بهینه‌سازی متغیرهای انتقال ژن توسط تفنگ ژنی با انتقال ژن کدکننده‌ی موتان‌سوکراز (gtfI) به نیشکر

نوع مقاله : مقاله پژوهشی

نویسندگان

1 دانشجوی دکتری، 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 دانشیار و مربی گروه زراعت و اصلاح نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

4 مربی گروه زراعت و اصلاح نباتات، دانشکده کشاورزی، دانشگاه لرستان، خرم‌آباد، ایران

چکیده

نیشکر (Saccharum officinarum) مهم‌ترین گیاه صنعتی تولیدکننده‌ی ساکارز در دنیا می‌باشد. کشت بافت و تراریختی این گیاه با چالش‌هایی مواجه است که نیازمند بهینه‌سازی است. در این تحقیق، ژن کدکننده‌ی موتان‌سوکراز (gtfI) از باکتری Streptococcus downei جداسازی، همسانه‌سازی و به نیشکر انتقال داده شد. در روش تفنگ ژنی فاصله 9 سانتی‌متر پرتاب ژن از ریزنمونه نسبت به فاصله 12 سانتی‌متر بهتر و اختلاف معنی‌داری (P≤0.01) را نشان داد. هم‌چنین استفاده از ترکیب سوربیتول و مانیتول برای حفظ فشار اسمزی کالوس‌ها درصد تراریختی را به‌طور معنی‌داری (P≤0.01) بالا برد. ترکیب هورمونی IBA و NAA بهترین کارآیی را در ریشه‌دار کردن گیاهان تراریخت نشان داد. تجزیه قند گیاهان تراریخت حاصل از دو مرحله زیرکشت، نشان داد که میانگین پارامتر پل (میزان ساکارز) در هر دو لاین تراریخت نسبت به شاهدهای مربوطه با کاهش قابل ملاحظه‌‌ای در حدود 30 درصد همراه بود. این موضوع نشان می‌دهد که آنزیم موتان‌سوکراز به خوبی در لاین‌های تراریخت نیشکر بیان شده و توانسته است این میزان قند را مصرف کند.

کلیدواژه‌ها


عنوان مقاله [English]

Optimization of Biolistic-mediated Gene Transfer Parameters by Transferring Mutansucrase (gtfI) Coding Gene in Sugarcane

نویسندگان [English]

  • Maryam Ahmadi 1
  • Farhad Nazarian Firoozabadi 2
  • Ahmad Ismaili 3
  • Bizhan Bajelan 4
1 PhD Student, Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Lorestan, Khoram Abad, Iran
2 Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Lorestan, Khoram Abad, Iran
3 Associate Professor, Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Lorestan, Khoram Abad, Iran
4 Lecturer, Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Lorestan, Khoram Abad, Iran
چکیده [English]

Sugarcane (Saccharum officinarum) is the most important industrial sugar producing plant. Both tissue culture practices and transformation are challenging and need to be optimized. A gene encoding a mutansucrase (gtfI) enzyme from Streptococcus downei bacterium was isolated and cloned in a binary expression vector and transferred to sugarcane cultivars, using gene gun and Agrobacterium-mediated transformation. Results of this study showed that Agrobacterium was not able to deliver gtfI gene to sugarcane plants cells, whereas Biolistic gene transfer was successful and resulted in almost 25.7% transformation efficiency. Distance between explant and rupture disc carrying gtfI construct had a significant effect on transformation efficiency with 9cm distance producing the higher number of transformants than 12 cm distance. A combination of sorbitol and mannitol osmoticums showed a profound effect on transformation efficiency. Furthermore, a combination of IBA and NAA auxins had a significant effect on root regeneration. Interestingly, mutansucrase was found active in transgenic sugarcane lines utilizing sucrose by almost 33%. Detailed sugar analysis of a few transgenic sugarcane plants revealed that transgenic plants had a significant lower sucrose (lower pol%) content than untransformed control plants.

