تشدید بیان ژن‌های شبه‌تایونین در گیاه آرابیدوپسیس و مقاومت به نماتد Heterodera schachtii

نوع مقاله : کامل علمی - پژوهشی

نویسنده

استادیار گروه گیاهپزشکی، دانشگاه زابل. زابل، ایران.

چکیده

تایونین‌ها و شبه‌تایونین، پپتیدهای گیاهی با وزن‌ملکولی کوچک و غنی از سیستئین با فعالیت ضدمیکروبی که نقش مهمی در مقاومت گیاهان در برابر عوامل بیماری‌زا دارند و از چندین گونه گیاهی جدا شده‌اند. هدف از این مطالعه تشدید بیان ژن‌های شبه‌تایونین (At1g12665 ​​و At1g20618) برای ایجاد مقاومت در برابر نماتد H. schachtii است. ژن‌های At1g12665 ​​(ThiL12) و At1g20618 (ThiL20) از گیاه آرابیدوپسیس جداسازی شده و گیاهان تراریخته از طریق باکتری اگروباکتریوم با استفاده از پلاسمید pMAA-red و با استفاده از روش آغشته سازی گل آذین تولید شدند. بیان ژنهای شبه تایونین در گیاهان تشدید بیان شده با روش RT-PCR تأیید و میزان تشدید بیان در بذرهای قرمزرنگ و هموزیگوت با روش RT-PCR کمی مقایسه گردید. به منظور بررسی مقاومت، ریشه گیاهان 12 روزه آرابیدوپسیس تحت شرایط استریل با 60-50 لارو نماتد به ازای هر بوته در محیط Knop تلقیح شدند. پانزده روز پس از تلقیح، هنگامی که لارو نر و ماده را می‌توان به‌وضوح تشخیص داد، تعداد لاروها شمارش شدند و نتایج با رقم وحشی (Col-0) مقایسه شد. نتایج نشان داد که مقاومت به H. schachtii در لاین‌های تشدید بیان شده در مقایسه با نوع وحشی افزایش یافته است. به هر حال تفاوت معنی‌داری در اندازه نماتد ماده و سلول های syncytia بین لاین‌ها به جز یک لاین مربوط به ژن At1g20618 مشاهده نشد.

کلیدواژه‌ها


عنوان مقاله [English]

Overexpression of thionin- like genes in and resistance to

نویسنده [English]

  • Sh. A. Sarani
Assistant professor of Plant protection department, Faculty of Agriculture, University of Zabol. Zabol, Iran.
چکیده [English]

Thionins and thionin-like peptides are small cysteine-reach antimicrobial plant peptides that play an important role in resistance to plant pathogens and have been isolated from several plant species. The overexpression of Thionins-like genes (At1g12665 and At1g20618) for resistance to H. schachtii was the purpose of this study in Arabidopsis plants. At1g12665 (ThiL12) and At1g20618 (ThiL20) genes were isolated from Arabidopsis thaliana and transgenic plants were generated through Agrobacterium-mediated using the pMAA-red vector and using the floral dip method. Expression of the thionin-like genes in overexpressed lines was confirmed by RT-PCR. In order to evaluate the resistance of the plants to H. schachtii, Arabidopsis roots of 12-day old seedlings were inoculated with about 50-60 juveniles of nematode per plant on Knop medium under sterile condition. Fifteen days after inoculation, when male and female larvae could be clearly distinguished, the number of larvae was counted and results were compared with the wild-type cultivar (Col-0). Results showed that the resistance to H. schachtii was increased in the overexpressed lines compared with the wild type. However, there was no significant difference in the size of female nematode and syncytia cells between the lines, except for one line related to At1g20618 gene.

کلیدواژه‌ها [English]

