Enzymatic Characteristics And Cloning Of Tyrosinase Of Suaeda Salsa L.

The shoot of S. salsa plant is red-violet throughout the whole growth period in the intertidal zone or in the flooding area, but green on highland and land far from the seaside. Our earlier results showed that the red pigments accumulated in S. salsa are betacyanins. As an euhalophyte, physiological and molecular responses of S. salsa to salinity stress have been extensively studied. However, the molecular mechanism and the enzymatic characteristics of relevant enzyme involving in betacyanin synthesis in S. salsa have not been elucidated. In the present study, the cloning of part of the gene MEL coding for the tyrosinase from S. salsa, which is the first enzyme of betalain synthesis, and its enzymatic characteristics were studied. These results are very important for isolation of the full length of gene MEL coding for the tyrosinase from S. salsa and study on the role of tyrosinase in betalain synthesis and its relations to environments.The results are showed as follows:In higher plants, tyrosinase is also called polyphenol oxidase. However, in the plants such as Suaeda salsa which betalain is synthesyzed, the tyrosinase is different from polyphenol oxidase of other higher plants which has no hydroxylase activity or little hydroxylase activity. Here, S. salsa seedlings grown in darkness for 3 days after the seeds sowed in the soil were used to study the technique of extraction and activity assays of tyrosinase, in particular, its enzymatic characteristics were studied in detail. For O-diphenol oxidation activity, the optimal temperature and pH are 35℃and 6.6, respectively Kinetic parameters Km and Vmax calculated from Lineweaver–Burk plots are 1.09 mmol/L and 71.43μmolg-1min-1. For the monophenol hydroxylation, the optimal temperature and pH are 40℃and 6.6, respectively. Kinetic parameters Km and Vmax calculated from Lineweaver–Burk plots are 3.16 mmol/L and 0.645μmolg-1min-1. Na2S2O3 can inhibit the activity of O-diphenol oxidation and monophenol hydroxylation.The activity of O-diphenol oxidation and monophenol hydroxylation can be significant activated when the copper concentration is 0.01 mmol/L, and 128.2 % and 126 % of controls, respectively. The results laied a foundations for further studying the offects of environmental factors on growth of and on the expression and activity of tyrosinase, and the role of tyrosinase in the process of betacyanin synthesis of Suaeda salsa.There is no information about tyrosinase gene sequences in higher plants, enven in Phytolacca americana and purslane in which betalain is synthesized.The genetic relations of Suaeda salsa tyrosinase to polyphenol oxidase in higher plants and tyrosinase in fungi and animals is unknown. Based on sequence homologies of the MEL genes from fungi and PPO genes from other plants, we used reverse transcriptase (RT)-PCR amplification to obtain the part of Ss MEL, which was 532 bp. BLAST search results showed that the sequence of Ss MEL was 71 % similar to polyphenol oxidase of Phytolacca americana in which betalain is synthesized and it is the sequence of Cu2+-binding. It establishes the foundation for isolation of the full length of gene MEL coding for the tyrosinase from S. salsa and study on the role of tyrosinase in betalain synthesis and its'relations to environments.High tyrosinase activity was detected in the seedlings of Suaeda salsa which cultured in darkness for 3 days after the seeds sowed in the soil. Surprisingly, there was no a satisfactory result for isolation of the full length of gene MEL coding for the tyrosinase through many times'attempts when we carried on 3'-race and 5'-race with extracted RNA reverse transcription from seedlings after germination 24 h, 36 h, 48 h, 72 h. This may be because the claim of the content of objective RNA in 3'-race and 5'-race is higher. So, we speculated that the tyrosinase was synthesyzed during the seed formation and stored in the seed for the ustalization of betalein synthesis during the germination of Suaeda salsa seeds. Therefore, we designd experiments to study the activity of tyrosinase in the seed of Suaeda salsa. Interestingly,high tyrosinase activity was detected in the seeds of Suaeda salsa at 0 h,0.5 h,5 h after imbibition. This indicates that the red-violet phenotype of 3 day-old of Suaeda salsa seedlings in the darkness was mainly due to the tyrosinase stored in the seeds rather than new syntheisis via gene expression. Therefore, 3 day-old of Suaeda salsa seedlings in the darkness is not suitable for gene MEL cloning. It lays the foundation for selecting materials of tyrosinase RNA extraction.Our previous experiments found that S. salsa callus can not synthesize betacyanins in the dark and only synthesyzed under light. And the gene of many fungi tyrosinase is also photoactivation. In this study, the activity of O-diphenol oxidation and monophenol hydroxylation increased at first and then decreased, when 2 day-old of Suaeda salsa seedlings in the darkness were transferred to light. This suggests that light really enhanced the Suaeda salsa tyrosinase activity, and the light treatment for too long will lead to the activity decrease.