fa استفاده از روش تراریزش توأم برای انتقال ژن کولین اکسیداز به برنج مهندسی ژنتیک گیاهی Plant پژوهشي Research <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">در</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">این</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">پژوهش</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">به</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">منظور</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">تولید</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">گیاه</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">برنج</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">تراریخته</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">با</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">ژن</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">کولین</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">اکسیداز</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">با</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">قابلیت</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">حذف</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">ژن نشانگر</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">انتخابی،</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">دو</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">پلاسمید</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">بیانی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">موسوم</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">به</span></span></strong> <strong><span dir="LTR"><span style="font-family:times new roman,serif;"><span style="font-size:12.0pt;">pABRII-Chl </span></span></span></strong>&nbsp;<strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">(ژن کولین اکسیداز حاوی پپتید راهنما برای تظاهر ژن در کلروپلاست) و</span></span></strong> <strong><span dir="LTR"><span style="font-family:times new roman,serif;"><span style="font-size:12.0pt;">pABRII-Cyt</span></span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">(</span></span></strong><strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">بدون</span></span></strong><strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;"> پپتید راهنما برای تظاهر ژن در سیتوپلاست) ساخته</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">شد</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">و</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">سپس</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">با</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">استفاده</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">از</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">روش</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">تراریزش</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">توأم،</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">به</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">همراه</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">پلاسمید </span></span></strong><strong><span dir="LTR"><span style="font-family:times new roman,serif;"><span style="font-size:12.0pt;">pTRA132</span></span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">حامل</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">ژن</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">مقاومت</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">به</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">هیگرومایسین</span></span></strong><strong><span dir="LTR"><span style="font-family:bzarbold;"><span style="font-size:13.0pt;">(</span></span></span></strong><strong><em><span dir="LTR"><span style="font-family:times new roman,serif;"><span style="font-size:12.0pt;">hph</span></span></span></em></strong><strong><span dir="LTR"><span style="font-family:bzarbold;"><span style="font-size:13.0pt;">) </span></span></span></strong><strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">،</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">به</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">کالوس های</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">جنین</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">زایی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">که</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">از</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">ناحیه</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">اسکوتلوم</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">بذور</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">رسیده</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">رقم</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">هاشمی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">منشا</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">گرفته</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">بودند،</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">به</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">روش</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">ریزپرتابی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">منتقل</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">شدند.</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">سلول</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">های</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">تراریخته</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">احتمالی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">از</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">بافت</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">های</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">بمباران</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">شده</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">پس</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">از</span></span></strong><strong><span dir="LTR"><span style="font-family:bzarbold;"><span style="font-size:14.0pt;"> 3 </span></span></span></strong><strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">دوره</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">گزینش</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">در</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">محیط</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">کالو</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">سزای</span></span></strong><strong><span dir="LTR"><span style="font-family:times new roman,serif;"><span style="font-size:12.0pt;">N6</span></span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">حاوی </span></span></strong><strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">هیگرومایسین</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">ب</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">گزینش</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">شدند.</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">به</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">صورتی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">که</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">غلظت</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">آنتی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">بیوتیک</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">از</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">60</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">میلی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">گرم</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">در</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">لیتر</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">به</span></span></strong><strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">80 </span></span></strong><strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">میلی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">گرم</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">در</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">لیتر</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">افزایش</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">یافت.