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Functional analysis of NQPT1 and NPQT2 promoters, and analysis using GUS activity the transcriptional control and strength of NQPT2 versus CaMV35S promoters

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20 pages
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  1. Introduction
  2. Materials and Methods
  3. Results
  4. Discussions
  5. Conclusion

Quinolinate phosphoribosyltransferase (QPT) links the pyridine nucleotide cycle and aspartate pathway by maintaining the availability of nicotinic acid important for de novo synthesis of NAD and alkaloid. QPT is encoded by QPT gene. Gene duplication leads to two different forms of QPT gene which are NQPT1 and NQPT2. The QPT promoters hence plays essential role in ensuring plant survival. This experiment aimed to investigate the functionality of NQPT1 and NQPT2 promoters in petunia, carrot, tobacco and tomato. This was performed through initial transfection with four different strains of Agrobacterium rhizogenes carrying the promoters and analyzed the presence of growth of differentiated transgenic roots. Plate tissues were first cultured at nutrient medium after initial infection and then transferred to medium containing cefotaxime. Number of emerging roots were counted and stained with X-Gluc. Moreover, this experiment also compared the strength of NQPT2 and CaMV35S promoters in transgenic Nicotiana tabacum. The effect of wounding on transcriptional expression of GUS activity by the NQPT2 and CaMV35S promoters were also examined. These were performed by first constructing calibration curve, GUS assay, protein quantification and then determine GUS specific activity, which indicated transcriptional activities of respective promoters.

It was found that petunia only had one out of eight transgenic roots for wild type and one unstained root for CaMV35S. Carrot only had 24 out of 39 roots stained for NQPT1 strain. Tobacco and tomato showed no roots growth. It was observed that the GUS specific activity of wounded tissues carrying CaMV35S promoters were insignificantly lower than their respective non-wounded counterparts. Similar observation were noted for NQPT2 but with larger difference.

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