بررسی بیان ژن 3- هیدروکسی 3- متیل گلوتاریل کوآنزیم A ردوکتاز (HMGR)و گاماترپینن سینتاز (TPS2) تحت تنش کم‌آبی در آویشن باغی (Thymus vulgaris L.)

نوع مقاله: علمی - پژوهشی

نویسندگان

1 دانشجوی کارشناسی ارشد بیوتکنولوژی کشاورزی، گروه زراعت و اصلاح نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

2 استاد، گروه زراعت و اصلاح نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

3 استادیار، گروه کشاورزی، دانشگاه پیام‌نور، تهران، ایران

4 استادیار، گروه بیوتکنولوژی، پژوهشکده گیاهان دارویی جهاد دانشگاهی، کرج، ایران

5 استادیار، گروه اصلاح نباتات، دانشکده کشاورزی، دانشگاه پیام‌نور، تهران، ایران

10.22055/ppd.2019.20928.1436

چکیده

چکیده
این آزمایش با هدف بررسی بیان ژن‌های دخیل در مسیر بیوسنتز مونوترپن‌ها و برخی صفات مورفولوژی (شامل وزن تر ریشه و اندام هوایی، ارتفاع بوته، طول ریشه و تعداد ساقه جانبی) در قالب طرح بلوک‌های کامل تصادفی با 4 تیمار شامل شرایط 100 درصد (T1، شاهد)، 70درصد (T2، تنش ملایم)، 40 درصد (T3، تنش متوسط) و 20 درصد (T4، تنش شدید) ظرفیت مزرعه‌ای در گلخانه تحقیقاتی پژوهشکده گیاهان دارویی جهاد دانشگاهی کرج در سال ۱۳۹۴ اجرا گردید. نتایج حاصل از صفات مورفولوژی نشان داد که با افزایش تنش کم‌آبی وزن تر ریشه، وزن تر اندام هوایی، ارتفاع بوته، طول ریشه و تعداد ساقه‌های جانبی کاهش یافت. نتایج بررسی بیان ژن‌ها نشان داد که بیشترین میزان بیان ژن‌های TPS2 و HMGR و بیشترین میزان تیمول و کارواکرول در تیمار T2 حاصل شد. روند تغییرات بیان ژن‌های TPS2 و HMGR با روند تغییرات میزان مونوترپن تیمول و کارواکرول در همه تیمارها مشابه بود؛ به‌طوری‌که بیشترین میزان مونوترپن‌ها و بیان ژن‌های TPS2 و HMGR در تیمار T2 و سپس در تیمار T1 مشاهده شد و کمترین نیز در تیمارهای T3 و T4 حاصل شد. در مجموع با توجه به نتایج حاصل از این پژوهش، تنش ملایم بر افزایش بیان ژن‌های TPS2 و HMGRمؤثر بود و منجر به تولید بیشتر مونوترپن‌ها گردید.

کلیدواژه‌ها

موضوعات


عنوان مقاله [English]

The Study on Gene Expression of 3-Hydroxy-3-Methylglutaryl-Coa Reductase (HMGR) and γ-Terpinene Synthesis in Thymus Vulgaris L. Under Water Deficit Stress

نویسندگان [English]

  • Fatemeh Mirzaei 1
  • Ahmad Ismaili 2
  • Foad Fatehi 3
  • Ardeshir Ghaderi 4
  • Soheila Afkar 5
1 M.Sc. Student of Agricultural Biotechnology, Department of Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
2 Professor, Department of Plant Breeding, Faculty of Agriculture, Lorestan University, Khorramabad, Iran
3 Assistant Professor, Department of Agriculture, Payame Noor University, Tehran, Iran
4 Assistant Professor, Department of Plant Breeding, Institute of Medicinal Plant Research, Jahad Daneshgahi, Karaj, Iran
5 Assistant Professor, Department of Plant Breeding, Faculty of Agriculture, Payame Noor University, Tehran, Iran
چکیده [English]

Abstract
 
Background and Objectives
Drought is a major abiotic stress that limits agricultural crop production. Thyme (Thymus vulgaris L.) is an aromatic and medicinal plant which is very important for the herbal industry. This study was aimed to investigate the variation some of the secondary metabolites and morphological traits as well as changes in expression of genes involved in the biosynthesis of thymol (including HMGR and TPS2) by real-time PCR. One of the main goals of the present study was to find out the relationship between the transcript of these genes and related metabolites in thyme.
 
