ايجاد ديرگلدهي در كاهو با استفاده از خاموشي ژن MSI4 به كمك RNA Silencing

Delayed flowering in lettuce using MSI4 gene silencing by RNAi

دﯾﺮﮔﻠﺪﻫﯽ در ﮐﺎﻫﻮ ﺑﺎﻋﺚ ﺗﻮﻟﯿﺪ ﺑﺮﮔﻬﺎي زﯾﺎد و ﺣﻔﻆ ﮐﯿﻔﯿﺖ ﺧﻮراﮐﯽ ﻣﺤﺼﻮل آن ﻣﯽﺑﺎﺷﺪ. ﺷﺮوع زﻣﺎن ﮔﻠﺪﻫﯽ در ﮔﯿﺎﻫﺎن ﭘﺪﯾﺪه اي اﺳﺖ ﮐﺎﻣﻼ ﻫﺪاﯾﺖ ﺷﺪه ﮐﻪ ﺑﻪ ﭘﺎراﻣﺘﺮﻫﺎي ﻣﺤﯿﻄﯽ و دروﻧﯽ ﮔﯿﺎه واﺑﺴﺘﻪ اﺳﺖ و ﺗﻮﺳﻂ ﺷﺒﮑﻪ ﺗﻨﻈﯿﻤﯽ ژﻧﻬﺎي زﯾﺎدي ﮐﻨﺘﺮل ﻣﯽﺷﻮد. ﯾﮑﯽ از ﻣﺸﮑﻼت در زﻣﯿﻨﻪ ﭘﺮورش و ﻋﻤﻠﮑﺮد ﺑﺎﻻي ﻣﺤﺼﻮﻻت زراﻋﯽ و ﺑﺎﻏﯽ ﻋﺪم ﺗﻄﺎﺑﻖ زﻣﺎن ﮔﻠﺪﻫﯽ ﺑﺎ ﺷﺮاﯾﻂ ﻣﻄﻠﻮب اﺳﺖ. ﺑﺎ اﻓﺰاﯾﺶ ﺟﻤﻌﯿﺖ، ﺗﻮﻟﯿﺪ ﮔﯿﺎﻫﺎن ﮐﺎرا و ﺑﺎ ﺑﯿﻮﻣﺎس ﺑﺎﻻﺗﺮ ﺿﺮوري ﻣﯽﺑﺎﺷﺪ. در ﺷﺒﮑﻪ ﺗﻨﻈﯿﻤﯽ ﮔﻠﺪﻫﯽ، Flowering Locus C (FLC)، ﯾﮏ ﻓﺎﮐﺘﻮر روﻧﻮﯾﺴﯽ، ﺟﻌﺒﻪ MADS اﺳﺖ ﮐﻪ ﻣﺎﻧﻊ از ﮔﻠﺪﻫﯽ ﻣﯽﺷﻮد. ﻣﺴﯿﺮ ﺧﻮدﻣﺨﺘﺎر ﺷﺮوع ﮔﻠﺪﻫﯽ را ﺑﻪ ﻃﻮر ﻣﺴﺘﻘﻞ از ﻃﻮل روز، از ﻃﺮﯾﻖ ﺳﺮﮐﻮﺑﯽ FLC، ﺗﺴﺮﯾﻊ ﻣﯽﮐﻨﺪ. ژن MSI4/FVE ﯾﮏ ژن ﮐﻠﯿﺪي ﻣﺴﯿﺮ ﺧﻮدﻣﺨﺘﺎر اﺳﺖ ﮐﻪ در ﮐﻨﺘﺮل زﻣﺎن ﺗﮑﺜﯿﺮ و ﺗﻤﺎﯾﺰ آﻏﺎزﯾﻦ ﺳﻠﻮل، رﺷﺪ اﻧﺪام و زﻣﺎن ﮔﻠﺪﻫﯽ درﮔﯿﺮ اﺳﺖ و ﻧﻘﺶ ﮐﻠﯿﺪي در ﺗﻌﯿﯿﻦ زﻣﺎن ﮔﻠﺪﻫﯽ دارد و ﺗﻮﺳﻂ ﺗﻐﯿﯿﺮات اﭘﯽژﻧﺘﯿﮑﯽ ﻣﻮﺟﺐ ﮐﺎﻫﺶ ﺑﯿﺎن ژن FLC ﻣﯽﮔﺮدد. ﻫﺪف اﯾﻦ ﺗﺤﻘﯿﻖ اﺳﺘﻔﺎده از ﺗﮑﻨﯿﮏ RNA Silencing ﺑﺮاي ﻣﻬﺎر ﮐﺮدن ژن MSI4 در ﺳﻄﺢ ﺑﻌﺪ از روﻧﻮﯾﺴﯽ در ﮔﯿﺎه ﮐﺎﻫﻮ ﻣﯽﺑﺎﺷﺪ. ﺑﺮاي ﺑﻬﯿﻨﻪ ﮐﺮدن ﮐﺸﺖ ﺑﺎﻓﺖ ﮔﯿﺎه ﮐﺎﻫﻮ در ﻣﺤﯿﻂ ﮐﺸﺖ MS از ﻫﻮرﻣﻮﻧﻬﺎي BAP، ﮐﺎﯾﻨﺘﯿﻦ و NAA و ﺟﻬﺖ ﺑﺎززاﯾﯽ ﻣﺴﺘﻘﯿﻢ از رﯾﺰﻧﻤﻮﻧﻪﻫﺎي ﮐﻮﺗﯿﻠﺪون و ﺑﺮگ در ﻣﺪت ﺳﻪ ﻫﻔﺘﻪ اﺳﺘﻔﺎده ﺷﺪ و ﺗﯿﻤﺎر ﻫﻮرﻣﻮﻧﯽ 0/1 ﻣﯿﻠﯽﮔﺮم در ﻟﯿﺘﺮ NAA و 0/01 ﻣﯿﻠﯽﮔﺮم در ﻟﯿﺘﺮ BAP ﺑﯿﺸﺘﺮﯾﻦ ﺑﺎززاﯾﯽ را ﻧﺸﺎن داد. ﺑﺮاي ﺳﻨﺘﺰ ﺳﺎﺧﺘﺎر ﺳﻨﺠﺎقﺳﺮي ﻣﻮرد ﻧﯿﺎز در ﺗﮑﻨﯿﮏ RNA silencing، ﻗﻄﻌﻪ اي از اﻧﺘﻬﺎي ژن MSI4 آراﺑﯿﺪوﭘﺴﯿﺲ ﺗﻮﺳﻂ PCR ﺗﮑﺜﯿﺮ و ﺑﺎ دو ﻫﻤﺴﺎﻧﻪ ﺳﺎزي ﻣﺘﻮاﻟﯽ در دو ﺟﻬﺖ ﺳﻨﺲ و آﻧﺘﯽﺳﻨﺲ درون وﮐﺘﻮر ﺧﺎﻣﻮﺷﯽ pFGC5941 ﻗﺮار داده ﺷﺪ. آﻧﮕﺎه ﺳﺎﺧﺘﺎر ﺳﻨﺠﺎقﺳﺮي در اﯾﻦ وﮐﺘﻮر ﻧﻮﺗﺮﮐﯿﺐ ﺑﻪ واﺳﻄﻪ آﮔﺮوﺑﺎﮐﺘﺮﯾﻮم ﺑﻪ ﮔﯿﺎه ﮐﺎﻫﻮ اﻧﺘﻘﺎل ﯾﺎﻓﺖ. ﮔﯿﺎﻫﺎن ﺗﺮارﯾﺨﺘﻪ ﺑﺪﺳﺖ آﻣﺪه ﺑﻪ ﻣﻨﻈﻮر ﺗﺎﺋﯿﺪ ﺣﻀﻮر ژن اﻧﺘﻘﺎﻟﯽ در آﻧﻬﺎ ﺗﻮﺳﻂ PCR ﺑﺮرﺳﯽ ﺷﺪ و ﻣﻄﺎﻟﻌﻪ روي ﻓﻨﻮﺗﯿﭗ ﮔﻠﺪﻫﯽ اﯾﻦ ﮔﯿﺎﻫﺎن ﺗﺮارﯾﺨﺖ ﻧﺸﺎن داد ﮐﻪ ﺧﺎﻣﻮﺷﯽ MSI4 در ﮐﺎﻫﻮ ﺑﺎﻋﺚ دﯾﺮﮔﻠﺪﻫﯽ اﯾﻦ ﮔﯿﺎه )ﺣﺪود ﯾﮏ و ﻧﯿﻢ ﻣﺎه ﺗﺎﺧﯿﺮ( و ﺗﻮﻟﯿﺪ ﺑﯿﺸﺘﺮ ﺑﺮگ در آن ﻣﯽﺷﻮد.

