Isolation Of Pectin Methylesterase Gene From Rice And Study On Its Expression

Pectin methylesterase (pme) widely exists in plant cells, playing a major role in modulating the amount of pectin formed on cell wall or between cells. Pme belongs to a gene family comprising various members, some of which express products in a spatio-temporal manner. To investigate how pme expression relates to the production of root border cells, as well as the tolerance ability of root tips suffering from environmental stresses. In order to investigate the relationship between expression of pme gene and the production and release of root border cells, and the farther effect on the environmental stress tolerance of root tip, the pme gene associated with the release of root border cells was isolated and expressed in E. coli and P. pastoris expression system, then transformed into rice and arabidopsis.The results are as follows:1. We isolated from rice a PME gene which had been reported to take effect in releasing root border cells, with its PI (pectin methylesterase inhibitor), PD (pectin methylesterase doman), AP (anti-pectin methylesterase) domains acquired The constructs pCAMBIA13011-OS-pme, pCAMBIA13011-OS-Anti-pme, pCAMBIA13011-OS-pmeI-Doman and pCAMBIA13011-OS-pme-Doman were intergrated into the genome of rice and arabidophsis through Agrobacterium-mediated transfection, and PCR-based identification.2. The E.coli expression vector pET28a-OS-pme was constructed followed by being transformed into an E.coli strain BL21. The molecular weight of candidate protein were validated to be 68 KDa by SDS-PAGE, indicating its existence at inclusion in E.coli. To further comfirm this product, western blotting was conducted using an anti-His antibody in terms of the region of His-tag within the vector of pET28a.3. Yeast expression vector pPIC9K-OS-pme was transformed into Pichia pastoris for the purpose of inducible secretion of candidate protein, Candidate protein was expressed when induced by methanol, and determined by SDS-PAGE to be 68 KDa as anticipated. We got slightly-glycosylated PME protein from supernatant of extration, the activitiy of which was therefore assayed.