Genetic Analysis Of Blades From Porphyra And The Expression Analysis Of PEPCK And PEPC In Different Stages Of Porphyra

Porphyra is an important marine crop and has a biphasic life-cycle consists with ahaploid gametophyte and a diploid sporophyte. Unlike higher plant, its gametophy ismacroscopic stage while its sporophy is microscopic stage. The meiosis in Porphyraoccurs during the germination of conchospores and the four resultant cells withdifferent genetic compositions form a blade together. Thus the balde is a geneticchimera and the results could be inaccurate if genetic analysis was done with suchblades. The blades live on static substrates in the intertidal zone and experienceemersion and submersion with tides while the conchocelis are endolithic algae livingin shells (e.g. oyster) in the subtidal zone. The different living environment may affectthe characteristic of photosynthesis. This study includes:1) Two types of blades from a single filament of P. yezoensis (40bladesgerminated from conchospores and88blades germinated from monospores) wereanalyzed by amplified fragment length polymorphism. In40blades fromconchospores, a total of538loci were get by15primer combinations, of which434(80.7%) were polymorphic. The genetic similarity between individuals ranged from0.47to0.93with an average of0.71. The88blades from monospores were analysisusing19primer combinations and a total of708DNA bands were scored, of which698(98.6%) were polymorphic. The genetic similarity between individuals rangedfrom0.43to0.78(average0.61). Constructed on the basis of genetic similarity data,the clustering maps of blades from conchospores and monospores contain two clades.A higher polymorphic loci ratio was detected in blades from monospores than thosefrom conchospores, and the average genetic similarity of blades from monospores waslower than that of blades from conchospores. These differences indicated that geneticanalysis using blades from monospores gives more accurate results.2) The relative expression levels of PEPCK and PEPC were analyzed by real-time quantification PCR between the gametophytes and sporophytes of P. haitanensis with18S and GAPDH as internal controls. Their enzyme activeities were assayed usingenzyme-coupled spectrophotometric method. P. haitanensis PEPCK and PEPC wereexpressed using the pMAL-c4X expression vector system. The antibody to PEPCKand PEPC were prepared using the expressed proteins, and protein content of PEPCKand PEPC in different generations of P. haitanensis was detected by western blotting.Both PEPCK and PEPC abundance and activity were higher in sporophytes than thosein gametophytes, suggesting that the carbon-fixation mechanism may different insporophytes and gamephytes of P. haitanensis and a C4-like carbon-fixationmechanism may be functional in P. haitanensis sporophytes.