بررسي عوامل موثر در انتقال ژن به سويا از طريق آگروباكتريوم

Study on the factors affecting Agrobacterium-mediated transformation of soybean

ﺗﺮارﯾﺨﺘﯽ ﺑﻪ واﺳﻄﻪ آﮔﺮوﺑﺎﮐﺘﺮﯾﻮم ﯾﮑﯽ از ﻣﻮﺛﺮﺗﺮﯾﻦ روشﻫﺎ ﺑﺮاي ﺗﻮﻟﯿﺪ ﮔﯿﺎﻫﺎن ﺗﺮارﯾﺨﺘﻪ ﺳﻮﯾﺎ ﺑﻪ ﺷﻤﺎر ﻣﯽآﯾﺪ. ﮐﺎراﯾﯽ ﺗﺮارﯾﺨﺘﯽ ﺳﻮﯾﺎ ﺑﺎ آﮔﺮوﺑﺎﮐﺘﺮﯾﻮم ﺑﻪ ﻋﻮاﻣﻞ ﻣﺨﺘﻠﻔﯽ ﺑﺴﺘﮕﯽ دارد. اﯾﻦ ﭘﮋوﻫﺶ ﺑﺎ ﻫﺪف ﺑﺮرﺳﯽ اﻣﮑﺎن ﺗﻮﻟﯿﺪ اﯾﻨﺘﺮﻓﺮون ﮔﺎﻣﺎي اﻧﺴﺎﻧﯽ در ﮔﯿﺎه ﺳﻮﯾﺎ اﻧﺠﺎم ﮔﺮﻓﺖ. ﺑﺪﯾﻦ ﻣﻨﻈﻮر ﺑﺮﺧﯽ ﻋﻮاﻣﻞ ﻣﻮﺛﺮ در ﺗﺮارﯾﺨﺘﯽ ﺳﻮﯾﺎ از ﻗﺒﯿﻞ ﻏﻠﻈﺖ آﮔﺮوﺑﺎﮐﺘﺮﯾﻮم )1 و 0/8 ،0/6 ،0/4 ،0/2=OD600nm(، ﻣﺪت زﻣﺎن ﺗﻠﻘﯿﺢ )40 ،20 ،10 و 60 دﻗﯿﻘﻪ(، ﻣﺪت زﻣﺎن ﻫﻢﮐﺸﺘﯽ )1، 4 ،3 ،2 و 5 روز( و ﺳﻄﻮح ﻣﺨﺘﻠﻒ اﺳﺘﻮﺳﯿﺮﯾﻨﮕﻮن )200 ،150 ،100 ،50 ،0 و 250 ﻣﯿﮑﺮوﻣﻮﻻر( ﻣﻮرد ﺑﺮرﺳﯽ ﻗﺮار ﮔﺮﻓﺘﻨﺪ. ﻧﺘﺎﯾﺞ ﻧﺸﺎن داد، ﺑﯿﺸﺘﺮﯾﻦ ﻣﯿﺰان ﺗﺮارﯾﺨﺘﯽ در ﻏﻠﻈﺖ 0/8 =OD600nm و ﮐﻤﺘﺮﯾﻦ ﻣﯿﺰان ﺗﺮارﯾﺨﺘﯽ در ﻏﻠﻈﺖ 0/2 =OD600nm ﺣﺎﺻﻞ ﺷﺪه اﺳﺖ. ﻫﻤﭽﻨﯿﻦ ﺑﯿﺸﺘﺮﯾﻦ ﻣﯿﺰان ﺗﺮارﯾﺨﺘﯽ در ﻣﺪت زﻣﺎن ﺗﻠﻘﯿﺢ 40 دﻗﯿﻘﻪ و ﻣﺪت زﻣﺎن ﻫﻢﮐﺸﺘﯽ 4 روز و ﮐﻤﺘﺮﯾﻦ ﻣﯿﺰان ﺗﺮارﯾﺨﺘﯽ در ﻣﺪت زﻣﺎن ﺗﻠﻘﯿﺢ 10 دﻗﯿﻘﻪ و ﻣﺪت زﻣﺎن ﻫﻢﮐﺸﺘﯽ 1 روز ﻣﺸﺎﻫﺪه ﺷﺪ. ﺣﻀﻮر اﺳﺘﻮﺳﯿﺮﯾﻨﮕﻮن اﺛﺮ اﻓﺰاﯾﺸﯽ در ﮐﺎراﯾﯽ ﺗﺮارﯾﺨﺘﯽ ﺳﻮﯾﺎ داﺷﺖ، ﺑﻪ ﻃﻮرﯾﮑﻪ ﺑﯿﺸﺘﺮﯾﻦ ﻣﯿﺰان ﺗﺮارﯾﺨﺘﯽ در ﻏﻠﻈﺖﻫﺎي 150 ﺗﺎ 200 ﻣﯿﮑﺮوﻣﻮﻻر اﺳﺘﻮﺳﯿﺮﯾﻨﮕﻮن ﺣﺎﺻﻞ ﺷﺪه اﺳﺖ. در ﻧﻬﺎﯾﺖ ﺣﻀﻮر ژن اﯾﻨﺘﺮﻓﺮون ﮔﺎﻣﺎي اﻧﺴﺎﻧﯽ در ﮔﯿﺎﻫﺎن ﺗﺮارﯾﺨﺘﻪ اﺣﺘﻤﺎﻟﯽ ﺑﻪ وﺳﯿﻠﻪ ﺗﮑﻨﯿﮏ PCR و آﻏﺎزﮔﺮﻫﺎي اﺧﺘﺼﺎﺻﯽ ﺗﺎﯾﯿﺪ ﺷﺪ. ﺷﺮاﯾﻂ ﺑﻬﯿﻨﻪ ﺑﺪﺳﺖ آﻣﺪه در اﯾﻦ ﻣﻄﺎﻟﻌﻪ ﻣﯽﺗﻮاﻧﺪ ﺑﺮاي اﻧﺘﻘﺎل ژنﻫﺎي ﻫﺪف ﺑﻪ اﯾﻦ رﻗﻢ ﺳﻮﯾﺎ اﺳﺘﻔﺎده ﺷﻮد.

Agrobacterium-mediated transformation is one of the most effective methods for producing the transgenic soybean plants. The efficiency of soybean transformation with Agrobacterium depends on the various factors. The aim of this study was investigating the possibility of human interferon gamma production in soybean. For this purpose, some effective factors in soybean transformation such as Agrobacterium concentration (OD600nm = 0.2, 0.4, 0.6, 0.8 and 1), inoculation time (10, 20, 40 and 60 minutes), co-cultivation time (1, 2, 3, 4 and 5 days) and different levels of acetosyringone (0, 50, 100, 150, 200 and 250 μM) were evaluated. The results showed that the highest percentage of transformation was observed in the OD600nm= 0.8 and the lowest percentage of transformation was obtained at the OD600nm= 0.2. Also, the highest percentage of transformation was obtained in the 40 minutes of inoculation and 4 days of co-cultivation and the lowest percentage of transformation was observed in the 10 minutes of inoculation and 1 day of co-cultivation. The presence of acetosyringone had the increasing effect on the soybean transformation efficiency, and the highest percentage of transformation was obtained at the 150 until 200 μM of acetosyringone. Finally, the presence of human interferon gamma gene in the probable transgenic plants was confirmed by the PCR technique and specific primers. The optimal conditions that obtained in this study can be used to transfer target genes to this soybean cultivar