Spatio Temporal Expression Pattern of an Insecticidal Gene ( c ry 2 A ) in Transgenic Cotton Lines

The production of transgenic plants with stable, high-level transgene expression is important for the success of crop improvement programs based on genetic engineering. The present study was conducted to evaluate genomic integration and spatio temporal expression of an insecticidal gene (cry2A) in pre-existing transgenic lines of cotton. Genomic integration of cry2A was evaluated using various molecular approaches. The expression levels of cry2A were determined at vegetative and reproductive stages of cotton at regular intervals. These lines showed a stable integration of insecticidal gene in advance lines of transgenic cotton whereas gene expression was found variable with at various growth stages as well as in different plant parts throughout the season. The leaves of transgenic cotton were found to have maximum expression of cry2A gene followed by squares, bolls, anthers and petals. The protein level in fruiting part was less as compared to other parts showing inconsistency in gene expression. It was concluded that for culturing of transgenic crops, strategies should be developed to ensure the foreign genes expression efficient, consistent and in a predictable manner.


Introduction
The application of biotechnology tools to agriculture has allowed scientists to transform plants without the need for sexual compatibility between species, thus establishing the possibility of rapidly producing new crop varieties with traits beneficial to human health and the environment.Plants have been transformed successfully to improve their pest and disease resistance, herbicide tolerance, nutritional qualities, and stress tolerance (Mackey and Santerre, 2000).
Pakistan is an important cotton and yarn producing country with the potential to become a key force in the global cotton and textile market place.Cotton and cotton products contribute about 10% to gross domestic product (GDP) and 55% to the foreign exchange earnings of the country (Bakhsh et al., 2010).
Genetically modified (GM) crops were cultivated on 148 million hectares globally in 2010.In Pakistan, insect Resistant cotton was grown on 2.4 million hectares out of 2.8 million hectares allocated land ( James, 2010).Bacillus thuringiensis (Bt) is perhaps, the most important source of insect resistant genes.Among them, transgenic cotton expressing insecticidal proteins from B. thuringiensis (Bt) has been one of the most rapidly adopted GM crops in the world (Barwale et al., 2004;Dong et al., 2005;James, 2002) containing cry gene(s) such as cry1Ac, cry1Ac + cry2Ab or cry1Ac + cry1F.Insect resistant cotton is considerably effective in controlling lepidopteran pests, and is highly beneficial to the grower and the environment by reducing chemical insecticide sprays and preserving population of beneficial arthropods (Gianessi and Carpenter, 1999;Tabashnik et al., 2002).
Transgenic cotton expressing Bt (Bacillus thuringiensis) toxins is currently cultivated on a large commercial but observations have shown that it behaves variably in toxin efficacy against target insects under field condition.For the insect resistant cotton to be sustainable, it is important that the toxin protein be expressed in adequate quantities in appropriate plant parts at the requisite time of the season to afford protection against major target insect pests.However, a number of studies conducted have indicated that the levels of toxin protein in cotton tissues fluctuate during the whole growing season, and may logically cause variation in efficacy of Bt cotton against lepidopteron pests (Bakhsh et al., 2010(Bakhsh et al., , 2011;;Benedict et al., 1996;Chen et al., 2000;Greenplate et al., 2001;Kranthi et al., 2005;Mahon et al., 2002).Understanding of the temporal and spatial variation in efficacy and the resulting mechanisms is essential for cotton protection and production.
The present study was conducted to evaluate the genomic integration and expression of insecticidal gene (cry2A) in pre-existing transgenic lines (T 5 Progeny) with the age of plants as well in different plant parts.The study was carried out at campus of National Centre of Excellence in Molecular Biology (CEMB), University of the Punjab, Lahore, Pakistan.ins was done by plotting absorbance values of cry2A test samples on the standard curve generated with purified cry2A standards on each of ELISA plates and expressed as nanogram of cry2A per gram of fresh tissue weight.Protein expression data of cry2A was further subjected to statistical analysis using SPSS software (version 11.0, SPSS Inc) to evaluate the differences among transgenic lines, sampling dates, interaction of transgenic lines and sampling dates at 5% level of significance.

