اﻧﺘﻘﺎل ژن ﻛﺎﻳﻤﺮ ﻛﻴﺘﻴﻨﺎز 42 ﺑﻪ ﮔﻴﺎه ﻛﻠﺰا ﺟﻬﺖ اﻳﺠﺎد ﻣﻘﺎوﻣﺖ ﻋﻠﻴﻪ ﻗﺎرچ Alternaria solani
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عنوان دوره: سیزدهمین کنگره زراعت و اصلاح نباتات ایران
نویسندگان
ﺗﻬﺮان، ﭘﮋوﻫﺸﮕﺎه ﻣﻠﻲ ﻣﻬﻨﺪﺳﻲ ژﻧﺘﻴﻚ و زﻳﺴﺖﻓﻨﺎوری
چکیده
ﻛﻠﺰا ﺑﻪﻋﻨﻮان ﻳﻜﻲ از ﻣﺤﺼﻮﻻت ﻣﻬﻢ زراﻋﻲ، ﻧﺴﺒﺖ ﺑﻪ ﺑﺴﻴﺎری از ﻗﺎرچﻫﺎی ﺑﻴﻤﺎریزا ﺣﺴﺎس ﻣﻲﺑﺎﺷﺪ. ﻳﻜﻲ از روشﻫﺎی ﻣﻤﺎﻧﻌﺖ از ﻧﻔﻮذ ﻗﺎرچﻫﺎی ﺑﻴﻤﺎریزا ﺑﻪ درون ﮔﻴﺎﻫﺎن، اﺳﺘﻔﺎده از آﻧﺰﻳﻢﻫﺎی ﻫﻴﺪروﻻزی ﻧﻈﻴﺮ ﻛﻴﺘﻴﻨﺎزﻫﺎ اﺳﺖ ﻛﻪ ﺑﺎﻋﺚ ﺗﺠﺰﻳﻪ ﻛﻴﺘﻴﻦ ﻣﻮﺟﻮد در دﻳﻮاره اﻳﻦ ﻗﺎرچﻫﺎ ﻣﻲﮔﺮدد. در اﻳﻦ ﺗﺤﻘﻴﻖ ﺟﻬﺖ اﻳﺠﺎد ﻣﻘﺎوﻣﺖ ﺑﻪ ﻗﺎرچ Alternaria solaniدر ﮔﻴﺎه ﻛﻠﺰا، ژن ﻛﻴﺘﻴﻨﺎز 42 ﺣﺎوی دﻣﻴﻦ ﻣﺘﺼﻞ ﺷﻮﻧﺪه ﺑﻪ ﻛﻴﺘﻴﻦ ﺟﺪا ﺷﺪه از ﻗﺎرچ Trichoderma atroviride ، ﺑﻪ اﻳﻦ ﮔﻴﺎه ﻣﻨﺘﻘﻞ ﮔﺮدﻳﺪه و ﻣﻴﺰان ﻣﻘﺎوﻣﺖ آن ﻣﻮرد ﺑﺮرﺳﻲ ﻗﺮار ﮔﺮﻓﺘﻪ اﺳﺖ. ﺑﺪﻳﻦ ﻣﻨﻈﻮر اﺑﺘﺪا ﺣﻀﻮر ژن ﻛﺎﻳﻤﺮ 42 در ﺳﺎزه ﺑﻴﺎﻧﻲ pBI121 ﺗﺤﺖ ﻛﻨﺘﺮل ﭘﺮوﻣﻮﺗﺮ CaMV 35S و ﺗﺮﻣﻴﻨﺎﺗﻮر ﻧﻮﭘﺎﻟﻴﻦ ﺳﻴﻨﺘﺎز )NOS( ﺑﺎ اﺳﺘﻔﺎده از اﻟﮕﻮی PCR و ﻫﻀﻢ آﻧﺰﻳﻤﻲ ﻣﻮرد ﺗﺄﻳﻴﺪ ﻗﺮار ﮔﺮﻓﺖ. ﺟﻬﺖ اﻧﺘﻘﺎل ژن ﻛﻠﻮن ﺷﺪه از tumefaciens
Agrobacterium ﺳﻮﻳﻪ LBA4404 و رﻳﺰﻧﻤﻮﻧﻪﻫﺎی ﻛﻮﺗﻴﻠﺪوﻧﻲ اﺳﺘﻔﺎده ﮔﺮدﻳﺪ. ﺟﻬﺖ ﺗﺄﻳﻴﺪ ﮔﻴﺎﻫﺎن ﺗﺮارﻳﺨﺖ از اﻟﮕﻮی PCR ﺑﺎ اﺳﺘﻔﺎده از آﻏﺎزﮔﺮﻫﺎی اﺧﺘﺼﺎﺻﻲ ژن اﺳﺘﻔﺎده ﺷﺪ. ارزﻳﺎﺑﻲ ﻣﻘﺎوﻣﺖ ﮔﻴﺎﻫﺎن ﺗﺮارﻳﺨﺖ در ﺑﺮاﺑﺮ ﻗﺎرچ Alternaria solani ﺑﻪ روش اﻧﺘﺸﺎر دﻳﺴﻚ و ﻣﻴﺰان رﺷﺪ اﺳﭙﻮرﻫﺎی ﻗﺎرچ اﻧﺠﺎم ﮔﺮﻓﺖ. آﻧﺎﻟﻴﺰ ﻧﺘﺎﻳﺞ ﺑﻪدﺳﺖ آﻣﺪه ﻧﺸﺎن داد ﻛﻪ ﮔﻴﺎﻫﺎن ﺗﺮارﻳﺨﺖ، ﻧﺴﺒﺖ ﺑﻪ ﮔﻴﺎه ﺷﺎﻫﺪ ﻛﻠﺰای ﻏﻴﺮ ﺗﺮارﻳﺨﺖ( در ﺑﺮاﺑﺮ ﻗﺎرچ A. solani از
