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Isolation And Characterization Of Photosystem Ⅱ Of Porphyra Yezoensis Udea And The Study Of Rubisco

Posted on:2006-08-24Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z Q GaoFull Text:PDF
GTID:1100360152985942Subject:Marine biology
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Porphyra yezoensis Udea(P. yezoensis) is a quite popular and delicious seaweed and widely cultured in China and most Asian countries. Four parts is composed of this research. (1) The thylakoid membranes were isolated and purified from gametophyte thallus of P. yezoensis by ultracentrifugation and differential speeds centrifugation. After the gametophyte thylakoid membranes were solubilized with SDS, the PSⅡ particles which had high DCIP photoreduction activity could be isolated by sucrose density gradient ultracentrifugation from thylakoid membranes collected by two methods above. The absorption spectra, room temperature fluorescence spectra of thylakoid membranes collected by two methods and PSⅡ particles were recorded. The activity of PS Ⅱ particles was determined with DCIP photoreduction reaction. The compositions of the purified PSⅡ particles and the compositions of thylakoid membranes collected by two methods were studied by SDS-PAGE. As a result, the thylakoid membranes collected by ultracentrifugation and differential speeds centrifugation had similar spectra character and same polypeptide compositions. D1 and D2 compound, CP47, CP43, 33kD protein, D1, D2, 20kD protein and cyt c-550 were identified in the PSⅡ particles. Two other new 16kD and 14kD proteins were found; But 12kDa protein that existed in other red algae and cyanobacteria was not detected. Among these extrinsic proteins, the 16kD and 14kD proteins were not reported previously. In this part, the separation and solubilization of thylakoid membrane, isolation of PSⅡ particles as well as dye of PSⅡ particles protein compositions were improved in a way on the basis of the traditional method. (2) The PS Ⅱ particles with high photoreduction activity were isolated from the filament sporophyte of P. yezoensis with the same method as above. The absorption spectra, room temperature fluorescence spectra, DCIP photoreduction activity and the activity of oxygen evolution of thylakoid membranes and PSⅡ particles were recorded. As a result, there were differences not only in spectra properties, between PSⅡ particles of the filament sporophytes and the thallus gametophytes, but also in PSⅡ particles compositions .Apart from the proteins existed in higher plants, a 102kD protein was detected. Cyt b559 and 12kD protein which was found in cyanobaterium and some red algae were distinguished . But cyt c-550, the 20kD, 14kD and 16kD proteins which were detected in thallus gametophytes PSⅡ particles, were not found in filamentous sporophyte PSⅡ particles. Therefore, it seems that the two different generations have different evolutionary positions in the life evolution history. To the authors' knowledge, the high molecular 102kD protein was not reported previously.(3) The 53kD protein which over expressed in the thallus gametophytes, but expressed much less in filamentous sporophyte was identified large subunit of Rubisco by N-end amino acid sequencing. The differential expression of Rubisco enzyme of the filamentous sporophyte and the thallus gametophytes was studied at the level of enzyme protein expression, the level of mRNA expression, the level of different carboxylase activity of Rubisco. As a result, whichever index was choosen as compared target, the quantity of Rubisco expressed in the thallus gametophytes was ten folds more than the quantity in the filamentous sporophytes at least, which suggested not only there were differences between the filamentous sporophytes and the thallus gametophytes carbon fixation efficiency and method, but also the different evolutionary position of the two generations in life evolution. There is no difference of the PEPC activity between the thallus gametophytes and thefilament sporophytes.
Keywords/Search Tags:red alga, Porphyra yezoensis Udea, thallus gametophytes, filament sporophytes, photosynthesis, photosysstem Ⅱ, Rubisco, differential expression, dissoluble proteins
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