کلیدواژه‌ها [English]

  • Sugar analysis
  • transformation
  • Sugarcane
  • Gene transfer
  • Glycosyltransferase
Altpeter, F. and Oraby, H. 2010. Sugarcane. in Genetic Modification of Plants, pp. 453-472. Springer.
Anonymous. 2016. Statistical book of agricultural of Iran. Iranian Statistical Centre. Tehran, Iran.
Arencibia, A. D., Carmona, E. R., Tellez, P., Chan, M-T., Yu, S-M., Trujillo, L. E. and Oramas, P. 1998. An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium tumefaciens. Transgenic Research, 7: 213-222.
Bauer, R., Basson, C. E., Bekker, J., Eduardo, I., Rohwer, J. M., Uys, L., van Wyk, J. H. and Kossmann, J. 2012. Reuteran and levan as carbohydrate sinks in transgenic sugarcane. Planta, 236: 1803-1815.
Behera, K. K. and Sahoo, S. 2009. Rapid in vitro micropropagation of sugarcane (Saccharum officinarum L. cv-Nayana) through callus culture. Nature and Science, 7: 1-10.
Bower, R. 1994. High efficiency production of transgenic sugarcane plants. in SRDC Project UQ12S. Sugar Research Australia Ltd, The University of Queensland.
Carsono, N. and Yoshida, T. 2008. Transient expression of green fluorescent protein in rice calluses: optimization of parameters for Helios gene gun device. Plant Production Science, 11: 88-95.
Falco, M., Tulmann Neto, A. and Ulian, E. 2000. Transformation and expression of a gene for herbicide resistance in a Brazilian sugarcane. Plant Cell Reports, 19: 1188-1194.
Frame, B. R., Zhang, H., Cocciolone, S. M., Sidorenko, L. V., Dietrich, C. R., Pegg, S. E., Zhen, S., Schnable, P. S. and Wang, K. 2000. Production of transgenic maize from bombarded type II callus: effect of gold particle size and callus morphology on transformation efficiency. In Vitro Cellular & Developmental Biology-Plant, 36: 21-29.
Gandonou, C. H., Errabii, T., Abrini, J., Idaomar, M., Chibi, F. and Senhaji, S. 2005. Effect of genotype on callus induction and plant regeneration from leaf explants of sugarcane (Saccharum sp.). African Journal of Biotechnology, 4: 1250-1255.
Heinz, D. J. 1987. Sugarcane improvement through breeding. Elsevier, Amsterdam, 11: 7-84.
Ingelbrecht, I. L., Irvine, J. E. and Mirkov, T. E. 1999. Posttranscriptional gene silencing in transgenic sugarcane. Dissection of homology-dependent virus resistance in a monocot that has a complex polyploid genome. Plant Physiology, 119: 1187-1198.
Iwase, A., Harashima, H., Ikeuchi, M., Rymen, B., Ohnuma, M., Komaki, S., Morohashi, K., Kurata, T., Nakata, M. and Ohme-Takagi, M. 2017. WIND1 promotes shoot regeneration through transcriptional activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis. The Plant Cell Online, 29: 54-69.
Jackson, M. A., Anderson, D. J. and Birch, R. G. 2013. Comparison of Agrobacterium and particle bombardment using whole plasmid or minimal cassette for production of high-expressing, low-copy transgenic plants. Transgenic Research, 22: 143-151.
Jagga-Chugh, S., Kachhwaha, S., Sharma, M., Kothari-Chajer, A. and Kothari, S. 2012. Optimization of factors influencing microprojectile bombardment-mediated genetic transformation of seed-derived callus and regeneration of transgenic plants in Eleusine coracana (L.) Gaertn. Plant Cell, Tissue and Organ Culture, 109: 401-410.