Leaf Biotoxicity Assay
Based on spatio-temporal results, leaf biotoxicity assays were performed to counter check the variation in efficacy of endotoxin cry2A against targeted insect pests after 30, 60 and 90 days of crop age.The five leaves from the upper, middle and the lower portion of each transgenic line along with the leaves from non-transgenic control line in triplicates were detached on moist filter papers in petri plates and 2 nd instar Heliothis larvae were fed to them.After 2-3 days, mortality rates of Heliothis larvae were recorded.The mortality rates were calculated as follows: %Mortality = No. of dead larvae/Total no. of larvae ×100

Confirmation of Gene Integration
Polymerase chain reaction (PCR) assays of transgenic lines confirmed the stable integration of cry2A gene to subsequent generations; gene specific primers amplified 600 bp internal fragment of cry2A gene (Fig. 1A) while no amplification was observed in non-transgenic plant sample (Negative control).
Few PCR positive plants were further subjected to southern blot to verify integration of cry2A gene in plant genome.Gene integration was detected by gene specific probe.Results revealed the integration of cry2A gene (~1.8 kb) in plant genome.Non-transformed CIM-482 plant DNA was used as negative control while that of plasmid DNA pk2Ac was used as positive control (Fig. 1B).

Temporal and Spatial Expression of cry2A Endotoxin
Temporal expression of cry2A gene was quantified in transgenic lines after 15 days interval.The result showed that the toxin level declined with the age of plant (Fig. 2) being maximum at vegetative stage, 30 days crop.As the crop progressed towards the maturity, a decline in expression level of cry2A was observed.Similar expression pattern was found in all these transgenic lines.Statistical analysis revealed that cry2A differences among transgenic lines, sampling dates and their interaction were significant at 5% level of significance.
To evaluate spatial expression, different plant parts i.e. leaves, square buds, bolls, anthers, petals and ovary were sampled from the field and subjected to ELISA assay.The result revealed that the expression of cry2A was variable in different plant parts, being maximum in the leaves fol-

Confirmation of Gene Integration
The various molecular approaches i.e.PCR and southern blot were performed to confirm the stable integration of insecticidal gene cry2A in transgenic lines.Genomic DNA was isolated from fresh cotton leaves using the method described by Dellaporta et al. (1983).PCR was run for the detection of integrated cry2A to amplify internal fragments of 600 bp by a modification of the method by Saiki et al. (1988) using forward 5'-AGATTACCCCAGTTC-CAGAT-3' , 5'-GTTCCCGAAGGACTTTCTAT-3' as reverse primers.DNA extracted from untransformed plants was used as negative control and that of plasmid pk2Ac as positive control.The PCR was performed at 94°C for 4 minutes 94°C for 1 minute 52°C for 1 minute and 72°C for 1 minute followed by 35 times.
Southern blot analysis was performed to confirm the integration of ~1.8 kb fragment of cry2A gene in representative plants of transgenic lines.Genomic DNA was digested with pvuII enzyme and rest of the procedure was followed as described by Southern (1975).Gene specific probe of cry2A was labeled using Fermentas Biotin De-caLabel™ DNA Labeling Kit (Cat #K0651).Detection procedure was followed as provided in Fermentas Biotin Chromogenic Detection Kit (Cat# K0661).

Temporal and Spatial Expression of cry2A Endotoxin
For spatio temporal studies, a single structure was selected for quantification of cry2A in transgenic lines.For each sample date and for all transgenic lines, a single mainstem terminal leaf was collected from five plants/transgenic line after every 15 days interval.For spatial expression, various plant parts i.e. leaf, anthers, pollens, square buds and bolls were selected at reproductive growth stage.The samples were transported to the laboratory and were processed the same day.
Expression of cry2A in transgenic lines was quantified by ELISA using Envirologix Kit (Cat # AP051).Negative and positive controls were added to the wells along with test samples.ELISA was performed according to procedure given in the kit and quantification of cry2A endotox-lowed by square buds, bolls and anthers (Fig. 3).Petals were showing less toxin expression as compared to other plant parts being only 8-10 nanograms per gram of fresh tissues weight while cry2A expression in ovary remained undetectable.

