Quantitative Analysis of Phaeodactylum tricornutum transcriptomes
Purpose:Using RNAseq to profile transcriptomes to compare wild type and PtTryp8 knockout lines across different phosphate conditions, aiming to further explore the molecular function of PtTryp8 in regulating cell growth and responses to P.Methods:Libraries for RNAseq were created from 1ug of mRNA from each culture. The resulting libraries were loaded into the patterned nanoarray, and single-end 50 bp reads were generated on BGIseq500 platform (BGI-Shenzhen, China), with a data output of about 22 M total clean reads for each library.Results:To identify the genes exhibiting the strongest interaction with PtTryp8 on the transcriptome level, cells were gathered under three conditions: HP, LP, and DIP, separately. KO5 and KO7 were chosen to be the present KOs, and KOC was regarded as the control strain. A total of 27 transcriptional libraries were constructed from nine combinations of genotype and P conditions (KOC_HP, KOC_LP, KOC_DIP; KO5_HP, KO5_LP, KO5_DIP; KO7_HP, KO7_LP, and KO7_DIP, each group had three biological replicates). An average of 42.5 million clean reads was yielded from each of the 27 RNA-seq samples. All clean reads data sets showed Q-20 values (error probability of 1%) >97.55% and total mapping genome ratios >87.98%. In this way, 11,531 unigenes (93.9%) were successfully annotated in at least one database, and 6,303 (51.3%) unigenes were annotated in the KEGG database. Differential gene expression analysis revealed 445 DEGs (229 upregulated and 216 downregulated) in the KO5_HP/KOC_HP comparison, 1125 DEGs (908 upregulated and 217 downregulated) in the KO7_HP/KOC_HP comparison, 653 DEGs (289 upregulated and 364 downregulated) in the KO5_LP/KOC_LP comparison, 670 DEGs (276 upregulated and 243 downregulated) in the KO7_LP/KOC_LP comparison, 670 DEGs (376 upregulated and 294 downregulated) in the KO5_DIP/KOC_DIP, and 501 DEGs (239 upregulated and 262 downregulated) in the KO7_DIP/KOC_DIP comparison. Since KO5 and KO7 are two different knockout strains, the differentially expressed genes do not overlap completely. The commonly differentially expressed genes for the two KOs under the three conditions were 198, 335, and 256 DEGs, respectively. Most importantly, 39 genes were differentially expressed under high phosphorus, low phosphorus, and phosphorus-restoring conditions and were consistent between the two mutant strains.Conclusions:These data establish PtTryp8 as a key component of diatom phosphorus physiology and uncover isozyme-level specialization within the diatom trypsin family, providing new insight into how marine phytoplankton regulate nutrient acquisition under fluctuating environmental conditions.