شناسايي SNPها در نواحي اگزوني واينتروني ژن هاي لينالول سينتاز و ژرماكرينD سينتاز در گياه ريحان

SNP discovery in exon and intron regions of Linalool synthase and Germacrene D synthase genes in basil

رﻳﺤﺎن )Ocimum basillicum L.( از ﻣﻬﻢ ﺗﺮﻳﻦ ﮔﻴﺎﻫﺎن داروﻳﻲ ﺣﺎوي ﺗﺮﻛﻴﺒﺎت ﺗﺮﭘﻨﻮﺋﻴﺪي ازﺟﻤﻠﻪ ﻣﻮﻧﻮﺗﺮﭘﻦ و ﺳﺰﻛﻮﺋﻲﺗﺮﭘﻦﻫﺎ ﻣﻲﺑﺎﺷﺪ. ژنﻫﺎي ﻟﻴﻨﺎﻟﻮل ﺳﻴﻨﺘﺎز و ژرﻣﺎﻛﺮﻳﻦ D ﺳﻴﻨﺘﺎز از ژن ﻫﺎي ﻛﻠﻴﺪي دﺧﻴﻞ در ﺳﻨﺘﺰ ﺗﺮﭘﻦﻫﺎ ﻣﻲﺑﺎﺷﻨﺪ. در ﻣﻄﺎﻟﻌﻪ ﺣﺎﺿﺮ ﺑﻪ ﻣﻨﻈﻮر ﺷﻨﺎﺳﺎﻳﻲ SNPﻫﺎ دراﻳﻦ دو ژن در 5 ﺗﻮده ﻣﺨﺘﻠﻒ رﻳﺤﺎن از ﻧﺸﺎﻧﮕﺮﻫﺎي CAPS و ﺗﻮاﻟﻲﻳﺎﺑﻲ اﺳﺘﻔﺎده ﺷﺪ. ﻗﻄﻌﺎﺗﻲ ﺑﻪ اﻧﺪازه 600 و 583 ﺟﻔﺖ ﺑﺎز از ﻧﻮاﺣﻲ ﻛﺪ ﻛﻨﻨﺪه اﻳﻦ دو ژن ﺗﻜﺜﻴﺮ و ﺑﺎ آﻧﺰﻳﻢ ﻫﺎي ﺑﺮﺷﻲ Pst1 و MseI ﻫﻀﻢ ﺷﺪ. ﺑﻌﺪ از ﺗﻮاﻟﻲ- ﻳﺎﺑﻲ و ﺑﺎزﻳﺎﺑﻲ ﻗﻄﻌﻪ ﺗﻜﺜﻴﺮي ﻫﺮ ژن، ﺷﻨﺎﺳﺎﻳﻲSNPﻫﺎ ﺑﺎ اﺳﺘﻔﺎده از ﻫﻢ ردﻳﻔﻲ ﺗﻮاﻟﻲ ﻫﺎي ﻫﺮ ژن در اﻓﺮاد ﻣﺨﺘﻠﻒ اﻧﺠﺎم ﮔﺮﻓﺖ. از ﻧﻪ SNPﺷﻨﺎﺳﺎﻳﻲ ﺷﺪه در ژن ﻟﻴﻨﺎﻟﻮل ﺳﻴﻨﺘﺎز، ﺑﻪ ﺗﺮﺗﻴﺐ ﭼﻬﺎر و ﭘﻨﺞ SNPدر ﻧﺎﺣﻴﻪ اﻳﻨﺘﺮون و اﮔﺰون ﻣﺸﺎﻫﺪه ﺷﺪ. از ﻛﻞSNP ﻫﺎي ﺷﻨﺎﺳﺎﻳﻲ ﺷﺪه دراﻳﻦ ژن، 77/8 درﺻﺪ آﻧﻬﺎ از ﻧﻮع ﻫﻢ ﺟﻨﺲ ﺑﺎ ﻓﺮاواﻧﻲ 44/4 درﺻﺪ A/Gو 33/3 درﺻﺪ T/C و 22/2 درﺻﺪ آﻧﻬﺎ از ﻧﻮع ﻧﺎﻫﻢ ﺟﻨﺲ ﺑﺎ ﻓﺮاواﻧﻲ ﻳﻜﺴﺎن ﻧﺎﺷﻲ از ﺗﺒﺪﻳﻼت C/G و C/A ﺑﻮد. از 28 SNP ﺷﻨﺎﺳﺎﻳﻲ ﺷﺪه در ژن ژرﻣﺎﻛﺮﻳﻦ D ﺳﻴﻨﺘﺎز،ﻫﻔﺖ SNPدر ﻧﺎﺣﻴﻪ اﮔﺰون و 21 SNP در ﻧﺎﺣﻴﻪ اﻳﻨﺘﺮون ﺷﻨﺎﺳﺎﻳﻲ ﺷﺪ ﻛﻪ 67/8 درﺻﺪ آﻧﻬﺎ از ﻧﻮع ﻫﻢ ﺟﻨﺲ ﺑﺎ ﻓﺮاواﻧﻲ 35/7 درﺻﺪ G/A و 32/1 درﺻﺪ C/T و 32/1 درﺻﺪ آﻧﻬﺎ از ﻧﻮع ﻧﺎﻫﻢ ﺟﻨﺲ ﺑﺎ ﻓﺮاواﻧﻲ 7/1 درﺻﺪ T/A، 14/3 درﺻﺪ G/C و 10/7 درﺻﺪ C/A ﺑﻮد. ﻧﺘﺎﻳﺞ ﻫﻢ ردﻳﻔﻲ ﺗﻮاﻟﻲ ژنﻫﺎ ﺑﻴﻦ اﻓﺮاد ﻧﺸﺎن داد ﻛﻪ ﺑﻴﺸﺘﺮﻳﻦ ﺟﻬﺶ در ژنﻫﺎي ﻟﻴﻨﺎﻟﻮل ﺳﻴﻨﺘﺎز و ژرﻣﺎﻛﺮﻳﻦ D ﺳﻴﻨﺘﺎز از ﻧﻮع ﺗﺒﺪﻳﻞ ﺑﺎزﻫﺎي ﻫﻢ ﺟﻨﺲ A/G و T/C ﻣﻲﺑﺎﺷﺪ.

Basil (Ocimum basillicum L.), one of the most important medicinal plants, contains monoterpene and sesquiterpene compounds. Linalool synthase (LIS) and Germacrene D synthase (GDS) are two key genes involved in terpenes biosynthesis. In the current investigation, cleaved amplified polymorphic sequence (CAPS) markers and sequencing were used to identify single nucleotide polymorphisms (SNPs) in both genes in five different basil populations. For this means, fragments of 600 and 583 bp from the coding sequences of these genes were amplified and digested by using Pst1 and Mse1 restriction enzymes. After retrieval of the sequences of the amplified gene fragments, SNPs were identified based on multiple sequence alignment in both genes in studied basil genotypes. Out of the nine SNPs identified in LIS gene, five occurred in exon region while the number of SNPs identified in intron was four. Of the total SNPs detected in this gene, the proportion of transition was 77.8%, with frequencies of A/G: 44.4% and T/C: 33.3% and C/G and C/A (transversion) with the same frequencies of 22.2%. In GDS gene, in total, 28 SNPs was detected (seven in exon and 21 in intron) of which 67.8% was transient with a frequency of G/A: 35.7% and C/T: 32.1%, and 32.1% transversion (T/A: 7.15%, G/C: 14.3% and C/A: 10.7%). The results of the sequence alignments revealed that the highest number of the identified SNPs in the studied genes are A/G and TC.