شناسايي ژن‌‌هاي كليدي درگير در رسيدگي ميوه ليموترش با استفاده از واكاوي داده‌‌هاي ريزآرايه

Identification of Key Genes Involved in Lemon (Citrus limon) Ripening using Microarray Data Analysis

ﻃﯽ دﻫﻪﻫﺎي اﺧﯿﺮ ﺗﻼشﻫﺎي ﻣﺘﻌﺪدي ﺑﻪﻣﻨﻈﻮر ﺗﺸﺮﯾﺢ ﻣﻮﻟﮑﻮﻟﯽ ﻣﮑﺎﻧﯿﺴﻢﻫﺎي اﺳﺎﺳﯽ رﺳﯿﺪن ﻣﯿﻮه اﻧﺠﺎم ﺷﺪه اﺳﺖ. درك ﺟﺎﻣﻊ ﺷﺒﮑﻪ ﺗﻨﻈﯿﻤﯽ رﺳﯿﺪﮔﯽ در ﺗﻨﻈﯿﻢ ﮐﯿﻔﯿﺖ ﻣﯿﻮه، ﺗﻐﯿﯿﺮ زﻣﺎن رﺳﯿﺪﮔﯽ و اﻓﺰاﯾﺶ ﻣﺎﻧﺪﮔﺎري ﺣﺎﺋﺰ اﻫﻤﯿﺖ ﻣﯽﺑﺎﺷﺪ. در ﭘﮋوﻫﺶ ﺣﺎﺿﺮ، ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ ارزش ﻏﺬاﯾﯽ و داروﯾﯽ ﻟﯿﻤﻮﺗﺮش، دادهﻫﺎي رﯾﺰآراﯾﻪ ﻣﺮﺑﻮط ﺑﻪ ﺑﺎﻓﺖ اﯾﻦ ﻣﯿﻮه در دو ﻣﺮﺣﻠﻪ ﻧﻤﻮي ﻧﺎرس و ﺑﻠﻮغ، ﺑﺎ ﻫﺪف ﺷﻨﺎﺳﺎﯾﯽ ﻣﺴﯿﺮﻫﺎ و ژنﻫﺎي ﻣﺮﺗﺒﻂ ﺑﺎ رﺳﯿﺪن، ﺗﻮﺳﻂ ﻧﺮماﻓﺰارﻫﺎي ﺑﯿﻮاﻧﻔﻮرﻣﺎﺗﯿﮏ از ﺟﻤﻠﻪ ﺑﺴﺘﻪ DAVID ،limma، STRING و iTAKﻣﻮرد ﺑﺮرﺳﯽ ﻗﺮار ﮔﺮﻓﺖ. ﻧﺘﯿﺠﻪﻫﺎ ﻧﺸﺎن داد ﺗﻌﺪاد 4255 ﭘﺮوبﺳﺖ در ﻣﺮﺣﻠﻪ ﻧﻤﻮي رﺳﯿﺪه ﻧﺴﺒﺖ ﺑﻪ ﻧﺎرس اﻓﺘﺮاق ﺑﯿﺎن ﻧﺸﺎن دادﻧﺪ. ﯾﮑﯽ از ﻣﺴﯿﺮﻫﺎي ﻏﻨﯽﺷﺪه KEGG ﺗﻮﺳﻂ اﯾﻦ ﭘﺮوبﺳﺖﻫﺎ، ﻣﺴﯿﺮ ﮐﺎروﺗﻨﻮﺋﯿﺪ ﺑﻮد. ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ اﻓﺰاﯾﺶ ﺑﯿﺎن ژنﻫﺎي ﻣﺴﯿﺮ زﯾﺴﺖﺳﺎﺧﺖ ﮐﺎروﺗﻨﻮﺋﯿﺪ در ﻣﺮﺣﻠﻪ رﺳﯿﺪﮔﯽ و ﻧﻘﺶ اﯾﻦ ﻣﺴﯿﺮ در ﺑﯿﻮﺳﻨﺘﺰ آﺑﺴﯿﺰﯾﮏ اﺳﯿﺪ )ABA(، ﻣﯽﺗﻮان ﺑﯿﺎن داﺷﺖ ﺳﻮﺧﺖ و ﺳﺎز و ﻋﻼﻣﺖدﻫﯽ ABA ﻧﻘﺶ ﮐﻠﯿﺪي در رﺳﯿﺪﮔﯽ ﻣﯿﻮه ﻟﯿﻤﻮﺗﺮش دارد. اﻓﺰون ﺑﺮ اﯾﻦ، اﻓﺰاﯾﺶ ﺑﺎﻻي روﻧﻮﺷﺖﻫﺎي ژنﻫﺎي ﮐﻠﯿﺪي درﮔﯿﺮ در ﻣﺴﯿﺮ زﯾﺴﺖﺳﺎﺧﺖ اﺗﯿﻠﻦ در ﻣﺮﺣﻠﻪ رﺳﯿﺪﮔﯽ، ﻣﯽﺗﻮاﻧﺪ ﻣﺆﯾﺪ ﻧﻘﺶ اﺗﯿﻠﻦ و ﻣﺴﯿﺮ اﻧﺘﻘﺎل ﭘﯿﺎم آن در رﺳﯿﺪﮔﯽ اﯾﻦ ﻣﯿﻮه ﺑﺎﺷﺪ. ﺑﺮ اﺳﺎس ﻧﺘﺎﯾﺢ ﭘﮋوﻫﺶ ﺣﺎﺿﺮ ژن رﻣﺰﮐﻨﻨﺪه آﻧﺰﯾﻢ1 -آﻣﯿﻨﻮ ﺳﯿﮑﻠﻮ ﭘﺮوﭘﺎن ﮐﺮﺑﻮﮐﺴﯿﻼت اﮐﺴﯿﺪاز 1 )aminocyclopropane-1-carboxylate oxidase 1-1( داراي ﺑﯿﺸﺘﺮﯾﻦ اﻓﺰاﯾﺶ ﺑﯿﺎن )13/05 ﺑﺮاﺑﺮ( در ﻣﺮﺣﻠﻪ رﺳﯿﺪﮔﯽ در ﻣﻘﺎﯾﺴﻪ ﺑﺎ ﻣﯿﻮه ﻧﺎرس ﻟﯿﻤﻮﺗﺮش ﺑﻮد. ﺟﺴﺘﺠﻮ ﺑﺮاي ﺗﻌﯿﯿﻦ ﻋﻮاﻣﻞ روﻧﻮﯾﺴﯽ ﺑﺎ اﻓﺰاﯾﺶ ﺑﯿﺎن ﻣﻨﺠﺮ ﺑﻪ ﺷﻨﺎﺳﺎﯾﯽ 41 ﻋﺎﻣﻞ روﻧﻮﯾﺴﯽ ﺷﺪ. ﻫﻢﭼﻨﯿﻦ واﮐﺎوي ﺷﺒﮑﻪ ﻣﺮﺗﺒﻂ ﺑﺎ ژنﻫﺎي ﺑﺎ اﻓﺰاﯾﺶ ﺑﯿﺎن، ﻣﻨﺘﺞ ﺑﻪ ﺷﻨﺎﺳﺎﯾﯽ 10 ژن ﺑﻪﻋﻨﻮان ﻧﻘﺎط ﮐﻠﯿﺪي ﺷﺒﮑﻪ از ﺟﻤﻠﻪ ﻋﺎﻣﻞ روﻧﻮﯾﺴﯽ HY5-like ﮔﺮدﯾﺪ. دﺳﺖورزي ژنﻫﺎي ﻣﻌﺮﻓﯽ ﺷﺪه ﻣﯽﺗﻮاﻧﺪ ﯾﮑﯽ از راﻫﮑﺎرﻫﺎي ﺑﻬﺒﻮد ﻣﺘﺎﺑﻮﻟﯿﺖﻫﺎي ﺛﺎﻧﻮﯾﻪ و ﺗﻐﯿﯿﺮ زﻣﺎن رﺳﯿﺪﮔﯽ ﻣﯿﻮه ﻟﯿﻤﻮﺗﺮش ﺑﺎﺷﺪ ﮐﻪ در ﺻﻮرت ﻣﻮﻓﻘﯿﺖ، ﺑﻪ ﺗﻮﻟﯿﺪ ارﻗﺎﻣﯽ ﺑﺎ زﻣﺎن رﺳﯿﺪﮔﯽ ﻣﻄﻠﻮب ﻣﯽاﻧﺠﺎﻣﺪ ﮐﻪ ﻣﯽﺗﻮاﻧﻨﺪ در ﻣﻨﺎﻃﻖ ﻣﺨﺘﻠﻒ ﻣﻮرد ﮐﺸﺖ ﻗﺮار ﮔﯿﺮﻧﺪ.

