تهیه سازه برای ایجاد مقاومت همزمان به سه ویروس ‏PLRV، ‏PVX‏ و ‏CMV‏ در سیب زمینی از طریق ‏RNA Silencing

نوع مقاله : مقاله پژوهشی

نویسندگان

1 کارشناسی ارشد، دانشکده کشاورزی دانشگاه ارومیه، ارومیه، ایران

2 استاد، دانشکده کشاورزی دانشگاه ارومیه، ارومیه، ایران

3 دانشیار، دانشکده کشاورزی دانشگاه شهید مدنی آذربایجان، تبریز، ایران

چکیده

سیب‌زمینی در جهان از نظر تولید پس از گندم، برنج و ذرت در جایگاه چهارم اهمیت قرار دارد. به دلیل تکثیر رویشی، ویروس‌ها از عوامل بیماریزای مهم در سیب­زمینی محسوب می­شوند. در گیاهان، خاموشی RNA یک ساز و کار دفاعی علیه ویروس‌هاست. تحقیقات زیادی برای ایجاد مقاومت به ویروس‌ها با خاموش کردن ژن­های خاص آنها صورت گرفته است، اما در نهایت به خاطر فعالیتِ پروتئین‌های ویروسیِ ممانعت کننده از خاموشیِ RNA، مقاومت گیاه شکسته شده و ویروس قادر به تکثیر و ایجاد خسارت می‌شود. هدف از این تحقیق ایجاد مقاومت هم‌زمان برای سه ویروس مهم PLRV، PVX و CMV در سیب­زمینی است. قطعاتی از ژن های پروتئین‌های سرکوبگر خاموشی RNA در ویروس‌های فوق، به ترتیب P0، P25 و 2b به کمک PCR با آغازگرهای اختصاصی تکثیر و به هم متصل شدند. قطعه نوترکیب ساخته شده در داخل پلاسمید pFGC5941 در جهت سنس و آنتی­سنس در دو طرف اینترون تحت پروموتر 35S بصورت تولید کننده RNA با ساختار سنجاق‌سری قرار گرفت. قسمت T-DNA پلاسمید حاصل به کمک آگروباکتریوم به سیب­زمینی رقم آگریا انتقال یافت. لاین­های تراریخته به‌دست‌آمده به‌منظور تأیید حضور ترانس­ژن، توسط PCR بررسی شدند و لاین‌های تراریخته انتخاب و تکثیر یافتند. در ادامه تحقیق قرار است مقاومت لاین­های منتخبِ تراریخته به سه ویروس سنجیده شود.

کلیدواژه‌ها

عنوان مقاله [English]

Preparation of a construct to establish simultaneous resistance against three PLRV, ‎PVX and CMV viruses in potato by RNA silencing method

نویسندگان [English]

  • Mona Bordar 1
  • Reza Darvishzadeh 2
  • Maqsoud Pazhouhandeh 3
1 M. Sc. Faculty of Agriculture, Urmia University, Urmia, Iran
2 Professor, Faculty of Agriculture, Urmia University, Urmia, Iran
3 Associate Professor, Faculty of Agriculture, Shahid Madani University, Tabriz, Iran
چکیده [English]

Potato is the world's fourth-largest food crop after maize, wheat, and rice. Potato is seriously affected by both single and mixed viral infections because of vegetative propagation. In plants, RNA silencing is a protective mechanism against viral infections. Some researchers have been used RNA silencing technique to silence viral genes, but ultimately, due to the activity of viral RNA silencing-suppressor proteins, the resistance of plants is broken down and the virus can replicate and make the damage. The purpose of this study was to provide simultaneous resistance to three important viruses including Potato X Virus, Potato Leafroll Virus, and Cucumber Mosaic Virus. From genes corresponding to proteins of suppressoer of RNA silencing in these viruses (P0, P25 and 2b, respectively), a fragment was amplified with specific primers by PCR and ligtaed to each other. The recombinant final fragment was cloned in the form of sense and antisense orientations with an intron between them, in pFGC5941 plasmid under 35S promoter to produce a hairpin RNA after transcription in plant. The T-DNA of recombinant hairpin vector was transformed into potato (cv. Agria) genome by Agrobacterium. The obtained transgenic plants were screened by PCR to confirm the presence of the transgenes. The resistance of selected tansgenic lines will be assessed against three viruses.

کلیدواژه‌ها [English]

  • Cloning
  • disease resistance
  • gene silencing
  • plant virus
  • transgenic plants‎

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علوم باغبانی ایران
دوره 53، شماره 2
شهریور 1401
صفحه 351-369

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سابقه مقاله

  • تاریخ دریافت: 21 اسفند 1398
  • تاریخ بازنگری: 17 مهر 1400
  • تاریخ پذیرش: 03 آبان 1400
  • تاریخ اولین انتشار: 18 تیر 1401

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