Study On Diversification Of Several Isoenzymes During Development And Senescence In Plant Fruit

Isoenzymes are very important substances for the life.In order to study the special character and the variation rule of the life during the development, the tendency with the cell life was analyzed and compared as followed:the same kind of isoenzymes were in the plant of the same species,and the same kind of isoenzymes were in the same plant of the different species,and the likely isoenzymes with character were in the same plant of the different species,and in different plants.The results indicted the universal rule in cell that isonezymes were the embodiment for the biology character during the course of life.These results were followed:With Dangshan pear as the experimental material,the change characters of POD were analyzed during the fruits’ growth,post-storage and browning.It was found that POD of various forms existed in the fruit.With the growth of fruit,the varieties of POD isoenzymes were reduced gradually and their activities changed with regularly.The activity of POD-A,larger molecular weight（MW 58.5 kDa） and high enzymatic activity content,was lowered gradually and the specific activity was decreased to 1/3 of that of original during the fruits’ development.However,the activity change in the browning of ripe fruit was not obvious.POD-A crystal was separated and purificated at the most appropriate reaction temperature（55℃） and pH value（5.4）.Through N-end sequence analysis,the first 5 amino acid sequences of POD-A N-end were Ala-Cys-Ile-Ala-Ile.POD-B,little molecular weight（MW 28 kDa） didn’t change much in the process of fruit development.However,the activity loosed rapidly during the browning process.The existence of two kinds of isozymes and their differences in change characters indicated the different metabolic characters of fruit during ripeness and consenescence.The results indicated that there had the relationship between the isoenzyme activity and their biological functions during development,and the negative relationship betwwen the enzymes activity and the course of fruit ripen.However,the change of POD-A activity was less influenced by the degree of the fruit brown during browning process.To be contrary,the POD-B loss its activity rapidly with brown and remained the stability level of enzyme activity during development.Then differences of POD in young fruit and ripe one were analyzed and compared with 2D-PAGE technology.There were POD isoenzyme molecules such as POD-A-b,c,d,e,f erc existed in young fruit,expect for POD-A-a and POD-A-g which also exited in the ripe fruit.Therefore,the lowering of POD isoenzyme activity was mainly resulted from the activity loss of POD-A-b,c, d,e,f etc.The results indicated that the existences of isozyme molecules with various forms were the reflection of different metabolic characters at different growth phases of plant organs and histiocytes.In order to investigate POD-A and POD-B in the biochemical functions of isoenzymes,with the PCR reactions of the same polynucleotide sequences,genes of POD-A（1.5 kB） and POD-B（0.8kB） were investigated.The result indicated that POD-A and POD-B had the same sequences of amino acids in N-terminal of protein. Predicting from the activity change trend of POD-A and POD-B,the POD-B might be the molecular of POD-A decompounded.The activity change law of polyphenoloxidase in the growth and storage process of Dangshan and XiZiLv pear with the application of polyacrylamide gel electrophoresis and gel with enzymatic activity coloration were analyzed and compared.The result indicated the variety and activity of polyphenoloxidase changed regularly along with the development of fruit: when the fruit was young,the PPO isoenzyme of A（PPO-A） and C（PPO-C） were the main component.With the development of fruit,the PPO activity concentrated in B（PPO-B）,C（PPO-C） and D（PPO-D） indicating the maturity of fruit.However,during the storage,the browning was mainly resulted from the rapid loss of activity of PPO-B,PPO-C and PPO-D.From the result,it could be consulted that PPO-B,PPO-C,PPO-D might be the key enzyme concerning the maturity of pear and the storable performance.So,it could act as a sign of alarm for estimating the quality of fruit and methods for storage by determining the PPO activity.The PPO was initially separated and purified through metal chelate affinity chromatography.In according with the research on the impact on enzymatic activity by activator and inhibitor,the quality of fruit processed with PPO inhibitor,such as 1%Vc,could be kept well.The research result was a certain importance in theory and application for fruit quality improvement and storage.With cinnamoin seed as the experimental material,the change characters of cinnamoin（MW 65 kDa） and Camphorin（MW 43 kDa） in the ripe seed of cinnamomin tree during ripeness and senescence were analyzed.It was fewer amounts of camphorin in the new seed of cinnamoin,and the amount to camohorin was increased with the time.The camphorin in extract cinnamoin added MgCl₂ was increased with the time.It was indicated the change of amount to camphorin and cinnamoin reflected the storage time of seed and conditions of ripeness and senescence.The A-chain of cinnamomin and Camphorin were purificated,and then the N-terminus sequences were determined respectively.5 amino acids sequence of N-terminus in the Camphorin were the same sequences from the NO.102 to the NO.106 amino acid of the cinnamoin.These results indicated that the amount of camphorin were positive relativity to senescence.A novel method for assaying the enzymatic activity of ribosome-inactivating proteins（RIPs） has been developed.The principle of the method was based on that RIP could remove some adenine bases from double-stranded supercoiled DNA molecules,subsequently,the deadenylated DNA was cleaved into nicked and linear form.After treatment with acidic aniline,the deadenylated DNA was degraded into many small fragments,and run out of the gel.The enzymatic activities of two RIPs（trichosanthin and cinnamomin） were tested using this method;the limits of sensitivity were about 50 ng（trichosanthin） and 5 ng（reduced cinnamomin）.It should be emphasized that the merit of this method was to avoid the preparation of ribosome.Summarizing these results above,different enzymes came from different material showed the same rule in change of biology function.All behaves of isoenzymes were not showed the same,and reflected the related between biology function and fructification in development.Those isoenzymes with many forms were elements to distinguish different spices,and were materialized biology characters during development and were the basic reason of acclimatize cell to the conditions.The amount to species of isoenzymes and character of suitable in cell were positive relativity.The changes of content, Such as:POD-A and POD-B,cinnamomin and camphorin,were reflected the cell character in different phases of development.So,those isoenzymes with many forms were positive relativity to its adaptability.Isoenzymes can reflect different in species and its special character during development,and can be distinguished the state of cell during storage and senescence,and can reflect the different treatment course by enzyme or non-enzyme with the content change of isoenzymes.All of these results indicated that those isoenzymes with many forms were related to the character of enzymes,and the amount about species of isoenzymes and character of suitable in cell were positive relativity.It indicated that we might find out distinguishes of the different cell,and different stage of the same cell, and special character in different environment with the change of isoenzymes. Just like the annual ring for the tree and the fingerprint for men,the code and stage of cell might be characterization by the change of their isoenzymes.