Transgene copy number estimation and analysis of gene expression levels in Populusspp. transgenic lines

Abstract

BackgroundThe genus Populus has certain important features, suchas a relatively small nuclear genome, it can be easilyregenerated easily in vitro and genetically transformedby Agrobacterium vector system, which make it ideal forgene transfer and molecular genetic studies in foresttrees [1]. Insect-tolerant poplars have been obtainedusing several types of insecticidal genes coding for Bacil-lus thuringiensis-toxins. Regenerated plants with insect-resistance were obtained in different studies. Agrobacter-ium-mediated transformation has been the favoredmethod for the introduction of foreign genes into plants.The effectiveness of insect-resistance in transgenicplants is related to the side effects of gene transfer (siteof gene insertion, copy number, gene silencing etc.).Moreover intransgenic plants, transgene copy numbercan greatly affect the expression level and genetic stabi-lity of the target gene, making estimation of transgenecopy numbers an important area of genetically modifiedplant research [2]. Thus molecular biological analysis oftransgenic plants, like real time PCR, widely used todetect and quantify DNA and cDNA [3], could repre-sent an useful tool to investigate the genetic stability oftransgenic forest trees having a long life cycleas well asfor determining copy number in transformed plants.Material and methodsThe present study was undertaken to investigatePopu-lus alba and P. tremula x P. tremuloides transgeniclines, obtained via Agrobacterium-mediated transforma-tion, carrying cry1Ab and nptII genes in the T-DNAregion. The plants were vegetatively propagated ingrowth chambers over 2 years. Ten individuals fromeach clone were planted in containers with “forest soil”,and grown in a climate chamber.Extraction of genomic DNA and RNA from leaves wasperformed for PCR and Real Time PCR (RT-PCR) ana-lysis to estimate the transgene copy number [4] as wellas expression of the inserted gene [5]in transgenicpoplar, respectively.Results and discussionAll lines contained one copy ofcry gene and two ofthem showed that the copy number was different forthe cry1Ab and nptII genes, suggesting rearrangementsor multiple but incomplete copies of the transferredDNA (Figure 1). The copy number was concordantamong the 3 individuals of each lines analysed and withthose determined from the same transgenic lines kept inmicropropagation for 2 years.The transcript levels from both genes were deter-mined in 3 individuals for each line growing in climaticchambers. High levels of mRNA expression weredetected with respect to the stable endogenousactingene for both transgenic lines (Figure 2). Comparing thetranscript level of inserted genes among lines, a signifi-cant low level of nptII gene (p = 0.005) in the line carry-ing 3 copies was observed.Preliminary results indicate a differential expression ofendogenous genes among transgenic lines and towardstheir isogenic form.ConclusionsThe evaluation of the copy number of the insertedgenes has indicated their stability after 2 years of