Intron - Mediated Enhancement of the Expression of Heterologous Protein in Plant

Abstract

Advances in the biotechnological approaches have led to exploration of various techniques and strategies for overexpression of heterologous proteins in plant system. One such strategy is intron-mediated enhancement (IME), which is being used for enhanced expression of recombinant proteins. Introns are the non-functional part of genomic DNA that is unable to code any protein. However, its presence in genomic DNA has attracted researchers to explore the mysterious nature of intron. Earlier studies in mammalian cells have revealed the regulatory role of intron in expression of gene. Further, the regulatory effect of intron on gene expression was also explored in plants. Subsequently, many studies were performed in plants for enhancement of transgene expression. In the present study, IME strategy was used to enhance the expression of erythropoietin (EPO) in Nicotiana tabacum plant using Synthetic 7 (Syn7) intron. Agrobacterium-mediated genetic transformation was used for transformation of N. tabacum. For the comparative analysis of EPO expression, two vector constructs namely control expression vector (without intron) and test expression vector (with intron) were prepared. Integration of EPO in genomic DNA of transgenic plants was analysed using PCR and southern blot analysis. Indirect confirmation of transgenic N. tabacum was done by studying the metabolic burden in transgenic plants using HPLC analysis of major alkaloid, Nicotine. Expression of EPO at transcriptional and translational level was analysed using Real time PCR and ELISA assay, respectively. The expression of EPO was increased 2-17 fold at mRNA level and 9 fold at protein level in the presence of intron Syn7 as compared to the intron less construct. Based on the results, it can be concluded that intron-mediated enhancement strategy was found efficient to enhance the expression of EPO in N. tabacum.