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Abstract   Introduction    Materials & Methods    Results    Discussion    Literature cited

Protein

 RuBisCO is a vital portion of the photosynthetic pathway.  Variegation is a genetic anomaly resulting in nonfunctional choloroplasts (Aluru, 2001). We hypothesize that the nonfunctional chloroplasts of the white leaf tissue will have no RuBisCO expression.  In order to compensate for these reduced RuBisCO levels, we expect to see an elevated expression of RuBisCo in the green leaf margins of variegated leaves in relation to wild-type green leaves.  An acrylamide gel was used to visualize the protein content of the leaf tissues.  As expected, the acrylamide gel exhibited an area of concentrated banding for both green leaf types at approximately 50 kDa.  This is evidence of functional RuBisCO within these plant tissues.  There were no clear areas of banding within the white leaf portion protein smear indicating the absence of functional RuBisCO.

            A western blot was used to ensure that the protein band at 50kDa could be identified as RuBisCO. Two bands were observed in the lanes containing proteins isolated from the green tissues.  No banding was observed in the white tissues from variegated leaves.  This is evidence that no RuBisCO is present in these tissues.  This is consistent with expected results.

            In the lanes containing green tissues two bands were observed. The presence of only one band was expected since the antibody should bind to RuBisCO. This is likely a result of not totally denaturing the RuBisCO protein. The larger band is likely the complete protein with all the subunits still attached. The smaller band is likely the denatured large subunit. To ensure that the large band is the undenatured RuBisCO protein, another trial should be done where the protein is boiled for longer to ensure complete denaturation.

DNA

Since variegation is due to a genomic mutation in the chloroplast, we expected that DNA would still be present in equal concentrations within all leaf samples since chloroplasts are still present. DNA collection, however, was relatively unsuccessful. DNA extraction was only successful for the wild-type green leaf sample. The failure of the DNA extraction for two of the three plant tissues was likely the result of an improperly prepared detergent (CTAB). The CTAB buffer did not allow for separation of the DNA from chloroform. Nanodrop analysis of the green leaf sample DNA revealed low  DNA concentration and high levels of protein contamination.  PCR amplification of this sample resulted in a wide range of Ct values.  Also amplification of our no DNA control resulted in more amplification than one of our samples.  This data does not let us reasonably conclude that there was sufficient DNA in our original sample for any PCR amplification to occur.  It is likely that the concentrations obtained were due to contaminations of the sample.  The agarose gel run of the PCR product produced no evidence of amplified DNA, but slight banding beyond the marker represented excess primer dimer. These results allow us to make no conclusions pertaining to the DNA concentrations relative to the three tissues. 

            In the future, we would like to repeat this experiment to confirm western blot data and obtain data about the RuBisCO gene presence in variegated and non-variegated tissues.  We are also interested in performing a microarray analysis of the two tissue types to compare mRNA RuBisCO expression.

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