جداسازي و توالي‌يابي ژن مربوط به آنتي‌پورتر هيدروژن/ سديم غشاي واكوئلي گياه هالوفيت (.Kochia scoparia L)

Isolation and Sequencing of Gene Related to Na+/H+ Anti-porter of Vacuolar Membrane Isolated from Halophytes Plant (Kochia scoparia L.)

ﭼﮑﯿﺪه در اﯾﻦ ﭘﮋوﻫﺶ از ﮔﯿﺎه دوﻟﭙﻪاي و ﺷﻮرزي ﮐﻮﺷﯿﺎ )Kochia scoparia( ﺑﻪﻋﻨﻮان ﻣﻨﺒﻊ ﺑﺮاي ﺟﺪاﺳﺎزي ژن اﺳﺘﻔﺎده ﺷﺪ. اﺑﺘﺪا ﺑﺎ اﺳﺘﻔﺎده از ﭘﺮاﯾﻤﺮﻫﺎي ﻃﺮاﺣﯽﺷﺪه در ﻣﻨﺎﻃﻖ ﺣﻔﺎﻇﺖﺷﺪة اﯾﻦ ژن در ﮔﯿﺎﻫﺎن ﻫﻢﺧﺎﻧﻮاده، ﻃﻮل 1605 ﻧﻮﮐﻠﺌﻮﺗﯿﺪي و ﺗﻮاﻟﯽ 535 اﺳﯿﺪآﻣﯿﻨﻪاي از ﺗﻮاﻟﯽ ﮐﺪﮐﻨﻨﺪة اﯾﻦ ژن ﺷﻨﺎﺳﺎﯾﯽ و ﺗﻮاﻟﯽﯾﺎﺑﯽ ﺷﺪ. ﺑﺎ اﺳﺘﻔﺎده از آﻧﺎﻟﯿﺰ BLAST ﻣﯿﺰان ﺷﺒﺎﻫﺖ اﯾﻦ ﺗﻮاﻟﯽ ﺑﺎ 87-%90% ﻫﻤﻮﻟﻮژي در ﺳﻄﺢ ﻧﻮﮐﻠﺌﻮﺗﯿﺪي و 98-%99% در ﺳﻄﺢ اﺳﯿﺪآﻣﯿﻨﻪاي ﺑﺎ ژنﻫﺎي NHX ﮔﯿﺎﻫﺎن ﺧﺎﻧﻮادة اﺳﻔﻨﺎﺟﯿﺎن ﺗﻌﯿﯿﻦ ﺷﺪ. آﻧﺎﻟﯿﺰﻫﺎي ﺗﻌﯿﯿﻦ ﺧﺼﻮﺻﯿﺎت ﭘﺮوﺗﺌﯿﻨﯽ و ارﺗﺒﺎط آن ﺑﺎ ﭘﺮوﺗﺌﯿﻦﻫﺎي دﯾﮕﺮ ﺑﺎ اﺳﺘﻔﺎده از ﻣﻄﺎﻟﻌﺎت ﺑﯿﻮاﻧﻔﻮرﻣﺎﺗﯿﮑﯽ اﻧﺠﺎم ﺷﺪ. ﺑﺮاﺳﺎس وﺟﻮد ﺳﺎﺧﺘﺎرﻫﺎي دوم ﭘﺮوﺗﺌﯿﻨﯽ ﻣﺸﺨﺺ ﺷﺪ ﮐﻪ در ﭘﻠﯽﭘﭙﺘﯿﺪ ﻣﻮردﺑﺮرﺳﯽ، 11 ﻣﺎرﭘﯿﭻ آﻟﻔﺎ در ﻧﺎﺣﯿﻪ ﮔﺬرﻧﺪه از ﻏﺸﺎ وﺟﻮد دارد و اﺳﯿﺪآﻣﯿﻨﻪﻫﺎﯾﯽ ﺑﺎ ﺧﺼﻮﺻﯿﺖ آبﮔﺮﯾﺰي در ﻧﺎﺣﯿﻪ ﮔﺬرﻧﺪه از ﻏﺸﺎ واﻗﻊ ﺷﺪه اﺳﺖ و در اداﻣﻪ اﻧﺘﻬﺎي ﮐﺮﺑﻮﮐﺴﯿﻠﯽ ﺑﺎ ﺗﻌﺪاد ﮐﻤﺘﺮي ﻣﺎرﭘﯿﭻ آﻟﻔﺎ و ﻣﯿﺰان آبدوﺳﺖ ﺑﻮدن ﺑﺎﻻ ﺑﻪﻋﻨﻮان ﻧﺎﺣﯿﮥ ﺳﯿﺘﻮﭘﻼﺳﻤﯽ ﭘﺮوﺗﺌﯿﻦ ﻣﻮردﻧﻈﺮ ﻣﻄﺮح ﻣﯽﺷﻮد. اﯾﻦ ﻧﺘﺎﯾﺞ ﻧﻘﺶ دﻓﻊ ﺳﺪﯾﻢ ﺗﻮﺳﻂ ﻧﺎﻗﻞﻫﺎي ﻏﺸﺎي ﭘﻼﺳﻤﺎﯾﯽ را ﺑﺮاي وﺟﻮد واﮐﻨﺶ ﻣﻘﺎوﻣﺖ ﺑﻪ ﺷﻮري در ﮔﯿﺎه ﮐﻮﺷﯿﺎ ﺗﺄﯾﯿﺪ ﻧﻤﻮد.

In this study Kochia scoparia, a halophyte and dicotyledonous was used as a source to gene isolation. First, using primers designed in conserved regions of this gene in plants of the same family, an approximate length of 1605 nucleotides and a sequence of 535 amino acids from the encoding sequence of this gene were identified and sequenced. Using BLAST analysis, the similarity rate of this sequence with 87%- 90% -87% homology at nucleotide level, and 98%- 99% homology at amino acid level with NHX genes of Chenopodiaceae family was determined. Analyses on protein characterization and its relationship with other proteins were conducted through bioinformatics studies. Based on the presence of second protein structures, it has been found that in the studied polypeptide, there are 11 alpha spirals in the region passing through the membrane, and the amino acids of the water-defect characteristic are located in the membrane passage region, while the carboxylic terminal with fewer alpha helix and high hydrophilic property can be introduced as the cytoplasmic region of the protein of interest. These results verified the role of sodium efflux for resistance response to salinity in Kochia using plasma membrane transporters.