The Preliminary Exploration Of Spermatangium Cell Differentiation Mechanism And Screening Of Resistance-related Internal Control Genes Of Pyropia

As the major cultivating red algae in China,Pyropia not only have high economic value,but also have high theoretical research value due to its unique biological characteristics.This thesis has focused on two species of Pyropia and carried on relevant research as follows:1.During the sexual reproduction stage,thallus of Pyropia yezoensisis is striped with yellowish spermatangia and rufous carpogonia.Under microscopic observation,thalli of different growth periods were obtained and the total soluble proteins were prepared respectively.And then,proteomic analysis was performed using iTRAQ labeling method.The developmental phase from vegetative cell to the spermatangium tissues was emphasized to explore the potential development mechanism and the relevant metabolic regulation.Compared with the vegetative cells,the up-regulated proteins in spermatangium cells were chromosome assembly and replication related,including molecular chaperone,microtubulin and so on.Some proteins might participate in the epigenetic regulation of transcription process through forming chromatin remodeling complex.Some subunits of 26 S proteasome also showed up-regulation during the development of spermatangium cells.The differentiation of the cells may speed up operation of the cell cycle via the ubiquitin-proteasome degradation pathway.Thus,protein recycling activities are active which provides the material basis for the transition from vegetative to reproductive growth.The up-regulation molecules such as kinase,phosphatase,calmodulin also indicated that the original metabolism network of cells has been adjusted during the development process.The energy metabolism that phosphate group participates provided the energy basis for the formation and release of germ cells.Meanwhile,the down-regulated proteins mainly participated in assimilation metabolism,including the subunits forming phycobiliprotein,oxygen evolving complex,electron carriers,which indicated that the energy-consuming assimilation process coupling with the photosynthesis declined at the same time.2.Based on the screening of the up-regulated phosphatase gene during the spermatangium cell development,we cloned the open reading frame and then constructed the recombinant expression vector pPICZ?A-pp2 c.By transformation it into pichia pastoris,the methylotrophic heterologous gene expression system was constructed.We also optimized the expression conditions of the target gene,however,the protein was not purified successfully.3.Pyropia haitanensis is one kind of cultivar exclusive to China,its annual production far exceeds the Pyropia yezoensis.And it is also considered to be the ideal material for study on the resistant mechanism of algae.Through absolute quantification and software analysis,we studied the expression level of seven genes,including 18 S,GAPDH,EF3,RPS,TubA,ACT and TubB,under different salinity and light intensity conditions.And then we screened genes with relatively stable expression under these stress conditions respectively.We proposed that EF3 and 18 S could be used as internal control genes to perform real-time quantitative pcr when the Py.haitanensis blades were subjected to salinity stress,and that TubA and 18 S could be used as the internal control genes when the stress was from strong light.This result laid the foundation for the follow-up study on the resistance mechanism of the Pyropia haitanensis.