Joyce, P., Hermann, S., O'Connell, A., Dinh, Q., Shumbe, L. and Lakshmanan, P. 2014. Field performance of transgenic sugarcane produced using Agrobacterium and biolistics methods. Plant Biotechnology Journal, 12: 411-424.
Joyce, P., Kuwahata, M., Turner, N. and Lakshmanan, P. 2010. Selection system and co-cultivation medium are important determinants of Agrobacterium-mediated transformation of sugarcane. Plant Cell Reports, 29: 173-183.
Kalantarhormozi, S., Siahpoosh, M. R., Rajabi Memari, H., Hamedi, H., Shomeili, M. and Hamoudi, J. 2015. Regeneration of two commercial sugarcane cultivars (CP48-103 and CP69-1062) from terminal leaves derived explants. Agricultural Biotechnology, 7: 155-174.
Kaur, A., Gill, M., Gill, R. and Gosal, S. 2007. Standardization of different parameters for ‘particle gun’mediated genetic transformation of sugarcane (Saccharum officinarum L.). Indian Journal of Bioteechnology, 6: 31-34.
Kim, J. Y., Gallo, M. and Altpeter, F. 2012. Analysis of transgene integration and expression following biolistic transfer of different quantities of minimal expression cassette into sugarcane (Saccharum spp. hybrids). Plant Cell, Tissue and Organ Culture, 108: 297-302.
Komor, E., Thom, M. and Maretzki, A. 1981. The mechanism of sugar uptake by sugarcane suspension cells. Planta, 153: 181-192.
Lakshmanan, P., Geijskes, R. J., Aitken, K. S., Grof, C. L., Bonnett, G. D. and Smith, G. R. 2005. Sugarcane biotechnology: the challenges and opportunities. In Vitro Cellular and Developmental Biology-Plant, 41: 345-363.
Lal, N. and Singh, H. 1994. Rapid clonal multiplication of sugarcane through tissue culture. Plant Tissue Culture, 4: 1-7.
Maniatis, T., Fritsch, E. F. and Sambrook, J. 1982. Molecular cloning: a laboratory manual. Cold Spring harbor laboratory Cold Spring Harbor, NY.
Manickavasagam, M., Ganapathi, A., Anbazhagan, V., Sudhakar, B., Selvaraj, N., Vasudevan, A. and Kasthurirengan, S. 2004. Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds. Plant Cell Reports, 23: 134-143.
Matroodi, S., Motallebi, M., Zamani, M., Mousavi, A., Davoodi, D. and Moghaddassi-Jahromi, Z. 2013. Sugarcane (NCo310) transient transformation using uidA reporter gene. Iranian Journal of Biotechnology, 1: 89-95.
Meng, Y., Moscou, M. J. and Wise, R. P. 2009. Blufensin1 negatively impacts basal defense in response to barley powdery mildew. Plant Physiology, 149: 271-285.
Merkle, S. A., Williams, E. G. and Bhojwani, S. S. 1990. Morphogenic aspects of somatic embryogenesis. (ed. PW Merkle SA, Williams EG, Bhojwani SS), p. 203. Elsevier.
Ming, R., Moore, P. H., Wu, K. D., Hont, A., Glaszmann, J. C., Tew, T. L, Mirkov, T. E., Da Silva, J., Jifon, J. and Rai, M. 2006. Sugarcane improvement through breeding and biotechnology. Plant Breeding Reviews, 27: 15.
Mousavi, M., Mousavi, A., Habashi, A. and Arzani, K. 2009. Optimization of physical and biological parameters for transient expression of uidA gene in embryogenic callus of date palm (Phoenix dactylifera L.) via particle bombardment. African Journal of Biotechnology, 8: 3721-37-30.
Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15: 473-497.
O'Connor, J. J. and Robertson, E. F. 2003. William Sealy Gosset. MacTutor History of Mathematics archive, University of St Andrews.
Pathak, S., Lal, M., Tiwan, A. and Sharma, M. 2009. Effect of growth regulators on in vitro multiplication and rooting of shoot cultures in sugarcane. Sugar Tech, 11: 86-88.
