشتی و متواد سوختگیها به طور خاص مورد استتفاده قترار متی گرفتته تمیزکننده مانند شامسوها، صتابون هتا و لوستیون هتا متورد استتت )Tajik and Shokohi Sabet Jalali, 2009( در استفاده قرار میگیرد لینتالول بته عنتوان حشتره کتش نیتز β گیاهان متابولیتهای ثانویته بته سته گتروه اصتلی تقستی دارای کاربرد است از نا های دیگتر آن - لینتالول، 3و6 Rispail et میشوند: 1 ترپنها 3 فنلها 2 آلکالوئیتدها ) دیمتیل 3و0 اکتا دی ان6- ال و لینالیتل الکتل متی باشتد Christianson, 2006( al., 2005( لینتتالول باعتت جتتذ حشتترات ( تمتتتا ترپنوئیتتتدها از اتصتتتال واحتتتدهای . ایزوپنتیتتتتلدیفستتتتفات )IDP( و ایزومتتتتر آلیلتتتتی آن گتتردهافشتتان مخصوصتتاً زنبورعستتل متتیشتتود فوایتتد دیمتیتتلآلیتتلدیفستتفات )DMADP( ستتنتز متتیشتتوند آرومتتاتراپی آن عبتتارت از ایجتتاد روحیتته شتتاد در انستتان Buchbauer et al., 1991 )Cheng et al., 2007( ترپنوئیدها معموﻻً بر اساس تعداد متتیباشتتد ) ( لینتتالول دارای Loizzo et al., 2007 واحدهای ایزوپرنوئید موجود در ستاختار ختود بته چنتد خاصتتیت ضتتدتوموری استتت ) ( و A دستتتتته از جملتتتته مونوترپنوئیتتتتدها، دیترپنوئیتتتتدها، همچنین بهعنوان یک حدواس مه در تولید ویتامین و Mercier et al., 1994 E سزکوئیترپنوئیدها، تری ترپنوئیدها، تترا ترپنوئیدها تقسی نیز استفاده میشود ) ( لینالول که A. میشوند )Cseke et al., 1998( ترپنها ماهیتتاً بت ه علتت از ترکیبات روغن اساسی بیشتر زیرگونههای بومادران ) millefolium طع و بوی خاص و همچنین کیفیتت هتای دارویتی شتان ( می باشد، قادر است مانع رشتد بتیش از 10 Pattnaik et al., 1997 ترکیبتتات بستتیار مهمتتی از نظتتر اقتصتتادی متتیباشتتند نوع باکتری و 17 نوع قارچ شود ) ( C H )Pichersky et al., 1994( مشتقات ترپنی همچنین بترای پیتتنن بتتا فرمتتول 16 17 و وزن مولکتتولی 126/326 Belletti et al., g/mol تولید متواد دارویتی مختلتف ماننتد داروی ضتد سترطان یک مونوترپن دوحلقته ای معطتر ) 2004 تاکسول و داروی ضتد ماﻻریتای آرتمیتزینین )از درمنته( ( از طبقه ترپن است آلفا و بتا- پینن از نظر صتنعتی نقتتش دارنتتد )Jennewein and Croteau, 2001( گونتته بستتیار مهتت بتتوده و در ستتاخت انتتواع معطرکننتتدههتتا، Achillea millefolium دارای ترکیتتتبهتتتایی از جملتتته ضدعفونیکنندهها و حشرهکشها، صابونها، کر ها، بخور، مونوترپن و سزکوئیترپنهای مختلتف بته عنتوان اجتزا پاککنندهها، رنگ، ﻻک، روکش، ﻻستیک و تهیه حﻻلهتا Downloaded from pgr.lu.ac.ir at 4:08 IRST on Tuesday October 5th 2021 [ DOI: 10.29252/pgr.2.1.23 ] Chalchat et al., 2001 اصلی میباشد اسان آن بیشتر در کرکهای ترشحی گیاه بهکار میروند ) ( Artemisia Pis که در برگ، ستاقه و بته ویتوه در گتل هتا موجتود استت، تتتاکنون ژن در