Extensive attempts have been made to clarify the molecular basis of fruit ripening mechanisms in recent decades. Regarding the effect of molecular and genetic processes in regulating fruit ripening, a comprehensive understanding of the ripening genetic network which is involved in fruit quality, ripening time, and shelf life of fruits is necessary. Due to the nutritional and medicinal value of lemon, microarray data of fruit juice vesicle tissue at two developmental stages (immature and mature) was analyzed in the present study. We aimed to identify genes and pathways related to ripening through bioinformatics tools, including Bioconductor Limma, DAVID, String, and iTAK. According to the results, 4,255 probes showed differential expression between the mature and immature stages. Based on the analysis, carotenoid biosynthesis was identified as one of the KEGG-enriched pathways. Considering the upregulated genes in the carotenoid biosynthesis pathway during maturation and its role in abscisic acid (ABA) biosynthesis, it can be stated that ABA signaling might play a vital role in the maturation of lemon fruit. Besides, enhanced expression of ethylene biosynthesis genes during the ripening stage might confirm the role of ethylene and its signal transduction in the ripening of this fruit. Based on the results of this study, the gene encoding 1-aminocyclopropane-1-carboxylate oxidase­ 1 enzyme showed the highest expression fold change (13.05 times) in the mature compared to immature lemon. A survey for the determination of upregulated transcription factors, resulted in the identification of 41 transcription factors. Also, upregulated genes network analysis led to the identification of 10 genes, such as the HY5-like transcription factor involved in the maturation process. Manipulation of these identified genes is introduced as one of the approaches to improve secondary metabolites, and change ripening time in lemon fruit which can lead to developing cultivars with favorable ripening time that can be grown in various areas.