شناسايي، توالي‌يابي و بررسي فيلوژني ژن ارتولوگ APETALA1 (AP1) در منداب (Eruca sativa)

Identification, sequencing and phylogeny of APETALA1 ortholog gene (AP1) in Eruca sativa (Brassicaceae)

ﮔﺬر از ﻓﺎز روﯾﺸﯽ ﺑﻪ زاﯾﺸﯽ ﯾﮏ ﻣﺮﺣﻠﻪ ي ﻧﻤﻮي ﻣﻬﻢ، ﺗﺤﺖ ﮐﻨﺘﺮل ﺷﺪﯾﺪ ژﻧﺘﯿﮑﯽ اﺳﺖ. اﯾﻦ ﺗﻐﯿﯿﺮ ﻓﺎز ﻧﯿﺎزﻣﻨﺪ ﻓﻌﺎﻟﯿﺖ ﻣﺠﻤﻮﻋﻪ اي از ژﻧﻬﺎ در ﻣﺮﯾﺴﺘﻢ رأس ﺷﺎﺧﺴﺎره اﺳﺖ ﮐﻪ ﺑﯿﺎن ﺳﺒﺐ ﺗﺒﺪﯾﻞ ﻣﺮﯾﺴﺘﻢ روﯾﺸﯽ ﺑﻪ زاﯾﺸﯽ ﻣﯽ ﺷﻮد. ژن APETALA1 (AP1) در ﭘﯿﺸﺒﺮد ﻣﺮﺣﻠﻪ ي ﮔﺬر و ﻧﯿﺰ ﺗﻌﯿﯿﻦ ﻫﻮﯾﺖ ﻣﺮﯾﺴﺘﻢ ﮔﻞ ﻧﻘﺶ ﻣﻬﻤﯽ دارد. ﺷﻨﺎﺳﺎﯾﯽ و ﻫﻢ ﺳﺎﺧﺘﯽ ﻫﻮﻣﻮﻟﻮژي( اﯾﻦ ژن و ﭘﺮوﺗﺌﯿﻦ اﺳﺘﻨﺒﺎﻃﯽ )Deduced Protein( در ﮔﯿﺎه ﻣﻨﺪاب )Eruca sativa( ﺑﺮرﺳﯽ ﺷﺪ. RNA ﮐﻞ از ﻏﻨﭽﻪ ﻫﺎي ﮔﻞ ﻣﻨﺪاب اﺳﺘﺨﺮاج و cDNA ﺳﺎﺧﺘﻪ ﺷﺪ. آﻏﺎزﮔﺮﻫﺎي اﺧﺘﺼﺎﺻﯽ ﺑﺮ اﺳﺎس ﻫﻢ راﺳﺘﺎﯾﯽ ﺗﻮاﻟﯽ ژن ﻫﺎي ﻫﻢ ﺳﺎﺧﺖ AP1 در ﮔﯿﺎﻫﺎن ﻫﻢ ﺧﺎﻧﻮاده، ﻃﺮاﺣﯽ و ﺑﺮاي واﮐﻨﺶ RT-PCR اﺳﺘﻔﺎده ﮔﺮدﯾﺪﻧﺪ. ﻗﻄﻌﻪ ﻣﻮرد ﻧﻈﺮ ﺑﻪ ﻃﻮل 782 ﻧﻮﮐﻠﺌﻮﺗﯿﺪ ﺑﻮد ﮐﻪEvsAP1 ﻧﺎﻣﯿﺪه و در ﭘﺎﯾﮕﺎه داده NCBI ﺑﺎ ﺷﻤﺎره KX524132.1 ﺛﺒﺖ ﺷﺪ. BLAST اﯾﻦ ﺗﻮاﻟﯽ، ﻧﺸﺎن داد ﮐﻪ ﻧﺰدﯾﮏ ﺗﺮﯾﻦ ﺗﻮاﻟﯽ ﻧﻮﮐﻠﺌﻮﺗﯿﺪي ﺑﻪEvsAP1 ﻣﺮﺑﻮط ﺑﻪ ﮔﯿﺎه ﺗﺮﺑﭽﻪ )Raphanus sativus( و ﺧﺮدل ﺳﻔﯿﺪ )Sinapis alba( ﺑﺎ %87 ﺷﺒﺎﻫﺖ اﺳﺖ(. ﭘﺮوﺗﺌﯿﻦ اﺳﺘﻨﺒﺎﻃﯽ ژن EvsAP1، داراي 256 آﻣﯿﻨﻮاﺳﯿﺪ اﺳﺖ ﮐﻪ %82 ﺷﺒﺎﻫﺖ ﺑﺎ ﺗﻮاﻟﯽ آﻣﯿﻨﻮاﺳﯿﺪي ﺗﺮﺑﭽﻪ ﻧﺸﺎن ﻣﯽ دﻫﺪ. ﻃﻮل ﮐﺎﻣﻞ ﭘﺮوﺗﺌﯿﻦ AP1 در اﮐﺜﺮ ﺗﻮاﻟﯽ ﻫﺎي ﭘﺮوﺗﺌﯿﻨﯽ ﺛﺒﺖ ﺷﺪه از اﯾﻦ ژن در ﺳﺎﯾﺖ NCBI ﮐﻪ ﻣﺘﻌﻠﻖ ﺑﻪ ﺧﺎﻧﻮاده ي ﺷﺐ ﺑﻮ ﻣﯽ ﺑﺎﺷﻨﺪ، 256 آﻣﯿﻨﻮ اﺳﯿﺪ اﺳﺖ ﮐﻪ ﺗﺎﯾﯿﺪي ﺑﺮاي ﺗﻮاﻟﯽ ﻣﻮرد ﻧﻈﺮ اﺳﺖ. ﻣﻄﺎﻟﻌﺎت درﺧﺖ ﻓﯿﻠﻮژﻧﯽ ﻧﺸﺎن داد ﮐﻪ ﮔﯿﺎﻫﺎن ﻫﻢ ﺧﺎﻧﻮاده Eruca sativa ، ﻫﻤﮕﯽ در ﺷﺎﺧﻪ ﻫﺎي )ﮐﻼدﻫﺎي( ﻣﺠﺎور ﻗﺮار ﮔﺮﻓﺘﻪ اﻧﺪ و ﺑﺎ ﺧﺮدل ﺳﯿﺎه )Brassica nigra( و ﺗﺮﺑﭽﻪ )Raphanus sativus( ﺷﺒﺎﻫﺖ ﺑﺎﻻﺗﺮي دارد ﺑﻄﻮري ﮐﻪ ﺑﺎ ﺗﺮﺑﭽﻪ در ﯾﮏ ﮐﻼد ﻗﺮار دارﻧﺪ ﮐﻪ ﺗﺄﯾﯿﺪي ﺑﺮ ﺻﺤﺖ ﺗﻮاﻟﯽ ﺧﻮاﻧﺪه ﺷﺪه ژنAP1 در اﯾﻦ ﮔﯿﺎه ﻣﯽ ﺑﺎﺷﺪ.

The transition from vegetative to reproductive phase is an important developmental under genetic control. This stage requires the activation of a set of genes in apical meristem, the expression of which transforms the meristem from vegetative to reproductive. The APETALA1 (AP1) gene plays an important role in transition phase and flower meristem identity. The identification and homology of this gene and its deduced protein in Eruca sativa was studied. Total RNA was extracted from flower buds and cDNA made. Specific primers were designed and then used for RT-PCR reaction. The results showed that the desired fragment of the gene contains 782 nucleotides (complete cds). This fragment was called EvsAP1 and recorded in the NCBI database (KX524132.1). BLAST of this sequence with other species, showed that Raphanus sativus and Brassica nigra have the highest similarity (87%) with EvsAP1. The deduced protein of the EvsAP1 gene contains 256 amino acids, which is 82% similar to Raphanus sativus. The total length of AP1 protein obtained from NCBI databases in most species of Brassicace is 256 amino acids confirming the sequence. Examination of location of the different species in the phylogenetic tree showed that all species of Brassicaceae place in clads close to Eruca sativa. The species has the highest similarity with Brassica nigra and Raphanus sativus so that the later and Eruca sativa located in one clade. These results confirm the accuracy of the obtained sequence for the AP1 gene in this specie.