گیاهتتانی ماننتتد درمنتته ) annua Jaimand and Rezaee, 2004( از خانواده Lu et al., 2002( Asteraceae(، مری تشکیل میشود ) ( Gambliel and Croteau, Salvia officinalis پیتتنن )Pinene( و لینتتالول )Linalool( از اجتتزای اصتتلی گلتتی ) ( ) McKay et al., Cicer arietinum 1982 تشکیلدهنده استان بومتادران هستتند ) ,.Rohloff et al (، نختتود ) ( ) Bohlmann et al., Abies grandis 2003 2007( کتتته از نظتتتر ستتتاختار شتتتیمیایی مونتتتوترپن (، نتتراد ) ( ) Solanum habrochaites 1997 )Monoterpene( متتیباشتتند و بتتهترتیتتب توستت آنتتزی (، گوجتتته وحشتتتتی ) ( Gonzales-Vigil et al., 2012 پیننسنتاز )Bohlmann et al., 1997( )Pinene synthase( ) ( و ژن لینتالول ستنتاز در Jia et al., و لینالول ستنتاز )Linalool synthase( از متابولیتت میتانی بستیاری از گونته هتای گیتاهی ماننتد درمنته ) 32 پژوهشهای ژنتیک گیاهی / جلد 2 / شماره 1 1999(، ریحتان )Iijima et al., ( )Ocimum basilicum گردیدند. مقایسه و ه ردیفی توالیهای پروتئینی و 2004(، سیب )Nieuwenhuizen et ( )Malus domestica نوکلئوتیدی متناظر با هریک از ژنهای موردنظر در al., 2013( و آرابیدوپستتی )Arabidopsis thaliana( گیاهان خانوادههای نزدیک به خانواده بومادران و بهویوه )Aubourg et al., 2002( جداسازی و توالییتابی گردیتده گیاهان ه خانواده اش مانند درمنه صورت گرفت و Asteraceae است همچنین از گیاهان خانواده جداستازی براساس نواحی حفاظتشده، یک جفت آغازگر هرز سایر ژنهای مسیرهای دارویی ماننتد ژن پارتنولیتد ستنتاز )Degenerate primer( مستقی و معکوس برای هر یک از Tanacetum Parthenolide synthase ) ( از بابونتته کبیتتر) ژنهای Pis و Lis برای تکثیر قطعههای مورد نظر از ژنو Germacrene A Liu et al., 2014 parthenium ( ) (، گیاه بومادران طراحی گردید )جدول 1( ساخت آغازگرها Cichorium intybus synthase ، کاستتتتتتتتتتنی ) ( توس شرکت Bioneer کره جنوبی صورت گرفت Kim Ixeris dentata Bouwmeester et al., 2002 ) ( و ) استخراجRNA کل: استخرا RNA از نمونههای برگ و Amorpha-4,11-diene synthase et al., 2005 ( و ژن گل با استفاده از دستورالعمل Rneasy Plant Mini kit Chang et al.,2000 (ADS) از گیتتاه درمنتته ) ( گتتزارش (Qiagen) بر اساس روش ارائه شده توس شرکت گردیتتدهانتتد تتتاکنون هتتی اطﻻعتتاتی در متتورد ژنهتتای سازنده کیت انجا گردید بهمنظور حذف DNA از RNA سنتزکننده لینالول و پیتنن در گیتاه بومتادران در بانتک ژن استخرا شده، از آنزی DNase شرکت Fermentas آلمان جهانی وجود ندارد. پووهش حاضر، بهمنظتور جداستازی استفاده شد در نهایت لولهی حاوی RNA برای استفاده Pis Lis ژنهای و با طراحی پرایمرهتای مناستب، از گیتاه طوﻻنی مدت به فریزر C° 17- منتقل گردید کیفیت و بومادران هزاربرگ صورتگرفت کمیت RNA استخراجی