Materials and Methods
This study was conducted in the greenhouse of Iranian Institute of Medicinal Plants of Jahad Daneshgahi (Karaj, Iran) in 2016. The experimental design was a randomized complete block design with 5 replication and 4 water deficit stress treatments, including 100% (T1, control), 70% (T2, mild stress), 40% (T3, moderate stress), and 20% (T4, severe stress) of field capacity (FC). Total RNA was extracted from leaves using TRIzol reagent following the manufacturer’s instructions. First strand synthesis of cDNA was performed immediately using 1 µg of total RNA with simultaneous use of oligo-dT (50 µM) primers and random hexamer and reverse transcriptase enzyme following the manufacturer protocol. In order to examine gene expression by real-time PCR, the method of fluorescence dye SYBR green (Fermentase, USA) was used. The amount of thymol and carvacrol were measured by HPLC.
 
Results
Results of morphological traits showed that increasing the water deficit stress caused a decrease in root biomass, shoot fresh weight, plant height, root length, and the number of side branches. Results of gene expression profiling showed that the highest expression of TPS2 and HMGR genes and the highest amount of thymol and carvacrol was obtained in treatment T2 (70% FC).
 
Discussion
TPS2 and HMGR gene expression changes were similar to the changes of monoterpenes thymol and carvacrol in all treatments and the highest number of monoterpenes and gene expression were obtained in mild water deficit stress treatment in comparison with control, moderate, and severe stresses. Altogether, according to the results of this study, mild stress had a significant effect on increasing the expression of HMGR and TPS2 and led to a higher production of monoterpenes.
 

کلیدواژه‌ها [English]

  • Carvacrol
  • Morphological traits
  • Thymol

References

Albouchi, A., Bejaoui, Z. and El Aouni, M. H. (2003). Influence of moderate or severe water stress on the growth of Casuarina glauca sieb. Science et Changements Planetaires/Secheresse, 14(3), 137-142.

Aziz, E. E., Hendawy, S. T., Azza, E. E. and Omer, E. A. (2008). Effect of soil type and irrigationintervals on plant growth, essential oil yield and constituents of Thymus vulgaris plant. Journal of Agriculture and Environment Sciences, 4(4), 443-450.

Azza, A., Ezz, E., Eman, E., Aziz, S. F., Hendawy, S. T. and Omer, E. A. (2009). Response of Thymus vulgaris L. to salt stress and Alar (B9) in newly Reclaimed Soil. Journal of Applied Sciences Research, 5(12), 2165-2170.

Babaee, K., Amini Dehaghi, M., Modares Sanavi, S. A. M. and Jabbari, R. (2010). Water deficit effect on morphology, prolin content and thymol percentage of thyme (Thymus vulgaris L.). Journal of Medicinal and Aromatic Plants, 26(2), 239-251. [In Farsi]

Baghallian, K. and Naghdibady, H. (2000). Essential oil plants. Iran: Andarz Publication. [In Farsi]

Bettaieb, I., Zakhama, N., Wannes, W. A., Kchouk, M. and Marzouk, B. (2009). Water deficit effects on Salvia officinalis fatty acids and essential oils composition. Scientia Horticulturae, 120(2), 271-275.

Braga, P. C., Culici, M., Alfieri, M. and Dal Sasso, M. (2008). Thymol inhibits Candida albicans biofilm formation and mature biofilm. International Journal of Antimicrobial Agents, 31(5), 472-477.

Carretero-Paulet, L., Ahumada, I., Cunillera, N., Rodriguez-Concepción, M., Ferrer, A., Boronat, A. and Campos, N. (2002). Expression and molecular analysis of the Arabidopsis DXR gene encoding 1-deoxy-d-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-d-erythritol 4-phosphate pathway. Plant Physiology, 129(4), 1581-1591.

Chomczynski, P. and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry, 162(1), 156-159.

Crocoll, C., Asbach, J., Novak, J., Gershenzon, J. and Degenhardt, J. (2010). Terpene synthases of oregano (Origanum vulgare L.) and their roles in the pathway and regulation of terpene biosynthesis. Plant Molecular Biology, 73(6), 587-603.

