تولید گیاه تراریخت فاقد نشانگر انتخابی با استفاده از بیان مشروط ژن ریکامبیناز در بذور تراریخت
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عنوان دوره: سیزدهمین کنگره زراعت و اصلاح نباتات ایران
نویسندگان
پژوهشگاه ملی مهندسی ژنتیک و زیست فناوری
چکیده
ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ وﺟﻮد ﻧﮕﺮاﻧﻲ ﻫﺎی ﻣﺘﻌﺪد در ﺣﻮزه اﻳﻤﻨﻲ زﻳﺴﺘﻲ ﺑﻪ دﻟﻴﻞ ﺑﺎﻗﻲ ﻣﺎﻧﺪن ژﻧﻬﺎی ﻧﺸﺎﻧﮕﺮ اﻧﺘﺨﺎﺑﻲ در ﮔﻴﺎﻫﺎن ﺗﺮارﻳﺨﺖ و ﺗﺎﺛﻴﺮ آن ﺑﺮ ﻛﺎﻫﺶ ﻣﻘﺒﻮﻟﻴﺖ ﻣﺤﺼﻮﻻت ﺗﺮارﻳﺨﺖ روﺷﻬﺎی ﻣﺘﻌﺪدی ﺑﺮای ﺣﺬف ژﻧﻬﺎی ﻧﺸﺎﻧﮕﺮ از ﮔﻴﺎﻫﺎن ﺗﺮارﻳﺨﺖ ﻣﻄﺎﻟﻌﻪ ﺷﺪه اﻧﺪ ﻛﻪ در ﺑﻴﻦ آﻧﻬﺎ روﺷﻬﺎی ﻣﺒﺘﻨﻲ ﺑﺮ اﺳﺘﻔﺎده از ﺳﻴﺴﺘﻢ ﻫﺎی ﻧﻮﺗﺮﻛﻴﺒﻲ در ﺟﺎﻳﮕﺎه وﻳﮋه ﺑﻪ دﻟﻴﻞ ﺳﺎدﮔﻲ، دﻗﺖ و ﻛﺎراﻳﻲ ﺑﺎﻻ ﻣﻮرد ﺗﻮﺟﻪ ﻣﻲ ﺑﺎﺷﻨﺪ. در اﻳﻦ ﺗﺤﻘﻴﻖ ﭘﺲ از ﻃﺮاﺣﻲ و ﺳﺎﺧﺖ ﻧﺎﻗﻞ ﺧﻮدﺑﺮش pG-synFFDD-creint-gusint ﻛﺎراﻳﻲ آن ﺑﺮای ﺗﻮﻟﻴﺪ ﮔﻴﺎﻫﺎن ﺗﺮارﻳﺨﺖ ﻓﺎﻗﺪ ﻧﺸﺎﻧﮕر ﻃﻲ ﻳﻚ ﻣﺮﺣﻠﻪ ﺗﺮارﻳﺨﺘﻲ ﺑﺮرﺳﻲ ﺷﺪه اﺳﺖ. در ﻧﺎﻗﻞ ﻣﺬﻛﻮر از ﺳﻴﺴﺘﻢ ﻧﻮﺗﺮﻛﻴﺒﻲ در ﺟﺎﻳﮕﺎه وﻳﮋه cre/loxP متعلق به باکتریوفاژ 1P استفاده شده است و راه انداز اختصاصی بذر متعلق به ژنBcNA از گیاه کلزا هدایت کننده بیان ژن ریکامبیناز (cre) می باشد . به این منظور پس از انتقال ناقل مذکور به ریز نمونه های برگی تنباکو و بازیابی و تایید گیاهان تراریخت اولیه کارایی آن از طریق آنالیزهای فنوتیپی و مولکولی شامل آزمون جوانه زنی بذور،آزمون هیستوشیمیایی بیان ژن PCR ،gus و توالی یابی در گیاهان نسل 1T بررسی شد. بر اساس نتایج به دست آمده قابلیت ناقل طراحی شده برای حذف ژن نشانگر انتخابی تایید شد و درصد حذف ژن نشانگر معادل 90 تا 100 درصد برآورد شده است.
کلیدواژه ها
Title
Generation of marker-free transgenic plant using conditional expression of recombinase in
transgenic seeds
Authors
Abstract
Remaining of selectable marker genes in transgenic crops has caused several concerns about safety of GM
plants. In order to decrease these concerns and simplify GM plants commercialization, elimination of marker
genes is more desirable. We developed an auto-excision binary vector was named as pG-synFFDD-creintgusint
based on cre/loxP system for elimination of nptII gene after transgenic identification. In our system
nptII and cre cassettes was placed between two directed loxP sites and cre was regulated by seed specific
BcNA1 promoter. Promoter-less gus gene was placed downstream and a pathogen inducible promoter was
located upstream of loxP flanked DNA. Transgenic tobacco plants analyzed in T1 generation for marker-free
plants recognition. Excision of nptII and recombines cassettes was verified by phenotypic and molecular
analysis. In four from 8 transgenic lines ratio of kanamaycine susceptibility was 100% and expression of gus
gene under control of inducible promoter was detected. Molecular analysis exhibited 90%- 20% for excision
ratio. This auto-excision strategy provides a promising approach to generate marker-free transgenic plants.
plants. In order to decrease these concerns and simplify GM plants commercialization, elimination of marker
genes is more desirable. We developed an auto-excision binary vector was named as pG-synFFDD-creintgusint
based on cre/loxP system for elimination of nptII gene after transgenic identification. In our system
nptII and cre cassettes was placed between two directed loxP sites and cre was regulated by seed specific
BcNA1 promoter. Promoter-less gus gene was placed downstream and a pathogen inducible promoter was
located upstream of loxP flanked DNA. Transgenic tobacco plants analyzed in T1 generation for marker-free
plants recognition. Excision of nptII and recombines cassettes was verified by phenotypic and molecular
analysis. In four from 8 transgenic lines ratio of kanamaycine susceptibility was 100% and expression of gus
gene under control of inducible promoter was detected. Molecular analysis exhibited 90%- 20% for excision
ratio. This auto-excision strategy provides a promising approach to generate marker-free transgenic plants.
Keywords
Marker-free transgenic plant, Auto-excision vector, cre/loxP recombination system