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Cloning of a functional mannose-6-phosphate reductase (M6PR) gene homolog from Egyptian celery plants (Apium graveolens): overexpression in non-mannitol producing plants resulted in mannitol accumulation in transgenic individuals

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Abstract

Salinity is a major limiting factor affecting crops production, survival and distribution worldwide. Engineering dehydration stress tolerance in commercial crops is a trait of economic importance, especially in saline-affected areas. In this work, we are reporting the cloning of the M6PR gene homolog (encoding a key enzyme, mannose-6-phosphate reductase, for mannitol biosynthesis in celery) from Egyptian celery plants. Using RACE technique, the full-length Egyptian-M6PR gene (1333 bp) was cloned into pRI-201AN plant expression vector. Analysis of the cloned gene revealed that both American and Egyptian clones had both start and stop codons in frame and was found to be 930 base long. The newly cloned EM6PR gene was found to be 126 base longer than its American counterpart at the non-coding region. Six differences at nucleotide level between the Egyptian and American sequences were observed, three of which in the coding region resulting in three polymorphic amino acids differences (tryptophan vs. leucine, glutamine vs. histidine and isoleucine vs. leucine). The newly cloned gene was introduced to tobacco via Agrobacterium and PCR analysis of T0 plants indicated the presence of the EM6PR gene into 10 out of 38 tobacco individuals. Moreover, RT-PCR analysis confirmed the presence of EM6PR transcripts in 9 out of the 10 PCR positive plants. GC/MS analysis of some RT positive individuals indicated the accumulation of mannitol in transgenics tobacco, while mannitol was absent in non-transgenic controls.

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Acknowledgements

The authors wish to thank the Agricultural Genetic Engineering Research Institute (AGERI), Agriculture Research Center (ARC), Giza for the support in achieving this work.

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  1. Shaimaa R. M. Khalil and Amr S. Ibrahim contributed equally for this article

Authors and Affiliations

  1. Oil Crops Biotechnology Lab, Agricultural Genetic Engineering Research Institute (AGERI), Agriculture Research Center (ARC), Giza, 12619, Egypt

    Shaimaa R. M. Khalil & Mohamed S. Tawfik

  2. Plant Genomic Laboratory, Agricultural Genetic Engineering Research Institute (AGERI), Agriculture Research Center (ARC), Giza, 12619, Egypt

    Amr S. Ibrahim

  3. Department of Genetics, Faculty of Agriculture, Cairo University, Giza, Egypt

    Basita A. Hussien & Ebtissam A. Hussien

Authors
  1. Shaimaa R. M. Khalil
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  2. Amr S. Ibrahim
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  4. Ebtissam A. Hussien
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  5. Mohamed S. Tawfik
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Correspondence to Mohamed S. Tawfik.

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The authors declare that there is no conflict of interests. The current work was partially funded through a Science and Technology Development Fund (STDF) Grant No. 400, Egyptian Ministry for Scientific Research.

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This work, in part, was funded through Grant I.D 400, from the Science and Technology Development Fund (STDF), Academy of Scientific Research, Ministry of Scientific Research, Egypt.

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Khalil, S.R.M., Ibrahim, A.S., Hussien, B.A. et al. Cloning of a functional mannose-6-phosphate reductase (M6PR) gene homolog from Egyptian celery plants (Apium graveolens): overexpression in non-mannitol producing plants resulted in mannitol accumulation in transgenic individuals. 3 Biotech 7, 341 (2017). https://doi.org/10.1007/s13205-017-0975-3

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