با بارگذاری بر روی ژل آگارز 1% و تعیین غلظت توس دستگاه اسسکتروفتومتر بر اساس مواد و روشها جذ نوری 317/367 مشخص گردید برای محاسبهی نمونههای گل و برگ بومادران مورد استفاده در این غلظت RNA استخرا شده از فرمول زیر استفاده شد )X تحقیق از باغ گیاهشناسی دانشکده داروسازی دانشگاه = غلظت RNA تک رشتهای در نمونه( علو پزشکی جندیشاپور در مرحله گلدهی جمعآوری X ng/l = OD گردیدند نمونهها پ از برداشت، با نیتروژن مایع )دمای 57 367 °C °C 136-( تثبیت شدند و به فریزر 17- منتقل Mega 5 جدول 1- توالی های آغازگرهای مستقی و معکوس طراحی شده به کمک نر افزار [ Downloaded from pgr.lu.ac.ir at 4:08 IRST on Tuesday October 5th 2021 [ DOI: 10.29252/pgr.2.1.23 Table 1. The sequences of forward and backward primers desined by Mega5 توالی هرز توالی آغازگر آغازگر ژن Gene Primer Primer sequence (5' - 3') Degenerate sequence Pis F-PIN GAGTTTGCSAAACTSGACTTCAA S [C/G] Pis R-PIN GTACCATACACATCRTAAACATCRTC R [A/G] Lis F-LIN ACTCTCGATGATGTYTAYGA Y [C/T] Lis R-LIN GTATCACCTCTYTCCATYTC Y [C/T] 35 …. جداسازی ژنهای لینالول سنتاز و پینن سنتاز از گیاه جاودان اصل و همکاران سنتز رشته اول cDNA: برای تبدیل RNA به DNA تکثیر ژن پینن سنتاز به روش واکنش زنجیرهای پلیمراز: بهعنوان قالبی برای شروع فرآیند PCR، سنتزcDNA با بهمنظور سنتز DNA دورشتهای و تکثیتر و جداستازی ژن استفاده از کیت cDNA synthetase شرکت QIAGEN پینن سنتاز از آغازگرهای هرز مستقی و معکوس )F-PIN توس آنزی نسخهبردار معکوس ) Reverse و R-PIN( استفاده شد که این کار با استفاده از آنزی Taq Omniscript )Transcriptase و پرایمر Oligo-dT انجا DNA Polymerase شرکت Roche و cDNA ستنتز شتده گردید از RNA استخرا شده از گل بومتادر ان )بته دلیتل داشتتن تکثیر ژن پینن سنتاز و لینالول سنتاز به روش PCR: به کیفیت بهتر نسبت بته بترگ( بته عنتوان الگتو در واکتنش منظور تکثیر ژنهای Pis و Lis به روش PCR از زنجیرهای پلیمراز صورتپذیرفت بهمنظور تعیتین دمتای آغازگرهای دژنتره مستقی )Forward primer( و معکوس اتصال دقیق برای تکثیتر جفتت آغتازگر، از برنامته شتیب PCR PCR )cDNA ،)Reverse primer سنتزشده گل به عنوان الگو و دمایی استفادهشد پ از انجا ، محصتول بتر آنزی Taq DNA Polymerase شرکت Roche و Biolabs روی ژل آگارز 1% الکتروفتورز گردیتد نتتای حا صتل از bp استفاده گردید )جدول 3( جهت تعیین دمای اتصال دقیق الکتروفورز، تکثیر قطعه موردانتظار با طول حدود 357 برای تکثیر جفت آغازگر، از برنامه شیب دمایی استفاده را برای ژن پینن سنتاز نشان داد )شکل 3( و دمتای بهینته شد سس ، محصول PCR جهت تعیین توالی به شرکت برای اتصال، جهت تکثیر این ژن بهوسیله آغازگرهای
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