Quinolinate phosphoribosyltransferase, pyridine nucleotide cycle
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.
[...] rhizogenes as well as those containing CaMV35S and QPT2 promoters roots were formed and 24 out of those were stained for carrots transfected with A. rhizogenes carrying QPT1 promoter. For tobacco and tomato, no transgenic roots were formed after infections with all four strains of A. rhizogenes. The highest number of stained roots relative to total number of roots counted was found to be in carrot roots where NQPT1 promoter was present. In this experiment, the wild type strain acted as negative control. [...]
[...] Functional analysis of NQPT1 and NPQT2 promoters, and analysis using GUS activity the transcriptional control and strength of NQPT2 versus CaMV35S promoters Summary: 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. [...]
[...] Alkaloids are nitrogen-containing secondary metabolites that are part of the plant's constitutive defense. Equipped with toxic properties, alkaloids must be localized to sites of protection and be accumulated at sufficient concentration to elicit defense effects (Shoji & Hashimoto 2011). Some commonly known alkaloids includes anatabine, nicotine and nornicotine. QPT transcripts are naturally high in alkaloids synthesizing roots and leaves (Ryan et al. 2012). When plant outer layer is breached, methyl jasmonate hormone released upon wounding and foliage damage stimulates signaling cascade which also increases synthesis rate of QPT transcripts. [...]
[...] tabacum under different treatment (CaMV35S or NQPT2 promoters and wounded or non-wounded) analyzed over 10 minutes intervals. Equation from figure 2 was used to calculate MU concentration. The concentration of MU produced increased with time for all AL (CaMV35S & non-wounded), BL (CaMV35S & wounded), CL (NQPT2 & non- wounded) and DL (NQPT2 & wounded). DL had larger increment as compared to AL, BL and CL, whereby the slower rate of production were quite similar. The average concentrations calculated would be used to plot a graph to compare the concentration of MU produced against time. [...]
[...] rhizogenes were used to infect petunia and carrot, followed by culturing in nutrient media with cefotaxine. The transgenic roots formed were to be stained with X-Gluc staining buffer, had the number of roots and stained roots were counted. No transgenic roots were observed for both petunia and carrots infected with all A. rhizogenes strains. Severe bacterial and fungi contamination however were observed. Class data was provided by laboratory demonstrators instead. Table 1 below showed the number of stained roots relative to the total number of roots extracted from explant of four different plant species infected with different strains of A. [...]
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