Petrillo, C. P., Carneiro, N. P., Purcino, A., Carvalho, C. H. S., Alves, J. D. and Carneiro, A. 2008. Optimization of particle bombardment parameters for the genetic transformation of Brazilian maize inbred lines. Pesquisa Agropecuaria Brasileira, 43: 371-378.
Porebski, S., Bailey, L. G. and Baum, B. R. 1997. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Molecular Biology Reporter, 15: 8-15.
Rae, A. L., Perroux, J. M. and Grof, C. P. L. 2005. Sucrose partitioning between vascular bundles and storage parenchyma in the sugarcane stem: a potential role for the ShSUT1 sucrose transporter. Planta, 220: 817-825.
Rein, P. 2007. Cane sugar engineering. Berlin: Verlag Dr. Albert Bartnes KG.
Santosa, D. A., Hendroko, R., Farouk, A. and Greiner, R. 2004. A rapid and highly efficient method for transformation of sugarcane callus. Molecular Biotechnology, 28: 113-119.
Sharafi, R. 2012. Cloning of a gene encoding mutansucrase enzyme (gtfi) from streptococcus dowen. Mfe 28 for expression in sugar beet plants. in Agriculture, p. 68. Lorestan, Lorestan University.
Shukla, R., Khan, A. and Garg, G. 1994. In vitro clonal propagation of sugarcane: optimization of media and hardening of plants. Sugar Cane 4.
Tadesse, Y., Sagi, L., Swennen, R. and Jacobs, M. 2003. Optimisation of transformation conditions and production of transgenic sorghum (Sorghum bicolor) via microparticle bombardment. Plant Cell, Tissue and Organ Culture, 75: 1-18.
Taparia, Y., Gallo, M. and Altpeter, F. 2012. Comparison of direct and indirect embryogenesis protocols, biolistic gene transfer and selection parameters for efficient genetic transformation of sugarcane. Plant Cell, Tissue and Organ Culture, 111: 131-141.
Tumer, N. E., Kaniewski, W., Haley, L., Gehrke, L., Lodge, J. K. and Sanders, P. 1991. The second amino acid of alfalfa mosaic virus coat protein is critical for coat protein-mediated protection. Proceedings of the National Academy of Sciences, 88: 2331-2335.
Van Engelen, F. A., Molthoff, J. W., Conner, A. J., Nap, J. P., Pereira, A. and Stiekema, W. J. 1995. pBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Research, 4: 288-290.
Wei, H., Wang, M. L., Moore, P. H. and Albert, H. H. 2003. Comparative expression analysis of two sugarcane polyubiquitin promoters and flanking sequences in transgenic plants. Journal of Plant Physiology, 160: 1241-1251.
Whalley, H. C. S. D. 1964. ICUMSA methods of sugar analysis : official and tentative methods recommended by the International Commission for Uniform Methods of Sugar Analysis (ICUMSA). Elsevier Pub. Co., Amsterdam.
Wu, H., Awan, F. S., Vilarinho, A., Zeng, Q., Kannan, B., Phipps, T., McCuiston, J., Wang, W., Caffall, K. and Altpeter, F. 2015. Transgene integration complexity and expression stability following biolistic or Agrobacterium-mediated transformation of sugarcane. In Vitro Cellular & Developmental Biology-Plant, 51: 603-611.
Zale, J. M., Borchardt-Wier, H., Kidwell, K. K. and Steber, CM. 2004. Callus induction and plant regeneration from mature embryos of a diverse set of wheat genotypes. Plant Cell, Tissue and Organ Culture, 76: 277-281.
Zhao, H., Li, M., Pei, Y., Guo, Y. and Dong, Y. 2000. Factors affecting wheat transformation efficiency by particle bombardment. Sichuan Daxue Xuebao, 38: 570-574.