Late flowering in lettuce produces additional leaves and maintains the edible quality of its product. Control of flowering time in plants is vital for plant fitness and for agricultural purposes and is an important factor in successful reproduction and seed crop yield and depends on the environmental and internal parameters of the plant and it is controlled by the regulatory network of genes. One of the problems in the field of crops and horticulture is the mismatch of flowering time with favorable environmental conditions. by increase in population, it is necessary to produce efficient plants with higher biomass. In the flowering regulatory network, Flowering Locus C (FLC), a MADS box transcription factor, prevents flowering. The autonomous pathway accelerates the onset of flowering independently of daylight by suppressing FLC. The MSI4/FVE gene is a key gene in the autonomous pathway. It is involved in controlling cell proliferation and early differentiation, organ growth and plays a key role in accelerating flowering by reducing the expression of FLC gene via epigenetic changes. in this study, using the RNA Silencing technique, MSI4/FVE gene expression was repressed at post transcriptional level. To this end, first, the tissue culture of lettuce in MS culture medium was optimized using BAP, Kinetin and NAA hormones for direct regeneration from cotyledon and leaf explants. The MS medium containing 0.1 mg/l NAA and 0.01 mg/l BAP has the highest regeneration rate for cotyledon explants. Then, for construction of silencing hairpin construct, a fragment of Arabidopsis thaliana MSI4 gene was amplified by PCR and then cloned in sense and in antisense directions into pFGC5941 vector by two consecutive cloning. The hairpin structure in this recombinant vector was then transferred to the lettuce plant by Agrobacterium. The obtained transgenic plants were tested by PCR to confirm the presence of the transgene and the study on the flowering phenotype of these transgenic plants showed that the silencing of MSI4 in lettuce caused the plant to flower late (about 1.5 months delay) and produce additional leaves