Davis, E. M., Ringer, K. L., Croteau, R. B. and Wildung, M. R. (2005). Menthol biosynthesis and molecular genetics. Naturwissenschaften, 92(12), 562-577.

Dunford, N. T. and Vazquez, R. S. (2005). Effect of water stress on plant growth and thymol and carvacrol concentrations in Mexican oregano grown under controlled conditions. Journal of Applied Horticulture, 7(1), 20-22.

Ghaedi, M. (2015). Application of optimized vortex-assisted surfactant-enhanced DLLME forpreconcentration of thymol and carvacrol, and their determination by HPLC-UV: Response surface methodology. Journal of Chromatographic Science, 53(7), 1222-1231.

Hall, T. A. (1999). BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41(1), 95-98.

Hampel, D., Swatski, A., Mosandl, A. and Wüst, M. (2007). Biosynthesis of monoterpenes and norisoprenoids in raspberry fruits (Rubus idaeus L.): The role of cytosolic mevalonate and plastidial methylerythritol phosphate pathway. Journal of Agricultural and Food Chemistry, 55(22), 9296-9304.

Hemmerlin, A., Harwood, J. L. and Bach, T. J. (2012). A raison d’être for two distinct pathways in theearly steps of plant isoprenoid biosynthesis. Progress in Lipid Research,51(2), 95-148.

Hemmerlin, A., Hoeffler, J. F., Meyer, O., Tritsch, D., Kagan, I. A., Grosdemange-Billiard, C., Rohmer, M. and Bach, T. J. (2003). Cross-talk between the cytosolic mevalonate and the plastidial methylerythritol phosphate pathways in tobacco bright yellow-2 cells. Journal of Biological Chemistry, 278(29), 26666-26676.

Horwath, A. B., Grayer, R. J., Keith-Lucas, D. M. and Simmonds, M. S. (2008). Chemical characterisation of wild populations of Thymus from different climatic regions in southeast Spain. Soil Biology and Biochemistry, 36(2), 117-133.

Jaleel, C. A., Manivannan, P., Wahid, A., Farooq, M., Jasim al-juburi, H., Somasundaram, R. and Panneerselvam, R. (2009). Drought stress in plants: A review on morphological characteristics and pigments composition. International Journal of Agriculture and Biology, 11(1), 100-105.

Lane, A., Boecklemann, A., Woronuk, G. N., Sarker, L. and Mahmoud, S. S. (2010). A genomics resource for investigating regulation of essential oil production in Lavandula angustifolia. Planta, 231(4), 835-845.

Lebaschy, M. H. and Sharifi Ashourabadi, E. (2004). Growth indices of some medicinal plants under different water stresses. Iranian Journal of Medicinal and Aromatic Plants Research, 20(3), 249-261. [In Farsi]

Liao, Y., Xu, F., Huang, X., Zhang, W., Cheng, H., Li, L., Cheng, S. and Shen, Y. (2015). Promoter analysis and transcriptional profiling of Ginkgo biloba 3- hydroxy-3- methylglutaryl coenzyme A reductase (GbHMGR) gene in abiotic stress responses. Notulae Botanicae Horti Agrobotanici, 43(1), 25-34.

Lichtenthaler, H. K. (1999). The 1-deoxy-D-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants. Annual Review of Plant Biology, 50(1), 47-65.

Lv, D. and Zhang, Y. (2017). Isolation and functional analysis of apple MdHMGR1 and MdHMGR4 gene promoters in transgenic Arabidopsis thaliana. Plant Cell Tissue and Organ Culture, 129(1), 133-143.

Mahmoud, S. S. and Croteau, R. B. (2002). Strategies for transgenic manipulation of monoterpene biosynthesis in plants. Trends in Plant Science,7(8), 366-373.

Malekzadeh, A., Majdi. M. and Maroufi, A. (2014). Gene expression analysis of γ-terpinene synthesis under salicylic acid, methyl jasmonate and Trans-cinnamic acid treatments in Thymus vulgaris L. 1st International and 13th Iranian Genetics Cogress, Isfahan. [In Farsi]

Mikio, Y. and Taeko, U. (1962). Biosynthesis of thymol. Chem Pharmaceu Bull, 10(1), 71-72.

Murashige, T. and Skoog, F. (1962). A revised mediumfor rapid growth and bio assays withtobacco tissue cultures. Physiologia Plantarum,15(1), 473-497.

