Purification And Characterization Of A Polygalacturonase Related To Abscission From Tomato Pedicel

The abscission of organs is a very widespread process in nature, it is the result of long-term adaptation of plants in the environment But in agricultural production, abscission causes flower and fruit dropping in tomato and other crops. It reduces the yield seriously and brinngs bad loss. It is proved that activity of polygalacturonase abscission was closely related to the activity of polygalacturonase. So in my study the pedicels explants of tomato in vitro treate with 20μl·L^-1 ethylene were used to extract and purify the polygalacturonase related to abscission in order to study the character and regulationg of the activity of polygalacturonasefather more. The results are as follows:1. It is clear that the effect of different extraction condition on acvitity of polygalacturonase from the crude enzyme extracts of tomato pecdicel, the optimun extraction condition is 50 mmol·L^-1 acetate buffer solution(pH5.5) with 0.1 M NaCl and 1 mM DTT to extract It is also proved that the ammonium sulfate graduation precipitation method not to be suitable for the polygalacturonase extraction.2. Established the purification condition of polygalacturonase of tomato pecdicel. Namely: first according to the difference of molecular weight the enzyme extracts which concentrated in low temperature are purified by Sephadex G-75 column equilibrated with flow rate of 0.2 mL·min^-1 and sample volume of 3.5mL .The extent of purification is 17.38-fold and the yield is 63.92%. Then the active fraction of polygalacturonase which concentrated in low temperature are further absorbed onto a CM sepharose CL-6B ionic exchange chromatographic with acetate buffer at pH5.5 and flow rate of 0.3 mL·min^-1. Finally, The extent of purification is 30.85-fold and the yield is 52.58%.3. It is clear that the molecular mass of the purified polygalacturonase of tomato pecdicel is 30.2 kD.4. It is clear that the characterization of polygalacturonase of tomato pecdicel, the pH optimum for activity is determined to be 5.0, temperature optima to be 40℃,thermal stabilities at 30℃is good, the activity is inhibited above 40℃and Km is 26.14 mg·ml^-1.5. It is clear that the effect of different metal ion on the activity of polygalacturonase. The result indicated that the enzyme activity was inhibited by 1 mmol·L^-1 Ca²⁺, Gu²⁺, Ba²⁺, Co²⁺ and Mn²⁺ and activated by 1 mmol·L^-1Mg²⁺, K⁺, Fe²⁺, Zn²⁺ and Fe³⁺. On the contrary, higher concentration Mg²⁺and K⁺ inhibited the activity of PG6. It is clear that the effect of different concentration of plant growth regulaters on the activity of polygalacturonase. The result indicated that GA and IAA can inhibite the activity of PG with the optimum concentration of 0.3 mg·mL^-1 and 0.1 mg·mL^-1 respectively. ABA can activate the activity of PG with the optimum concentration of 0.3 mg·mL^-1. Lower concentration ZT can activate the activity of PG, but not distinct. When the concentration came up, ZT inhibite the activity of PG with the optimum concentration of 0.2 mg·mL^-1.7. In this study, the activity of polygalacturonase is regulated in order to inhibite the abscission of tomato flowers for the first time. It is a new way to avoid the abscission of flower and fruit of tomato and other crops.