گیاهان برنج تراریخته ی احتمالی به روش آنالیز زنجیره ای پلیمراز، آنالیز سادرن و </span></span></strong><strong><span dir="LTR"><span style="font-family:times new roman,serif;"><span style="font-size:12.0pt;">RT-PCR</span></span></span></strong><strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;"> مورد بررسی قرار گرفتند و حضور ژن و بروز آن مورد تایید قرار گرفت.</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">فراوانی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">قابل</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">قبول</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">گیاهان</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">تراریخته</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">حاصل</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">از</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">روش</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">تراریزش</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">همزمان</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">در</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">این پژوهش</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">نشان</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">داد</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">که</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">روش</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">مورد</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">استفاده</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">یک</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">روش</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">تکرار</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">پذیر</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">و</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">نسبتاً</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">کارا</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">برای</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">انتقال</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">پایدار ژن</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">های</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">مفید</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">و</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">تولید</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">گیاهان</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">تراریخته</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">عاری</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">از</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">ژن</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">انتخابگر</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">در</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">مقیاس</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">وسیع</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">است.</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">انتقال</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">ژن مقاومت</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">به</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">آنتی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">بیوتیک</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">در</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">ساختار</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">جداگانه</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">امکان</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">جداسازی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">این</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">ژن</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">از</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">محصول</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">نهایی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">را</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">در نتیجه</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">تفرق</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">فراهم</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">می</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">آورد</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">که</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">از</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">جنبه</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">ایمنی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">زیستی</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">حائز</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">اهمیت</span></span></strong> <strong><span style="font-family:b mitra;"><span style="font-size:14.0pt;">است</span></span></strong><strong><span dir="LTR"><span style="font-family:bzarbold;"><span style="font-size:14.0pt;">.</span></span></span></strong> In order to produce a marker-free transgenic rice with improved tolerance to salinity and drought stresses, expression vectors pABRII-Chl and pABRII-Cyt containing &quot;<em>choline oxidase</em>&quot; gene (with or without leader sequence respectively) were constructed from pChl and pCyt and pTRA132 for co-transformation. The pChl and pCyt vectors were digested with <em>Hin</em>dIII-<em>Bam</em>H and <em>Bam</em>HI<em>Eco</em>RI enzymes. Then the resulting sequences were ligated and inserted into expression vector pTRA132, in which the <em>Hind</em>III-<em>Eco</em>RI fragment (<em>hph </em>gene) had been deleted. The constructs pABRII-Chl or pABRII-Cyt and pTRA132 (containing <em>hph </em>gene) were introduced into embryogenic calli derived from the mature seeds of a rice cv. Hashemi by biolistic transformation method. Then putative transformants were screened after 3 rounds of selection on N6 medium containing increasing concentrations of Hygromycin B from 60 to 80 mg/L. Finally, Hygromycin resistant calli were regenerated on MS medium supplemented with 50 mg/L Hygromycin B. Putative transgenic rice plants were analyzed by polymerase chain reaction PCR. Then, four of the transgenic plants were analyzed using Southern blotting. Each transgenic plant received one copy number of both <em>choline oxidase </em>and <em>hph </em>genes. Expression of the transgene was confirmed by reverse transcription PCR. The high frequency of transformation rate in this study showed that co-transformation method is a reliable method for stable transformation with the goal to make markerfree transgenic plants in subsequent steps. برنج هاشمی, تراریزش توام, ریزپرتابه, ژنhph , کولین اکسیداز, کولین اکسیداز, گلایسین, بتائین Choline oxidase, Glycine Betaine, Co-transformation, Rice, hph Gene, Biolistic. 37 42 http://gebsj.ir/browse.php?a_code=A-10-252-1&slc_lang=fa&sid=1 Saeideh Keyarsalan سعیده کی ارسلان saeideh.keyarsalan@gmail.com 10031947532846001752 10031947532846001752 Yes کارشناس ارشد بیوتکنولوژی و اصلاح نباتات Elyas Mortazavi الیاس مرتضوی Mortazavi@abrii.ac.ir 10031947532846001753 10031947532846001753 No پژوهشکده بیوتکنولوژی کشاورزی ایران Behzad Ghareyazie بهزاد قره یاضی ghareyazie@yahoo.com 10031947532846001754 10031947532846001754 No پژوهشکده بیوتکنولوژی کشاورزی ایران Sakineh Mehranie سکینه مهرانی 10031947532846001755 10031947532846001755 No کارشناس ارشد بیوتکنولوژی و اصلاح نباتات