Agrobacterium ﺳﻮﻳﻪ LBA4404 و رﻳﺰﻧﻤﻮﻧﻪﻫﺎی ﻛﻮﺗﻴﻠﺪوﻧﻲ اﺳﺘﻔﺎده ﮔﺮدﻳﺪ. ﺟﻬﺖ ﺗﺄﻳﻴﺪ ﮔﻴﺎﻫﺎن ﺗﺮارﻳﺨﺖ از اﻟﮕﻮی PCR ﺑﺎ اﺳﺘﻔﺎده از آﻏﺎزﮔﺮﻫﺎی اﺧﺘﺼﺎﺻﻲ ژن اﺳﺘﻔﺎده ﺷﺪ. ارزﻳﺎﺑﻲ ﻣﻘﺎوﻣﺖ ﮔﻴﺎﻫﺎن ﺗﺮارﻳﺨﺖ در ﺑﺮاﺑﺮ ﻗﺎرچ Alternaria solani ﺑﻪ روش اﻧﺘﺸﺎر دﻳﺴﻚ و ﻣﻴﺰان رﺷﺪ اﺳﭙﻮرﻫﺎی ﻗﺎرچ اﻧﺠﺎم ﮔﺮﻓﺖ. آﻧﺎﻟﻴﺰ ﻧﺘﺎﻳﺞ ﺑﻪدﺳﺖ آﻣﺪه ﻧﺸﺎن داد ﻛﻪ ﮔﻴﺎﻫﺎن ﺗﺮارﻳﺨﺖ، ﻧﺴﺒﺖ ﺑﻪ ﮔﻴﺎه ﺷﺎﻫﺪ ﻛﻠﺰای ﻏﻴﺮ ﺗﺮارﻳﺨﺖ( در ﺑﺮاﺑﺮ ﻗﺎرچ A. solani از
کلیدواژه ها
Title
Transgenic canola plants harboring chimeric chitinase 42 gene inhibit mycelia growth of Alternaria solani
Authors
Abstract
Canola (Brassica napus L.) an agro-economically important crop in the world, is sensitive to many fungal pathogens. One way to combat fungal diseases are using genetic engineering to transfer genes encode hydrolyses enzymes such as chitinase that causes the breakdown of chitin in the wall of fungi. In the present study chit 42 (containing chitin binding domain) from Trichoderma atroviride for resistance to the fungus Alternaria solani transferred in Brassica napus. For this purpose, the chimer 42 gene under the control of the CaMV 35S constitutive promoter and NOS terminator in pBI121 was confirmed by PCR and digestion manner. Transformation of cotyledonary petioles was achieved via Agrobacterium tumefaciens LBA4404. The presence of chimer 42 in putative transgenic lines confirmed by PCR using specific primers. Also, antifungal activity was detected in crude protein extracts from transgenic canola plants when compared to non-transgenic canola using disk diffusion assay. This comparison shows that transgenic plants have a greater resistance against fungi.
Keywords
Canola, chimeric chitinase 42, gene transfer, Alternaria solani