Leaf Biotoxicity Assay
Laboratory biotoxicity assays with 2 nd Instar Heliothis larvae were conducted to confirm the variation in expression of the insecticidal gene at 30, 60 and 90 days of crop age.The results showed that transgenic lines had a varying mortality rate of Heliothis larvae that ranged between 60-90% at different growth stages.The varying mortality rates indicated a variation in cry2A protein levels.Transgenic lines that showed 100 mortality rate of Heliothis larvae at 30 days crop age, showed varying 60-80% mortality rate at 90 days of crop age (Fig. 4) while in case of non-transformed control CIM-482, no any larval mortality was recorded.

Discussion
The present study was undertaken to evaluate the genomic integration and expression of insecticidal gene (cry2A) in pre-existing transgenic lines with the age of plants as well in different plant parts.Result showed that gene integration remained stable in subsequent genera-tions as it was confirmed by PCR and Southern blot analysis of the transgenic plants (Fig. 1).Spatio temporal studies revealed that expression level of cry2A declined during the crop growth with toxin level falling to 15-20 nanogram per gram of fresh tissue weight (Fig. 2).These results are in agreement with previous studies conducted by Fitt et al. (1998) Bakhsh et al. (2011Bakhsh et al. ( , 2012) ) who have reported inconsistency in insecticidal gene expression over the crop growth period.Expression levels of insecticidal gene also remained variable in different plant parts (Fig. 3).The leaves of transgenic cotton were having maximum expression as compared to fruiting parts (Adamczyk et al., 2001;Bakhsh et al., 2010Bakhsh et al., , 2012;;Gore et al., 2001;Greenplate, 1999;Greenplate et al., 2000).Furthermore, laboratory biotoxicity assays with 2 nd Instar Heliothis larvae were conducted to confirm the variation in expression of the insecticidal gene at 30, 60 and 90 days of crop age.The results obtained from biotoxicity assays at different intervals also confirmed the variation in cry2A expression (Fig. 4).Study of toxin titer in cotton plant is very crucial as it must be in sufficient quantity to protect the crop against lepidopterans especially boll worms.A gradual decline in endotoxins expression was found along the passage of time of plant growth and most importantly, the expression level Gene expression varies with the nucleotide sequence of the gene, promoter, and the insertion point of the gene in the DNA of the transgenic variety, transgene copy number, the internal cell environment, as well as several external factors in the environment (Guo et al., 2001;Hobbs et al., 1993;Rao, 2005).Therefore, investigation at molecular, genetic, as well as physiological levels should help in understanding the differential expression of transgenes and the quantitative changes in insecticidal proteins in insect resistant cotton plants.
The mechanisms of variation in endotoxin protein content in plant tissues are rather complicated.The reduction in Bt protein contents in late-season cotton tissues could be attributed to the overexpression of the Bt gene at earlier stages, which leads to gene regulation at post-transcription levels and consequently results in gene silencing at a later stage.Methylation of the promoter may also play a role in the declined expression of endotoxin proteins.This has triggered research in finding possible new promoters that will induce more consistent production of insecticidal genes throughout the life of the cotton plant (Bakhsh et al., 2012).Therefore, efforts should also focus on evolving new transgenic cotton varieties with tissue-specific promoters to enhance the expression of toxin genes in fruiting parts that are susceptible to attack.
Thus, the variations in the efficacy of insecticidal genes in transgenic cotton and the involved mechanisms need to be understood fully, so as to plan rational resistance management strategies to retard the rate of the development of resistance, and to control target pests effectively by enhancing endotoxin expression through genetic or agronomic management.It can be concluded that developing new cotton varieties with more powerful resistance, applying certain plant growth regulators and maintenance of general health of the transgenic crop are important in realizing the full transgenic potential in transgenic Bt cotton.

Fig. 4 .
Fig. 4. Mortality % of 2 nd Heliothis Larvae in different transgenic lines at 30, 60 and 90 days of crop plants.(A) Heliothis larvae feeding on the leaf of control plant while Heliothis larvae were dead after feeding on leaves of transgenic plants, (B) Graph shows the mortality percentage of Heliothis larvae.Lines showed 100% mortality of larvae at 30 days crop stage while this percentage varied at 90 days observation while no any larval mortality was recorded in non-transgenic control cotton cultivar 'CIM-482'