خاموشي ژن bZIP14 با استفاده از CRISPR/Cas9 و بررسي موتانت ها در پاسخ به تنش شبكه آندوپلاسمي در گياه Marchantia polymorpha

Knocking out bzip14 gene using CRISPR/Cas9 and analyzing the mutant line in response to induced ER stress in Marchantia polymorpha

اﻧﺒﺎﺷﺘﻪ ﺷﺪن ﭘﺮوﺗﺌﯿﻦﻫﺎي ﺗﺎﻧﺨﻮرده و ﺑﺪ-ﺗﺎﺧﻮرده ﺳﺒﺐ ﺗﻨﺶ ﺷﺒﮑﻪ آﻧﺪوﭘﻼﺳﻤﯽ ﻣﯽﺷﻮد. در ﭘﺎﺳﺦ ﺑﻪ ﺗﻨﺶ ﺷﺒﮑﻪ آﻧﺪوﭘﻼﺳﻤﯽ، ﭘﺎﺳﺦ ﺑﻪ ﭘﺮوﺗﺌﯿﻦ ﺗﺎﻧﺨﻮرده )UPR( ﺗﻮﺳﻂ ﺣﺲﮔﺮﻫﺎي ﺗﺮاﻏﺸﺎﯾﯽ ﺷﺒﮑﻪ آﻧﺪوﭘﻼﺳﻤﯽ راهاﻧﺪازي ﻣﯽﺷﻮد. اﯾﻦ ﻣﺴﯿﺮ ﻣﺠﻤﻮﻋﻪاي از ﺳﯿﮕﻨﺎلﻫﺎي ﭘﯿﺎمرﺳﺎن ﯾﮑﭙﺎرﭼﻪ اﺳﺖ ﮐﻪ ﺑﺮاي ﺑﺎزﮔﺮداﻧﺪن ﭘﺎﯾﺪاري ﺷﺒﮑﻪ آﻧﺪوﭘﻼﺳﻤﯽ ﻃﺮاﺣﯽ ﺷﺪه اﺳﺖ. دو ﻓﺎﮐﺘﻮر روﻧﻮﯾﺴﯽ bZIP ﺑﻪ ﻧﺎمﻫﺎي bZIP14 و bZIP7 ﻣﺴﯿﺮ UPR را در ﮔﯿﺎه Marchantia polymorpha ﺗﻨﻈﯿﻢ ﻣﯽﮐﻨﻨﺪ. در اﯾﻦ ﻣﻄﺎﻟﻌﻪ ﻻﯾﻦّﻫﺎي ﻣﻮﺗﺎﻧﺖ bzip14 ﺑﺎ اﺳﺘﻔﺎده از ﻓﻨﺎوري CRISPR/Cas9 در ﮔﯿﺎه ﻣﺎرﮐﺎﻧﺘﯿﺎ ﺟﻤﻊآوري ﺷﺪه اﺳﺖ. ﺑﺪﯾﻦ ﻣﻨﻈﻮر ﺗﻮاﻟﯽ DNA ژن bZIP14 در ﭘﺎﯾﮕﺎه اﻃﻼﻋﺎﺗﯽ Marchantia.info ﺑﻪ ﮐﻤﮏ Blastp ﺑﺮاي ﮔﯿﺎه ﻣﺎرﮐﺎﻧﺘﯿﺎ ﺷﻨﺎﺳﺎﯾﯽ ﺷﺪ. ﺑﺪﯾﻦ ﻣﻨﻈﻮر، ﺗﻮاﻟﯽ DNAي ژن bZIP14 ﺑﺎ اﻧﺠﺎم ﺑﻼﺳﺖ ﭘﺮوﺗﺌﯿﻨﯽ در وﺑﮕﺎه Marchantia.info ﺷﻨﺎﺳﺎﯾﯽ ﺷﺪ. ﺗﻮاﻟﯽ اﮔﺰون ﺑﻪ ﻣﻨﻈﻮر ﺷﻨﺎﺳﺎﯾﯽ sgRNAﻫﺎ در ﭘﺎﯾﮕﺎه /http://crispor.tefor.net وارد و از ﻣﯿﺎن sgRNAﻫﺎي ﭘﯿﺸﻨﻬﺎدي، ﺳﻪ ﮐﺎﻧﺪﯾﺪ اﻧﺘﺨﺎب ﺷﺪﻧﺪ. sgRNAﻫﺎ درون ﺳﺎزة اﺧﺘﺼﺎﺻﯽ CRISPR/Cas9 ﺑﺮاي ﮔﯿﺎه ﻣﺎرﮐﺎﻧﺘﯿﺎ ﺑﻪ ﻧﺎم pMpGE013 وارد و ﭘﺲ از اﻧﺘﻘﺎل ﺑﻪ اﮔﺮوﺑﺎﮐﺘﺮي ﺳﻮﯾﮥ GWB303+pSOUP ﺑﻪ ﮔﯿﺎه اﻧﺘﻘﺎل داده ﺷﺪ. از ﮔﯿﺎﻫﺎن ﺑﺎززا ﺷﺪه در ﻣﺤﯿﻂ اﻧﺘﺨﺎﺑﯽ داراي ﻫﺎﯾﮕﺮوﻣﺎﯾﺴﯿﻦ، اﺳﺘﺨﺮاج DNA اﻧﺠﺎم و ﭘﺲ از ﺗﮑﺜﯿﺮ ﺑﺎ ﭘﺮاﯾﻤﺮﻫﺎي اﺧﺘﺼﺎﺻﯽ، ﺑﺮاي ﺗﻮاﻟﯽﯾﺎﺑﯽ ارﺳﺎل ﺷﺪ. ﻫﺸﺖ ﻻﯾﻦ از 11 ﻻﯾﻦ ﺑﺎ ﺗﻮاﻟﯽ ﺻﺤﯿﺢ، ﺗﻐﯿﯿﺮات ﻧﻮﮐﻠﺌﻮﺗﯿﺪي ﺣﺬف و اﺿﺎﻓﻪ در ﺗﻮاﻟﯽ DNA ﻧﺸﺎن دادﻧﺪ. ﯾﮏ ﻻﯾﻦ ﻣﻮﺗﺎﻧﺖ اﻧﺘﺨﺎب و ﺑﺮاي ارزﯾﺎﺑﯽ ﻓﻨﻮﺗﯿﭙﯽ و آزﻣﻮن زﻧﺪهﻣﺎﻧﯽ ﺑﻪ ﻣﺤﯿﻂ ﺣﺎوي ﺗﺮﮐﯿﺐ ﺗﻮﻧﯿﮑﺎﻣﺎﯾﺴﯿﻦ، ﻣﺎدة ﺷﯿﻤﯿﺎﯾﯽ اﻟﻘﺎ ﮐﻨﻨﺪة ﺗﻨﺶ ﺷﺒﮑﻪ آﻧﺪوﭘﻼﺳﻤﯽ اﻧﺘﻘﺎل ﯾﺎﻓﺖ. ﺑﺮرﺳﯽﻫﺎ ﻧﺸﺎن داد ﮐﻪ bZIP14 ﺗﻨﻬﺎ ﻫﻮﻣﻮﻟﻮگ ژنﻫﺎي bZIP17 و bZIP28 در ﮔﯿﺎه آراﺑﯿﺪوﭘﺴﯿﺲ اﺳﺖ. ﻫﻤﭽﻨﯿﻦ ﺗﺤﺖ ﺗﻨﺶ ﺷﺒﮑﻪ آﻧﺪوﭘﻼﺳﻤﯽ، ﮔﯿﺎﻫﺎن ﻣﻮﺗﺎﻧﺖ در ﻣﻘﺎﯾﺴﻪ ﺑﺎ ﮔﯿﺎﻫﺎن وﺣﺸﯽ ﺗﻔﺎوت ﻣﻌﻨﺎداري در ﺳﻄﺢ5 درﺻﺪ ﻧﺸﺎن داده اﻧﺪ.

