Ubiquitin Protein Extraction And Purification And Functional Verification Of Chlamydomonas Reinhardtii

As fossil resources increasingly depleted,microalgae was used as a raw material for the development of biomass energy,it has broad prospects for development,especially the control of nutrient deficiency stress greatly promoted the content of micro algae cell energy storage material.Thenitrogen stress can stimulatethe redistribution of nitrogen compounds directly.Including the selective protein degradation,and the typical process is ubiquitin mediated proteolysis.Chlamydomonas reinhardtii is a unicellular eukaryotic algae,it can carry out heterotrophic growth and autotrophic growth.Chlamydomonas reinhardtii can carry out photosynthesis.Because of the feature of simple structure.Chlam.ydomonasreinhardtiibecomes a research model of photosynthesis.This paper set up a corresponding protein spectrum research method,which can identify the Chlamydomonas reinhardtii ubiquitin proteinunder nitrogen stress and speculate its biological significancepreliminary.First of all,the C.reinhardtii wild strain of CC-124 was used in this test.We measured the cell's chlorophyll fluorescence kinetics parameter Fv/Fm,total fatty acid,biomass,dry weight under the condition of nutrient-rich and nitrogen stressFv/Fm,etal.According to the variation of the above parameters,we determine the mass spectrometry determination of sampling time point.Aiming at the shortcomings of the traditional Sample Preparation of FASP proteomics,we import by the moderate denaturant and ultrasonic broken into the test.We established the method of the microalgae specificity mFASP(Modified Filter Aided Sample Preparation Method)microalgae proteomic sample preparation methods.The MS data was searched in C.reinhardtii + TAIR10 database,identified 1354 proteins,with the features of easy operation,high yield,can be the characteristics of the parallel processing a large number of samples.Only the A.K.Peatel's result is higher than this test.We collect the samples by the method of mFASP after processing.We use UPLC/MS/MS identifythe different nutrition group samples.148 ubiquitin proteins and 52 kinds of ubiquitin proteinwereidentified,and the functionhad annotation.We found LHCII and Glutamate synthase,(NADH-dependent)with ubiquitin characteristics,they are all the key proteins in the pathway of photophosphorylation electron transport chain,it will continue to explore in a follow-up study of ubiquitin modifications to participate in the energy storage mechanism.