Genetically Modified Herbicide-Tolerant Crops and Sugar Beet—Environmental and Health Concerns - Sweeteners: Nutritional Aspects, Applications, and Production Technology - page 383

genes)arethattheyneedtobeabundant,remainconstantinproportiontothetotalRNA,andbe
unaffectedbytheexperimentaltreatments.Thebestchoicesproposedtobeusedasnormalizersof
isolatedmRNAquantitiesaremainlyRNAsproducedfromglyceraldehyde-3-phosphatedehydro-
genase(GAPDH;Bhatiaetal.1994),β-actin(Kreuzeretal.1999),tubulin(Brunneretal.2004),or
rRNA(Bhatiaetal.1994).However,ingeneral,dependingonthedevelopmentalstageorenviron-
mentalstimuli,theexpressionofcertainreferencegenesiseitherup-ordown-regulated.
TheuseofGAPDHmRNAasanormalizerisrecommendedwithcaution,asithasbeenshown
thatitsexpressionmaybeup-regulatedinproliferatingcells(Zhuetal.2001).Theusageof18S
RNAasanormalizerisnotalwaysappropriate,asitdoesnothaveapolyAtailandthusprohib-
itsthesynthesisofcDNAwitholigo-dT.Additionally,18SRNA,beingofribosomalorigin,may
notalwaysberepresentativeoftheentirecellularmRNApopulationandisinsuchoverwhelming
quantitiesrelativetoraremessagethatcompetimersmustfrequentlybeemployedtoobtainrelevant
normalization,makingtheresultsmorecomplex.Theexpressionofactinortubulinoftendepends
ontheplantdevelopmentalstage(Diaz-Caminoetal.2005;Czechowskietal.2005)andisaffected
uponenvironmentalstresses(Jinetal.1999),makingtheiruseasnormalizersinappropriate.
Argyropoulos et al. (2006) proposed an alternative method for an internal control that would be
applicable to different organisms without prior knowledge of genomic sequences, assuming that one
wantstonormalizeagainstthetotalmRNA.Inthepresentedmethodology,syntheticDNAmolecules
(adapters)ofknownsequencetailcDNAduringRTandareusedinsteadofinternalreferencegenes.
12.4.1.7 Nuclear Magnetic Resonance
Sobolevetal.(2010)usedthe 1 HNMRmethodologyinthestudyofGMfoods.Transgeniclet-
tuce ( Lactuca sativa cv Luxor) overexpressing the Arabidopsis KNAT1 gene was presented as a
casestudy.Twenty-twowater-solublemetabolitespresentinleavesofconventionalandGMlettuce
weremonitoredbyNMRandquantiiedattwodevelopmentalstages.NMRspectradidnotreveal
any difference in metabolite composition between the GM lettuce and its wild-type counterpart.
Statisticalanalysisofmetabolitevariableshighlightedmetabolismvariationasafunctionofleaf
development aswell asthetransgene. Amain effect of thetransgene wasthealterationof sugar
metabolism.
12.4.2 Metabolic engineering and Biotransformation
Metabolicengineeringofplantcellshasbeenemployedforthebiotransformationofhesperidin
extractedfromorangepeels,by-productsoftheorangejuiceindustry,intoneohesperidin,asub-
stratefortheproductionofthelow-caloriesweetenerandlavorenhancerneohesperidindihydro-
chalcone(Frydmanetal.2005).Threestepswereused:(1)extractionofhesperidinfromorange
peels;(2)hydrolysisofsugarmoieties;and(3)biotransformationofhesperidinhydrolysisproducts
intoneohesperidinusingmetabolicallyengineeredplantcellcultures(transgenictobaccoandcar-
rot)expressingarecombinantlavanone-7- O -glucoside-2- O -rhamnosyltransferase.
FongChongetal.(2007)engineeredsugarcanetosynthesizesorbitolbyintroducingthegene
from Malus domestica encodingtheenzymesorbitol-6-phosphatedehydrogenase(whichcatalyzes
thereductionofglucose-6-phosphatetosorbitol-6-phosphate)andthegenefrom Zymomonas mobi-
lis encodingtheenzymeglucokinase.Motivatedbytheatypicaldevelopmentoftheleavesinsome
sorbitolcane,thepolarmetaboliteproilesintheleavesofthoseplantswerecomparedtoagroupof
controlsugarcaneplants.Eighty-sixpolarmetabolitesweredetectedintheleafextracts.Principal
component analysis of the metabolites indicated that three compounds were strongly associated
with sorbitolcane. Two were identiied as sorbitol and gentiobiose, and the third was unknown.
Gentiobioseandtheunknowncompoundwerepositivelycorrelatedwithsorbitolaccumulation.The
unknowncompoundwasasorbitol-glucoseconjugate(FongChongetal.2010).