Transcriptome Sequencing And Expression Analysis Of Nitrate Assimilation-Associated Genes In Pyropia Yezoensis Under N-sufficient Conditions

As an important marine economic crop,Pyropia yezoensis has great economic value,which is mainly cultivated in the coastal areas of Jiangsu and Shandong in China.Based on previous studies,the concentration of dissolved NO3--N in the cultivation area of P.yezoensis ranged from 7.4 to 95μM due to the aggravation of seawater eutrophication and the invasion of marine green tide,and the annual variation of nitrogen concentration was rapid and intense.Therefore,it is important to explore the physiological metabolic reaction of P.yezoensis under the condition of nitrogen enrichment for studying the adaptation and evolution process of algae.In this experiment,P.yezoensis was used as a sample and cultured in control medium with 0.168mM nitrate and N-sufficient medium with 1.68mM nitrate respectively.On the second day,the phenotypic traits,growth rate and photosynthetic pigment content of the N-sufficient group were significantly down-regulated,and transcriptional sequencing analysis was conducted.The transcriptome size of P.yezoensis was 38.6M,and the GC content was 67.34%.The predicted total Unigene number was 21489.Through GO enrichment analysis of differential genes,it was found that the differential genes were mainly concentrated in cell parts,catalytic activity,binding and cellular processes.KEGG annotation analysis showed that the up-regulated genes were mainly concentrated in carbon metabolism,folding,sorting and degradation processes.The down-regulated genes were mainly distributed in energy metabolism,carbon metabolism,cofactors and vitamin metabolism.Through transcriptome differential gene analysis,it was found that the expression levels of nitrate assimilation-associated genes in P.yezoensis were all down-regulated in the N-sufficient group.Based on transcriptional sequencing data,the full-length sequences of PyNR and PyNiR genes and partial fragments of PyGS were successfully cloned in P.yezoensis for the first time.The full-length of PyNR was 2532 bp,and Genebank accession number was MW410947.The full length of PyNiR sequence was 1347 bp,and Genebank accession number was MW410948.The partial PyGS is 795bp,and the Genebank accession number is MW683969.This study fills in blank of the gene cloning of nitrate assimilation-associated genes in P.yezoensis,and provided information for further studies on the regulation of N-sufficient/N-deprivation.The nitrate uptake rate of P.yezoensis under N-sufficient condition and control condition was measured to study the variation of nitrate uptake by thalli.After further extracting and detecting the changes of intracellular nitrate and nitrite contents,it was found that saturation values and critical values of nitrate accumulation was in P.yezoensis.When intracellular nitrate content reached 800mg/kg,nitrate uptake rate decreased,growth rate slowed down,and photosynthetic pigment content did not increase.When the intracellular nitrate content reached 1000mg/kg,the nitrate assimilation was seriously inhibited,the growth was stopped,and the photosynthetic pigment content decreased,which was not conducive to the growth of P.yezoensis.According to the statistics,the nitrogen utilization efficiency decreased under the N-sufficient condition.In the control group,nitrogen accounted for 3.36%in the increased biomass,while under the N-sufficient condition,it reached 6.2%,resulting in the loss of nitrogen in the cultivation process.In addition,under the N-sufficient condition,the contents of nitrate and nitrite in the P.yezoensis were greatly accumulated.The nitrate content was close to the safe value of 1500mg/kg set by the World Health Organization,while the nitrite content exceeded the limit of 15.5mg/kg set by China’s food safety guidelines.The cultivation of P.yezoensis with high concentration of nitrogen not only caused the loss of nitrogen,which is not conducive to economic benefit and environmental safety,but also led to the accumulation of nitrate and nitrite in thalli,which is not conducive to food safety.The mRNA expression levels of nitrate transporter gene NRT2 and nitrate assimilation-associated genes PyNR,PyNiR,PyGS and PyGOGAT were detected by qPCR.It was found that under the condition of N-sufficient culture,the expression levels of NRT2,PyNR,PyNIR and PyGOGAT were rapidly up-regulated,and nitrate was rapidly assimilated and fixed.When the intracellular nitrate reached the critical value,the gene expression would be rapidly inhibited,and the mRNA expression level was maintained at a very low level at 24h,and the absorption and metabolism of nitrate stopped.Compared with the nitrogen enrichment group,the expression level of key enzyme genes for nitrogen absorption in the control group treated with appropriate nitrogen concentration was always maintained at a stable level.The expression of nitrogen absorption-related genes was strictly regulated by nitrogen concentration.