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

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 homolo...

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Bibliographic Details
Published in:	3 Biotech Vol. 7; no. 5; pp. 341 - 12
Main Authors:	Khalil, Shaimaa R. M., Ibrahim, Amr S., Hussien, Basita A., Hussien, Ebtissam A., Tawfik, Mohamed S.
Format:	Journal Article
Language:	English
Published:	Berlin/Heidelberg Springer Berlin Heidelberg 01-10-2017 Springer Nature B.V
Subjects:	Accumulation Agricultural economics Agriculture Amino acids Bioinformatics Biomaterials Biosynthesis Biotechnology Cancer Research Celery Chemistry Chemistry and Materials Science Cloning Codons Crops Dehydration Economic importance Gene expression Glutamine Histidine Homology Isoleucine Leucine Mannitol Mannose Original Original Article Plants (botany) Polymerase chain reaction Reductase Stem Cells Stress concentration Tobacco Transgenic plants Tryptophan RACE technique M6PR gene Celery Mannitol production Mannose-6-phosphate reductase
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Summary:	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 T 0 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.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	2190-572X 2190-5738
DOI:	10.1007/s13205-017-0975-3