POLY PHENOL OXIDASE

        The work envisages cloning of Poly Phenol Oxidase (PPO) gene for over-expressionin potato and isolation, and cloning of commercially important enzymes like catalase. 

                               A putative PPO gene fragment was amplified and cloned from tea (Chinary type) using conserved sequences. Expression analysis across clones showed unequivocal presence of transcript. To clone full-length genes, RACE was performed (Fig. 6.5). RTPCR based expression analysis showed highest expression in vegetative bud and first leaf followed by stem, second, third and fourth leaves (Fig. 6.6). PPO activity was also studied across various clones in response to different treatments at biochemical and molecular levels. Treatments such as PEG, ABA or jasmonate did not significantly alter the expression of PPO (Fig. 6.7). Wounding, however, enhanced the expression of PPO (Fig. 6.8).

Fig. 6.5 Various stages to clone full-length cDNA of polyphenol oxidase from tea. A) Amplification with 5’ RACE B) 3’ RACE C) Full length PPO gene.

Fig. 6.6 RT-PCR based expression analysis of PPO in different parts of tea shoot

Fig. 6.7 Effect of treatments onthe expression of PPO.

Fig. 6.8 Effect of wounding on expression of PPO in tea.

Expression of Polyphenol Oxidase in yeast system:

The complete coding sequence of gene encoding polyphenol oxidase (PPO) tea cloned by 5´ and 3´ RACE  was placed in E. coli  expression system.  Further,  PPO was cloned in yeast system. Approaches similar to that for SOD gene expression in yeast system was followed for expression of PPO in yeast. Colony PCR with PPO gene specific primers resulted in amplification of 1.8 kb fragment (Fig 7).

Fig-7: Colony PCR of P. pastoris transformants : Lanes 1, 2 and 3 are positive for PPO insert of ~ 1.8 kb. Lane L is fast ruler DNA ladder (100, 200, 300, 600, 1000, 1500, 2000, 2500, 3000 bp).