Functional analysis of NQPT1 and NPQT2 promoters, and analysis using GUS activity the transcriptional control and strength of NQPT2 versus CaMV35S promoters
- Materials and Methods
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. [...]
[...] In plants chloroplast, aspartate is converted to quinolinate and QPT catalyzes the formation of nicotinic acid mononucleotide, inorganic phosphate and carbon dioxide in the presence of Mg2+ ions from quinolinic acid and phosphoribosyl 1-pyrophosphate. The nicotinic acid is then transported out to the cytoplasm where it becomes intermediate compounds for NAD synthesis. Besides that, nicotinic acid will condense with other nitrogen-containing metabolites like N-methyl pyrrolinium to form alkaloids (Ashihara et al. 2012). Thus, this enzyme links the pyridine nucleotide cycle and aspartate pathway by maintaining the availability of nicotinic acid which ultimately regulates the synthesis of NAD and alkaloids. It indirectly acts as coenzyme for metabolic oxidation reaction in organisms (Eads et al. [...]
[...] Both wounded and non-wounded leafs tissues transformed with NQPT2 promoters were of higher GUS specific activity than that of CaMV35S. Table Absorbance readings at 595nm (units), concentration of whole proteins (µg/mL), amount of protein GUS activity (pMoles/min) and GUS specific activity for AL (CaMV35S & non-wounded), BL (CaMV35S & wounded), CL (NQPT2 & non-wounded) and DL (NQPT2 & wounded). Example calculations for AL (CaMV35S & non-wounded): Protein concentration Absorbance readings at 595nm = 0.0004 (concentration of BSA standards) 0.090 = 0.0004 (protein concentration) Protein concentration = 0.090 / 0.0004 = 225.0 µg/mL Amount of protein quantified in 20µL original tissue extract Protein concentration = 225.0 µg/mL = 225.0 /1000 = 0.225 µg/µL Amount of protein in 20µL = 0.225 x 20 = 4.5 µg GUS activity in 20µL original tissue extract Volume of original extract corresponding to amount of MU detected in 1mL = (100µL/1500µL) x 60µL = 4µL Equation from figure 3 for AL is Average concentration = 20.222 (time) + 44.366 Gradient of graph = 20.222 pMoles/mL MU per minute. [...]
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