  • Arabidopsis
  • Resistance
  • Thionin-like
  • Sugar beet cyst nematode
Ali MA, Shah KH, Bohlmann H. pMAA-Red: a new pPZP-derived vector for fast visual screening of transgenic Arabidopsis plants at the seed stage. BMC Biotechnology. 2012; 12; DOI: 10.1186/1472.6750.12.37
Almaghrabi B.  Characterization of thionin-like genes from Arabidopsis thaliana (PhD thesis). Austria, Vienna: University of Natural Resources and Life Sciences; 2012.
Bohlmann H. The role of thionins in plant-protection. Critical Reviews in Plant Sciences. 1994; 13:1-16.
Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal. 1998; 16: 735–743.
Chan YL, Yang AH, Chen JT, Yeh KW, Chan MT. Heterologous expression of taro cystatin protects transgenic tomato against Meloidogyne incognita infection by means of interfering sex determination and suppressing gall formation. Plant Cell Reports. 2010; 29: 231-238.
Egorov TS, Odintsova T. Defense peptides of plant immunity. Russian Journal of Bioorganic Chemistry. 2012; 38: 7-17.
Garcia-Olmedo F, Molina A, Alamillo J, Rodriguez-Palenzuela P. Plant defense peptides. Biopolymers. 1998; 47: 479-491
Hancock REW, Falla TJ, Brown M. Cationic antimicrobial peptides. Advances in Microbial Physiology. 1995; 37: 136-175.
Holsters M, Dewaele D, Depicker A, Messens E, Vanmontagu M, Schell J. Transfection and transformation of Agrobacterium tumefaciens. Molecular Genetics and Genomics. 1978; 163: 181-187.
Jürgensen K. Untersuchungen zum Assimilat- und Wassertransfer in der Interaktion zwischen Arabidopsis thaliana und Heterodera schachtii (PhD thesis). Kiel, Germany: Christian-Albrechts Universität; 2001.
Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2-ΔΔCt Method. Methods. 2001; 25: 402–408.
Logemann E, Birkenbihl RP, Ulker B, Somssich IE. An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol. Plant Methods. 2006; 16: 1-5.
Mao Z, Zheng J, Wang Y, Chen G, Yang Y, Feng D, Xie B. The new CaSn gene belonging to the snakin family induces resistance against root-knot nematode infection in pepper. Phytoparasitica. 2011; 39: 151–164.
Muller J. The economic importance of Heterodera schachtii in Europe. Helminthologia. 1999; 36: 205-213.
Norouzi P, Mahmoudi SB, Taleghani D, Rajabi A, Sadeghzadeh Hemayati S, Aghaiezadeh M, Soltani Idliki J, Darabi S, Babaee B, Fasahat P, Vahedi S. Development and phenotypic and molecular evaluation of sugar beet hybrids for resistance to rhizomania and root-knot nematode (Meloidogyne spp.). Journal of Sugar Beet. 2021; 36(2) 117- 128. (in Persian with English abstract)
Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain terminating inhibitors. Proceedings of the National Academy of Sciences. 1977; 74: 5463-5467.
Siddique S, Endres S, Atkins JM, Szakasits D, Wieczorek K, Hofmann J, Blaukopf C, Urwin PE, Tenhaken R, Grundler FM, Kreil DP, Bohlmann H. Myo-inositol oxygenase genes are involved in the development of syncytia induced by Heterodera schachtii in Arabidopsis roots. New Phytologist. 2009; 184: 457-472
Sijmons PC, Grundler FMW, Vonmende N, Burrows PR, Wyss U. Arabidopsis thaliana as a new model host for plant parasitic nematodes. Plant Journal. 1991; 1: 245-254.
Silverstein KA, Moskal WA, Wu HC, Underwood BA, Graham MA, Town CD, Vanden Bosch KA. Small cysteine-rich peptides resembling antimicrobial peptides have been under - predicted in plants. Plant Journal. 2007; 51: 262-280.
Taveira GB, Mathias LS, da Motta OV, Machado OLT, Rodrigues R, Carvalho AO, Teixeira-Ferreira A, Perales J, Vasconcelos IM, Gomes VM. Thionin-like peptides from Capsicum annuum fruits with high activity against human pathogenic bacteria and yeasts. Peptide Science. 2014; 102: 30-39.
Taveira GB, Carvalho AO, Rodrigues R, Trindade FG, Da Cunha M, Gomes VM. Thionin-like peptide from Capsicum annuum fruits: mechanism of action and synergism with fluconazole against Candida species. BMC Microbiology. 2016; 16: 1-13.
Van Loon LC. Systemic induced resistance. In AJ Slusarenko, RSS Fraser, LC Van Loon, eds, Mechanisms of resistance to plant diseases. Kluwer Academic Publishers, Dordrecht, The Netherlands, 2000; pp. 521-574.
Van Loon LC, Rep M, Pieterse CMJ. Significance of inducible defense-related proteins in infected plants. Annual Review of Phytopathology. 2006; 44: 135-162.