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Title |
In silico Construction of phosphomannose isomerase (PMI) transformation vectors and evaluation of the effectiveness of vectors in tobacco (Nicotiana tabacum L.) |
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Authors |
Bohari Bahariah1, Ghulam Kadir Ahmad Parveez1*, Mat Yunus Abdul Masani1 & Norzulaani Khalid2 |
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Affiliation |
1Advanced Biotechnology and Breeding Centre Division, Malaysian Palm Oil Board (MPOB), P. O. Box 10620, 50720 Kuala Lumpur, Malaysia; 2Institute of Biological Sciences, Faculty of Science, Universiti Malaya, 50603 Kuala Lumpur, Malaysia.
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parveez@mpob.gov.my; *Corresponding author
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Article Type |
Hypothesis
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Date |
Received January 12, 2012; Accepted January 17, 2012; Published February 03, 2012
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Abstract |
Phosphomannose isomerase (pmi) gene isolated from Escherichia coli allows transgenic plants carrying it to convert mannose-6-phosphate (from mannose), a carbon source that could not be naturally utilized by plants into fructose-6-phosphate which can be utilized by plants as a carbon source. This conversion ability provides energy source to allow the transformed cells to survive on the medium containing mannose. In this study, four transformation vectors carrying the pmi gene alone or in combination with the β-glucuronidase (gusA) gene were constructed and driven by either the maize ubiquitin (Ubi1) or the cauliflower mosaic virus (CaMV35S) promoter. Restriction digestion, PCR amplification and sequencing were carried out to ensure sequence integrity and orientation. Tobacco was used as a model system to study the effectiveness of the constructs and selection system. PMI11G and pMI3G, which carry gusA gene, were used to study the gene transient expression in tobacco. PMI3 construct, which only carries the pmi gene driven by CaMV35S promoter, was stably transformed into tobacco using biolistics after selection on 30 g 1-1 mannose without sucrose. Transgenic plants were verified using PCR analysis.
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Keywords |
Transformation vectors, phosphomannose isomerase, gusA, biolistics, tobacco.
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Abbreviations |
PMI/pmi - Phosphomannose isomerase, Ubi1 - Maize ubiquitin promoter, CaMV35S - Cauliflower mosaic virus 35S promoter, gusA - β-glucuronidase GUS reporter gene.
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Citation |
Bahariah et al.
Bioinformation 8(3): 151-157 (2012) |
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Edited by |
P Kangueane
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ISSN |
0973-2063
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Publisher |
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License |
This is an Open Access article which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. This is distributed under the terms of the Creative Commons Attribution License. |