Omidbygy, R. (2005). Production and processing of medicinal plants (volume 3). Tehran: Astan Quds
Razavi Press. [In Farsi]

Pirzad, A., Alyari, H., Shakiba, M., Zehtab-Salmasi, S. and Mohammadi, A. (2006). Essential oil content and composition of German chamomile (Matricaria chamomilla L.) at different irrigation regimes. Journal of Agronomy, 5(3), 451-455.

Poulose, A. and Croteau, R. (1978). Biosynthesis of aromatic monoterpenes: Conversion of γ-terpinene to p-cymene and thymol in Thymus vulgaris L. Archives of Biochemistry and Biophysics, 187(2), 307-314.

Rahimi, Y. (2013). Effects of water deficit stress on genes expression menthol biosynthesis pathway in Mentha piperita. Master Thesis of Science In Breeding Plant, Tehran university, Iran.

Ramak, P., Kazempour Osaloo, S., Ebrahimzadeh, H., Sharifi, M. and Behmanesh, M. (2014). Expression of gene 1-deoxy-D-xylulose 5-phosphate reductoisomerase and its relation to monoterpene carvacrol biosynthesis in Satureja Khuzestanica. Iranian Journal of Biology, 27(4), 622-634. [In Farsi]

Rohdich, F., Zepeck, F., Adam, P., Hecht, S., Kaiser, J., Laupitz, R., Grawert, T., Amslinger, S., Eisenreich, W. and Bacher, A. (2003). The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH protein. Proceedings of the National Academy of Sciences, 100(4), 1586-1591.

Sauret-Gueto, S., Botella-Pavia, P., Flores-Perez, U., Martinez-Garcia, J. F., San Roman, C., Leon, P., Boronat, A. and Rodriguez-Concepcion, M. (2006). Plastid cues posttranscriptionally regulate the accumulation of key enzymes of the methylerythritol phosphate pathway in Arabidopsis. Plant Physiology, 141(1), 75-84.

Schilmiller, A. L., Schauvinhold, I., Larson, M., Xu, R., Charbonneau, A. L., Schmidt, A., Wilkerson, C., Last, R. L. and Pichersky, E. (2009). Monoterpenes in the glandular trichomes of tomato are synthesized from a neryl diphosphate precursor rather than geranyl diphosphate. Proceedings of the National Academy of Sciences, 106(26), 10865-10870.

Schmittgen, T. D. and Livak, K. J. (2008). Analyzing real-time PCR data by the comparative CT method. Nature Protocols, 3(6), 1101-1108.

Schuhly, W. (2004). Pharmacognosy: Phytochemistry, medicinal plants. Phytomedicine: International Journal of Phytotherapy and Phytopharmacology, 11(1), 90-91.

Schuhr, C. A., Radykewicz, T., Sagner, S., Latzel, C., Zenk, M.H., Arigoni, D., Bacher, A., Rohdich, F. and Eisenreich, W. (2003). Quantitative assessment of crosstalk between the two isoprenoid biosynthesis pathways in plants by NMR spectroscopy. Phytochemistry Reviews, 2(1), 3-16.

Seyyedan, P., Daneshian, J., Mirza, M., Maleki, A. and Alireza, S. (2014). The effect of nitrogen chemical fertilizer and zinc sulfate application on yield and its components of nigella sativa L. under different humidity conditions. Bulletin of Environment, Pharmacology and Life Sciences, 3(2), 92-99.

Sharifi, P. and Mohammadkhani, N. (2017). Effects of drought stress on enzymatic and non-enzymatic antioxidants in flag leaf and spikes of tolerant and sensitive wheat genotypes. Plant Productions, 41(3), 37-50.

Viera, H., Bergamaschi, H., Angelocci, L. and Libardi, P. (1991). Performance of two bean cultivars under two water availability regimes. II. stomatal resistance to vapour diffusion, transpiration flux density and water potential in the plant. Pesquisa Agropecuaria Brasileira, 24(9), 1045-1053.

Zhang, L., Yan, X., Wang, J., Li, S., Liao, P. and Kai, G. (2011). Molecular cloning and expression analysis
of a new putative gene encoding 3-hydroxy-3-methylglutaryl-CoA synthase from Salvia miltiorrhiza. Acta Physiologiae Plantarum, 33(3), 953-961.