Browsing by Subject "pEAQ-HT"

Transient expression is a preferred way to produce proteins compared to stable transformation because it is suitable for rapid screening of constructs. pEAQ-HT vectors are easy and quick vectors without the need for viral replication, and they are developed for transient expression. They promote high expression levels and allow the insertion of multiple expression cassettes in a single plasmid. Agroinfiltration is a widely used method to induce transient gene expression in plants because it is efficient and straightforward. This study aimed to improve heterologous protein expression using Gateway compatible pEAQ-HT-DEST vectors. The genes G2PS1, 2 and 3 were subcloned to these and pK2GW7 derived expression vectors and infiltrated into tobacco (Nicotiana benthamiana) leaves. pK2GW7 derived expression vectors were used as a reference when samples were analyzed by western blotting, ELISA and HPLC. Tobacco should produce 6-methyl-4-hydroxy-2-pyrone and 4,7-dihydroxy-5-methylcoumarin when one of these genes is expressed, but they both have not yet been observed at the same time. Higher expression levels could allow us to see that. That would be important for further studies because if the synthesis could get started under agroinfiltration, co-transformation with reductases can lead to gerberin, parasorboside and 4-hydroxy-5-methylcoumarin biosynthesis, thus proving function. Western blotting was successful and showed a stronger signal with all pEAQ-HT derived expression vectors compared to pK2GW7 derived expression vectors. Samples collected seven days after agroinfiltration showed a stronger signal than samples collected two days after infiltration. Results from ELISA also showed more protein from pEAQ-HT vectors even though quantitative data was not obtained. HPLC showed only 6-methyl-4-hydroxy-2-pyrone but not 4,7-dihydroxy-5-methylcoumarin. Previous studies have also shown increased expression using pEAQ-HT vectors. Hence, using pEAQ-HT vectors does increase transient gene expression compared to conventional binary vectors. Even though pEAQ-HT vectors are proved to work efficiently, there are already studies about improving them.