Cloning, expression and enzymatic characterization of a cystatin gene involved in herbivore defense in tea plant (Camellia sinensis)

Plant cystatins play crucial roles in the process of plant defense against herbivorous insects. A cDNA clone, designated CsCPI2, was isolated from Camellia sinensis cv. Longjing 43 using 5 '-/3 '-RACE extension. The full-length cDNA gene is 618 bp in size, encodes 205 amino acid residues, and has a deduced molecular weight of 23.07 kDa. CsCPI2 was different from the already published CsCPI1, as the two cDNA sequences share 69.31% and 60.4% pairwise identity at the nucleic acid and amino acid levels, respectively. The CsCPI2-MBP fusion protein was over-expressed in Escherichia coli. We found that the inhibitory effect of CsCPI2-MBP on ficin was 1.17, 3.46, and 3.73 times the effect on papain, chymopapain, and bromelain, respectively. The highest inhibitory activity of CsCPI2-MBP was found at pH 7 at 40 degrees C, and CsCPI2-MBP was stable at temperature below 60 degrees C. Moreover, CsCPI2-MBP was found to be more responsive to acidic environment than to alkaline one. Higher cystatin inhibitory activity was found in the tea leaves infested by Myllocerinus aurolineatus than that in intact healthy leaves. Finally, we found that CsCPI2 was a competitive inhibitor of the cysteine proteinases in tea weevil's gut, and leaves treated with CsCPI2-MBP incurred compensatory damage, and the weight gain of tea weevils fed on CsCPI2-MBP treated leaves was significantly higher than that of MBP treated leaves, but did not cause mortality. Taken together, our research highlights potential defensive proteins for future tea engineering approaches, but more in vivo experiments are needed to confirm their effectiveness.