Accumulation of unfolded and mis-folded proteins cause ER stress. In response to ER stress, the unfolded protein response (UPR) is triggered by ER transmembrane sensor. UPR, a set of integrated signaling pathways, designed to restore ER homeostasis. Two bZIP transcriptional factors regulate the UPR pathway in Marchantia Polymorpha: bZIP14 and bZIP7. In this study, bzip14 mutant lines were generated using CRISPR/Cas9. In this order, the bZIP14 DNA sequence was identified by Blastp in Marchantia.info, a database website for M. polymorpha. Exons are implied to find out the proper sgRNAs as candidates in http://crispor.tefor.net/. Among all suggested sgRNAs, three of them were selected as candidates. sgRNAs were introduced into pMpGE013, a specific CRISPR/Cas9 vector in M. polymorpha. After transformation to agrobacteria GWB303+pSOUP, it was transformed into the plants. DNA was extracted from plants regenerated in selective media containing hygromycin, amplified using PCR by specific primers, and sent to sequencing. Eight out of 11 lines that were sequenced properly showed deletion and insertion in the DNA sequence. One mutant line was chosen for phenotypic assay and a survival test in the media containing tunicamycin, ER stress inducer. Our result showed that there are two isoforms of bZIP17 and bZIP28 in Arabidopsis thaliana, as homologs with bZIP14 in M. polymorpha. Also, a significant difference (p-value ≤ 0.05) was observed between bzip14 mutant and wild type under ER stress conditions.