Cloning, expression and characterization of aquaporins from wheat in insect cells using a baculovirus expression system

Aquaporins are water transport proteins that possess important roles in plantwater relationship. The allopolyploid nature of wheat makes this staple cereal crop more difficult to sequence and understand the diversity of aquaporin proteins. Most of the aquaporins in rice, Arabidopsis and maize shows tissue specific expression. This study aimed to identify, express and characterize root specific aquaporin proteins of wheat and investigate their specific functions. Two full length cDNAs of root specific aquaporins from common wheat (T.aestivum L. cv. Cranbrook) Triticum aestivum were isolated. The newly isolated sequences were identified as a PIP2 and PIP1 aquaporin family members by comparison with the Rice and existing wheat aquaporin genes in the database. Additionally one possible non-functional (alternate spliced) PIP2 aquaporin was isolated. This study found that the PIP2 gene was highly expressed at the early seedling stage (8 day old plant) of the plants than at the heading and flowering stage. This correlates with the higher water demand for the growing plant at early stages. In this study the insect cell/baculovirus system was used to express the identified full length PIP1 and PIP2 genes. The functional analysis of the wheat and human AQP proteins was performed by subjecting the whole insect cell to hypo-osmotic conditions and the size of the whole insect cell was measured using ImageJ software. Results indicate that the water treatment for the infected and uninfected insect cells was unsuccessful because of the technological problems and time constraints. Further glcerol transport and mercry sensitive aquaporins can be tested using the proposed method. This will be the first plant aquaporin expressed in the baculovirus system to demonstrate water transport activity and substrate specificity.