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Expression studies of chitinase gene in transgenic potato against Alternaria solani

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Abstract

Plant chitinases are strong antifungal enzymes, and their genes can be utilized to engineer crop plants, including potatoes, with tolerance against fungal pathogens. The purified recombinant chitinase protein significantly inhibited the growth of phytopathogenic fungi, including Alternaria solani, in a qualitative invitro antifungal assay. Furthermore, transgenic potatoes (variety Desiree) over-expressing the endo-chitinase gene were successfully developed using the Agrobacterium-mediated transformation method. The transformation efficiency was 21% based on preliminary molecular and biochemical analyses, including histochemical GUS staining, PCR, and Southern blotting. Crude protein extracts from transgenic potato plants inhibited the hyphal growth of A. solani from 39.5 to 60.5% in a quantitative in vitro assay. The in-vitro bioassay showed that transgenic potato plants exhibit strong resistance against A. solani infection as these plants remains green and healthy, while non-transgenic control plants turned yellow and eventually died 3-week post infection. In the detached leaf assay, the leaves of transgenic plants challenged with A. Soltani showed a decrease in susceptibility compared to the control. The number of necrotic spots in the control leaves was significantly higher compared to the number in the transgenic leaves of potato plants infected with A. solani. Furthermore, A. solani infection triggered a gradual increase in mRNA expression of the transgene in transgenic plants compared to non-transgenic plants as revealed in the qRT-PCR assay. mRNA expression increased to 7.34-fold higher at 72-h post infection compared to the control, and mRNA expression was directly correlated with the abundance of specific transgene transcripts.

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Abbreviations

SDS-PAGE:

Sodium dodecyl suplhate; poly acrylamide gel electrophoresis

kDA:

Kilo Daltons

bp:

Base pair

GUS:

β-Glucuronidase

qPCR:

Quantitative polymerase chain reaction

mRNA:

Messenger RNA

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Author contributions

AK: All the research work/experiments were carried out by this author. IN: The study was planned and designed by this author. He is the advisor of Mr. Anwar Khan. BT: This author helped in the experiments including realtime assays and cloning, expression studies. Also research article is being edited by this author. KA: SDS–PAGE has been performed by this author. MT: This author carried out protein purification and chitinase activity assay. AQR: The author helped with experiments.

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Authors and Affiliations

  1. Centre of Excellence in Molecular Biology, University of the Punjab, 87-West Canal Road, Thokar Niaz Baig, Lahore, 53700, Pakistan

    Anwar Khan, Idrees Ahmad Nasir, Bushra Tabassum, Khadija Aaliya, Muhammad Tariq & Abdul Qayyum Rao

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  1. Anwar Khan
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  2. Idrees Ahmad Nasir
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  3. Bushra Tabassum
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  4. Khadija Aaliya
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  5. Muhammad Tariq
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  6. Abdul Qayyum Rao
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Correspondence to Anwar Khan.

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Khan, A., Nasir, I.A., Tabassum, B. et al. Expression studies of chitinase gene in transgenic potato against Alternaria solani . Plant Cell Tiss Organ Cult 128, 563–576 (2017). https://doi.org/10.1007/s11240-016-1134-y

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  • DOI: https://doi.org/10.1007/s11240-016-1134-y

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