The Draft Map Of A Marine Diatom Thalassiosira Pseudonana Proteome And Its Proteogenomic Study

Diatoms are one of the main phytoplankton groups in the ocean with high diversity and wide distribtion.Diatoms are also the major primary producers of marine ecosystem,contributing approximately 40%of marine primary productivity and are of great importance to ecosystems.The ecological success of diatoms suggests that diatoms has evolved diverse strategies and mechanisms in response to environmental changing.As the first model diatom species with whole-genome sequencing,Thalassiosira pseudonana is widely used in the study of important ecological functions and environmental changing response.Although the whole genome of T.pseudonana has been reported,it still lacks a high-quality genomic annotation.In addition,there have been many studies involved in the mechanism of physiological and molecular response to environmental nutrient changeing in diatoms,but most are focused on the specific response to one nutrient changing.Little is known about the common and specific response to different nutrient deficiencies.In this study,T.pseudonana strain CCMP 1335 was used as the model for proteomic and proteogenomic study.Samples from three growth phases and three different macronutrient(nitrogen,phosphorus and silicon)deficiency conditions were analyzed using a deep proteomic approach,proteogenomic analysis strategy,iTRAQ quantitative proteomic and quantitative PCR methods,combined with the bioinformatic analysis,to obtain a draft map of T.pseudonana proteome,to improve the annotation of T.pseudonana genome,and to explore the common and specific responses to different macronutrient deficiencies.The main results of this study were as follows:(1)Using multiple protein extraction methods,sequential extraction and enrichment strategy,a deep proteomic analysis of samples from three growth phases of T.pseudonana was performed using a high resolution mass spectrometry to obtain a drapt map of T.pseudonana proteome.In total,9263 proteins were identified,accounting for about 79%of the predicted coding protein genes.The translational expressions of most predicted genes from the genome of T.pseudonana was verified by our data.An average of six peptides per identified protein were mapped and an average coverage of 12%per protein sequence was identified.There were 6149 proteins with functional annotations,accounting for 83%of the predicted genes with functional annotations.In addition,comparative analysis of differentially expressed proteins among three growth phases showed that each growth phase had its distinct metabolic characteristics.(2)The integration of our MS data from three growth phases,reported RNA-seq data and genomic data were analyzed using the proteogenomic method.1235 novel genes(30 of them were mapped in the region of pseudogene),979 revised genes,104 alternative splice proteins and 234 single amino acid variants(including 19 new proteins,32 revised proteins and 183 annotated proteins)were found.The annotation of the T.pseudonana genome was greatly supplemented and improved by our study.These results can provide valuable database resource and basic support for future diatom research.(3)The differentially expressed proteins and physiological responses to nitrogen,phosphorus and silicon deficiencies in T.pseudonana were analyzed.The results showed that the proteins involved in nutrient transport and utilization were highly expressed under each individual macronutrient deficiency condition,and utilization of organic nitrogen or organic phosphorus was enhanced respectively under nitrogen or phosphorus deficiency.Macronutrient deficiency depressed carbon fixation and photorespiration but promoted neutral lipid and carbohydrate accumulation.Under both nitrogen and phosphorus deficiencies,photosynthesis,carbon fixation,chlorophyll and protein biosynthesis were down-regulated,which resulted in the reduction of chlorophylla,protein and cellular nitrogen contents,while carbohydrate metabolism was up-regulated.In addition,intranuclear process was up-regulated under nitrogen deficiency,resulting in the increase of cellular phosphor content.However,the silicon deficiency caused a significantly different response compared with nitrogen and phosphorus deficiencies:light harvesting proteins,chloroplastic ATP synthase,as well as transcription and protein synthesis in the chloroplast were up-regulated,which increased the contents of chlorophyll a,cellular nitrogen and phosphorus.Overall,our study revealed the common and specific responses to three different macronutrient deficiencies in T.pseudonana.In this study,we successfully obtained a high-quality draft proteome map of T.pseudonana,which is the most complete protein identification in the current reported proteomics research,providing reference and support for future diatom research;the genome annotation of T.pseudonana was supplemented and improved using proteogenomic analysis,providing basic support for obtaining a more complete draft proteome map of T.pseudonana and its molecular mechanism research.