جداسازی ژن Phosphoglycerate Dehydrogenase از گیاه هالوفیت Aeluropus littoralis و بررسی عملکرد جهش یافته T-DNA آن در گیاه مدل Arabidopsis thaliana

نوع مقاله : علمی پژوهشی

نویسندگان

1 استادیار گروه مهندسی ژنتیک و بیولوژی، پژوهشکده ژنتیک و زیست‌فناوری کشاورزی طبرستان، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری

2 دکتری اصلاح‌نباتات، دانشگاه گیلان، گیلان

3 گروه ژنتیک مولکولی، موسسه ژنتیک گیاهی و تحقیقات گیاهان زراعی لیبنیز (IPK)، آلمان

چکیده

تحلیل داده‌های زیستی نقش مهمی در بررسی ژن‌ها در پاسخ به تنش‌ها دارد. هالوفیت Aeluropus littoralis، منبع ژنتیکی ارزشمندی در شناسایی ژن‌های دخیل در تحمل گیاه به تنش‌ها می‌باشد. در مطالعه حاضر، ژن فسفوگلیسرات دهیدروژناز (PGDH) به عنوان اولین آنزیم مؤثر در تولید سرین، بر اساس توالی EST ثبت شده از گیاه آلوروپوس در شرایط تنش شوری و با استفاده از روش RLM-RACE جداسازی شد. با همپوشانی توالی ناحیه َ3 و EST، قطعه‌ای به طول bp 1506 شامل ناحیه ORF به طول 1268 و ناحیه 3UTR به طول 238 نوکلئوتید بدست آمد. بررسی فیلوژنتیکی AlPGDH با دیگر ژن‌های اورتولوگ در گیاهان مختلف انجام و همولوگ‌های ژن PGDH شناسایی شد. درخت فیلوژنتیکی بیانگر قرابت بالای ژن AlPGDH با گیاهان تک‌لپه و خانواده غلات مانند سورگوم، ارزن دم‌روباهی و برنج بود. بررسی شبکه هم‌بیانی ژن AtPGDH، بیانگر نقش مؤثر ژن PGDH در مسیرهای بیوسنتزی از جمله تولید آمینو اسیدها، تولید متابولیت‌های ثانویه و مسیر متابولیسم گلایسین، سرین و ترئونین بوده و آنالیز بیانی این‌سیلیکو آن نیز حاکی از افزایش بیان ژن AtPGDH در تنش‌های مختلف می‌باشد. بررسی فنوتیپی جهش‌یافته خاموش شده (آرابیدوپسیس) این ژن در تنش‌های کلرید سدیم و PEG حاکی از کاهش شدید خصوصیات رشدی گیاهان جهش‌یافته نسبت به کنترل بود که می‌تواند تأییدی بر نقش ژن در تنظیمات پاسخ به تنش‌های شوری و خشکی باشد. یافته‌های این تحقیق، خصوصیات عملکردی ژن PGDH، تغییرات فنوتیپی گیاهان جهش‌یافته pgdh در مواجهه با تنش شوری و خشکی و نقش احتمالی آن را در افزایش تحمل گیاه به تنش‌ها ارائه می‌نماید.

کلیدواژه‌ها

موضوعات

عنوان مقاله [English]

Isolation Phosphoglycerate Dehydrogenase gene from Aeluropus littoralis halophyte plant and functional analysis of T-DNA mutant in Arabidopsis thaliana

نویسندگان [English]

  • Seyyed Hamidreza Hashemi-Petroudi 1
  • Hamidreza Ghorbani 2
  • Markus Kuhlmann 3
1 Genetic Engineering and Biology Department, Genetic and Agricultural Biotechnology Institute of Tabarestan (GABIT), Sari Agricultural Sciences and Natural Resources University (SANRU), Sari, Iran,
2 Ph.D. in Plant Breeding, Faculty of Agricultural Sciences, University of Guilan, Guilan, Iran.
3 RG Abiotic Stress Genomics/ RG Heterosis, Department Molecular Genetics, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). Germany.
چکیده [English]

