Intron mediated enhancement of the expression of heterologous proteins in plants

Abstract

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