Bioinformatic analysis plays an important role in the study of genes and the prediction of their function in response to stresses. Halophyte Aeluropus littoralis, a valuable genetic resource for identifying genes involved in plant tolerance to abiotic stresses. In this study, Phosphoglycerate Dehydrogenase (PGDH) gene as the first important enzyme in the synthesis of serine, was Isolated based on EST sequence from plant Aeluropus littoralis in salinity using by the RLM-RACE method. By overlapping the 3’ and EST sequences, a 1506 bp fragment including the ORF region (1268 nucleotides) and 3’UTR region (238 nucleotides) were obtained. The phylogenetic analysis of AlPGDH was done with other ortholog genes in different plants and its homologs were identified. Based on phylogram, the high degree of homology was observed between AlPGDH gene and other homologous genes from monocot cereals such as sorghum, foxtail millet and rice. The AtPGDH co-expression network analysis showed the important role of the PGDH gene in biosynthetic pathways, including amino acid synthesis, secondary metabolites synthesis and the pathway of glycine, serine and threonine metabolism, and its expression analysis indicated that the expression was increased in different stresses. The Phenotyping of the Arabidopsis knockout mutants for PGDH gene in NaCl and PEG stress condition indicated that the growth characteristics were significantly reduced in compared to the control plant, which could be confirmed the role of this gene in the response to salt and drought stress. The findings of this study reveal the functional characteristics of AlPGDH gene, phenotypic changes in AtPGDH mutant plants in exposure to salt and drought stress, and its possible role in increasing plant tolerance to stress.

کلیدواژه‌ها [English]

  • Abiotic Stress
  • Halophyte
  • PGDH Gene
  • Serine
  • T-DNA

مراجع

Emrich S, Barbazuk WB, Li L, Schnable PS (2007) Gene discovery and annotation using LCM-454 transcriptome sequencing. Genome res. 17: 69-73.
Goff SA, Ricke D, Lan TH, Presting G, Wang R, Dunn M, Glazebrook J, Sessions A, Oeller P, Varma H, Hadley D, Hutchison D, Martin C, Katagiri F, Lange BM, Moughamer T, Xia Y, Budworth P, Zhong J, Miguel T, Paszkowski U, Zhang S, Colbert M, Sun WL, Chen L, Cooper B, Park S, Wood TC, Mao L, Quail P, Wing R, Dean R, Yu Y, Zharkikh A, Shen R, Sahasrabudhe S, Thomas A, Cannings R, Gutin A, Pruss D, Reid J, Tavtigian S, Mitchell J, Eldredge G, Scholl T, Miller RM, Bhatnagar S, Adey N, Rubano T, Tusneem N, Robinson R, Feldhaus J, Macalma T, Oliphant A, Briggs S (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Sci. 296: 92-100.
Hashemi SH, Nematzadeh G, Askari H, Ghahary S (2014) Involvement of Cytosine DNA methylation in different developmental stages of Aeluropus littoralis. J. Plant Mol. Breed. 2: 56-67.
Ho CL, Saito K (2001) Molecular biology of the plastidic phosphorylated serine biosynthetic pathway in Arabidopsis thaliana. Amino Acids. 20: 243-259.
Hopper DW, Ghan R, Schlauch KA, Cramer GR (2016) Transcriptomic network analyses of leaf dehydration responses identify highly connected ABA and ethylene signaling hubs in three grapevine species differing in drought tolerance. BMC Plant Biol. doi:10.1186/s12870-016-0804-6.
Kawabata S, Terao Y, Hamada S. (2000) Molecular cloning, sequence and characterization of a novel streptococcal phosphoglycerate dehydrogenase gene. Oral Microbiol. Immunol. 15: 58-62.
Kito K, Tsutsumi k, Rai V, Theerawitaya C, Cha-um S, Yamada-Kato N, Sakakibara S, Tanaka Y, Takabe T (2017) Isolation and functional characterization of 3-phosphoglycerate dehydrogenase involved in salt responses in sugar beet. Protoplasma. 254: 2305-2313.
Kobayashi Y, Sadhukhan A, Tazib T, Nakano Y, Kusunoki K, Kamara M, Chaffai R, Iuchi S, Sahoo L, Kobayashi M, Hoekenga OA, Koyama H (2016) Joint genetic and network analyses identify loci associated with root growth under NaCl stress in Arabidopsis thaliana. Plant Cell Environ. 39(4): 918-934.
Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Lu F, Marchler GH2, Song JS, Thanki N, Wang Z, Yamashita RA, Zhang D, Zheng C, Geer LY, Bryant SH (2017) CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 4(45): 200-203.
Moschen S, Higgins J, Di Rienzo JA, Heinz RA, Paniego N, Fernandez P (2016) Network and biosignature analysis for the integration of transcriptomic and metabolomic data to characterize leaf senescence process in sunflower. BMC Bioinformatics. doi:10.1186/s12859-016-1045-2.
Nath Radhamony R, Mohan Prasad A, Srinivasan R (2005) T-DNA insertional mutagenesis in Arabidopsis: a tool for functional genomics. Electronic Journal of Biotechnology. 8(1): 82-106.
Okamura E, Hirai MY (2017) Novel regulatory mechanism of serine biosynthesis associated with 3-phosphoglycerate dehydrogenase in Arabidopsis thaliana. Scientific Reports. 7: 3533. DOI: 10.1038/s41598-017-03807-5.
Omranian N, Kleessen S, Tohge T, Klie S, Basler G, Mueller-Roeber B, Fernie AR, Nikoloski Z (2015) Differential metabolic and coexpression networks of plant metabolism. Trends Plant Sci. 20(5): 266-268.
Serin EAR, Nijveen H, Hilhorst HWM, Ligterink W (2016) Learning from Coexpression Networks: Possibilities and Challenges. Front. Plant Sci. doi:10.3389/fpls.2016.00444.
Takehisa H, Sato Y, Antonio B, Nagamura Y (2015) Coexpression network analysis of macronutrient deficiency response genes in rice. Rice. doi:10.1186/s12284-015-0059-0.
Tantong S, Pringsulaka O, Weerawanich K, Meeprasert A, Rungrotmongkol T, Sarnthima R, Roytrakul S, Sirikantaramas S (2016) Two novel antimicrobial defensins from rice identified by gene coexpression network analyses. Peptides. 84: 7-16.
Toujani W, Muñoz-Bertomeu J, Flores-Tornero M, Rosa-Téllez S, Anoman AD, Alseekh S, Fernie AR, Ros R (2013a) Functional Characterization of the Plastidial 3-Phosphoglycerate Dehydrogenase Family in Arabidopsis. Plant Physiol. 163: 1164-1178.
Toujani W, Muñoz-Bertomeu J, Flores-Tornero M, Rosa-Téllez S, Anoman AD, Ros R (2013b) Identification of the phosphoglycerate dehydrogenase isoform EDA9 as the essential gene for embryo and male gametophyte development in Arabidopsis. Plant Signal. Behav. 8:11. e27207, DOI: 10.4161/psb.27207.
Waditee R, Bhuiyan NH, Hirata E, Hibino T, Tanaka Y, Shikata M, Takabe T (2007) Metabolic engineering for betaine accumulation in microbes and plants. J. Biol. Chem. 282: 34185-34193.
Wei Y, Guangmin X, Daying Z, Huimin Ch (2001) Transfer of salt tolerance from Aeleuropus littorulis sinensis to wheat (Triticum aestium L.) via asymmetric somatic hybridization. Plant Sci. 161: 259-266.
Xu J, Tian YS, Peng RH, Xiong AS, Zhu B, Jin XF, Gao F, Fu XY, Hou XL, Yao QH (2010) AtCPK6, a functionally redundant and positive regulator involved in salt/drought stress tolerance in Arabidopsis. Planta. 231: 1251-1260.
Zhang B, Horvath S (2005) A general framework for weighted gene coexpression network analysis. Stat. Appl. Genet. Mol. Biol. doi: 10.2202/1544-6115.1128.
Zhang H, Mao X, Wang C, Jing R (2010) Overexpression of a common wheat gene TaSnRK2.8 enhances tolerance to drought, salt and low temperature in Arabidopsis. PLoS One. 5(12): e16041.
Zhang JC, Zheng HY, Li YW, Li HJ, Liu X, Qin HJ, Dong LL, Wang DW (2016) Coexpression network analysis of the genes regulated by two types of resistance responses to powdery mildew in wheat. Sci. Rep. doi: 10.1038/srep23805.
Zhang Z, Schwartz S, Wanger L, Miller W (2000) A greedy algorithm for aligning DNA sequences. J. Comput. Biol. 7: 203-214.
 
 

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