The Physiological Basis Of Sugarcane Responsive To Nitrogen Treatment And Mining Of Its Differentially Expressed Genes

Sugarcane is the most important sugar crop in the worldwide.Nitrogen（N）is one of the most essential macronutrient for sugarcane growth.Due to the important role of nitrogen on the yield and quality,excessive amounts of nitrogen fertilizer is used in sugarcane cultivation,which not only increases the cost of production but also pollutes the environment.Therefore,improving the nitrogen use efficiency（NUE）and reducing the application levels of nitrogen fertilizer are necessary for the sustainable development of the sugarcane industry.Tissue culture seedlings of two sugarcane（Saccharum spp.hybrid）genotypes of ROC22 with high NUE and Badila（Saccharum officinarum）with relative low NUE were used in this study.Seedlings were cultured in normal and low concentrations of nitrogen in climatron,and then the comparision of their phenotypes,physiological and biochemical parameters,and the transcriptome sequencing data obtained from seedlings under low nitrogen concentration stress.Meanwhile,a Mediator subunit gene,named ScMED7,were isolated from sugarcane based on analysis of sequencing data and RT-PCR,and its characteristics and biological functions were furtherly analysed.The main results and conclusions are as follows.1.After treatment for 16 d under normal and low N concentrations respectively,non obvious phenotypical difference was observed in ROC22.While,the phenotype of Badila seedlings was short and small,along with the nitrogen deficiency symptoms of yellowing and chlorosis,and the chlorophyll content was markedly reduced comparing with the normal N treatment.At the view of physiological and biochemical changes,the total nitrogen content and the nitrogen accumulation in ROC22 have no significant difference under different nitrogen levels,whereas obvious difference was observed in Balida.The activity of two nitrogen metabolism enzymes of glutamate dehydrogenase and glutamine synthetase kept at a high level in ROC22,and both were remarkablely higher than those in Badila.Besides,the responses of nitrogen metabolism related genes,including reductase（nitrate reductase and nitrite reductase）genes,transporter（ammonium transporter 1.1,nitrate transporter 1 and nitrate transporter 1.1）genes and synthetase（glutamine synthetase 1,glutamine synthetase 2,ferredoxin glutamate synthase and NADH glutamate synthase）genes,under low N stress in ROC22 were more quickly than that in Badila.2.The seedlings（three biological replicates with total of 12 samples）of ROC22（high NUE）and Badila（relative low NUE）were collected for Illnmina RNA-seq at time points of 0 h（control group）and 6 h（treatment group）after low N stress.The results of RNA-seq were as follows.（1）In total,125.76 Gb data and 838,298,078 clean reads were generated from 12 sugarcane seedling samples through transcriptome sequencing.The total of 409,142 transcripts were obtained by de novo assembly,which N50 was 1,236 bp and contained 194,850（47.62%）transcripts at length of more than 500 bp and 99,037（24.21%）transcripts at length of more than 1.0 kb.After merging,272,220 Unigenes were generated,which N50 was 1,433 bp and contained 190,436 and 98,966 Unigenes,which length were more than 500 bp and 1.0 kb.Comparing Unigene with Nr,Nt,Pfam,KOG,Swiss-Prot,GO and KEGG databases,209,644 bioinformatics annotation results of Unigenes were found.In addition,62,576 Unigenes（22.99%）had not been commented.We conjecture that these sequences may be new transcripts,which need further analysis and research without comments.At the same time,the gene structure was analyzed.Then,45,382 SSR loci and 81 transcription factor families were obtained.（2）The gene expression levels in different groups of sugarcane samples were analyzed,and then qRT-PCR was occupied for validation of some differently expressed genes observed in sequencing.The results suggest that:① Under low nitrogen stress for 6 h,10,960 genes were differentially expressed between different groups of ROC22.Among them,5,638 genes were up regulated and 5,322 genes were down regulated.While,less genes（4,344）were differentially expressed in Badila.Among them,1,665 genes were up regulated,taking 38.33%,and 2,679 genes were down regulated which taking 61.67%.A total of 2,397 differentially expressed genes（DEGs）were common in both genotypes,and the specific DEGs for ROC22 and Badila were 1,665 and 1,947,respectively.These specific DEGs may be more important candidate genes for research on the improvement of NUE.②All of the DEGs are related to various biological functions and involved in multiple metabolic pathways,including cellular nitrogen compound metabolic process,organic substance metabolic process,primary metabolic process,single-organism process,intracellular,intracellular part,cytoplasm,cell,cell part and atalytic activity,etc.KEGG enrichment analysis showed that,these DEGs involved in nitrogen metabolism,carbon fixation in photosynthetic organisms,photosynthesis and glyoxylate and dicarboxylate metabolism,etc.Visibility,low nitrogen stress involved in not only the pathway of nitrogen metabolism but also the pathways related to carbon metabolism,and the molecular mechanism of nitrogen utilization was regulated by multiple gene network system.③A total of 20 DEGs related to nitrogen metabolism,carbon fixation in photosynthetic organisms and transcriptional regulation were randomly selected for qRT-PCR validation.The result showed that the relative expression trend of 18 DEGs in qRTPCR were consistent with that in RNA-seq,except glutamate dehydrogenase and aspartate transaminase genes.It suggested that the results of RNA-seq is reliable.3.Based on the data of RNA-seq,43 transcripts encoding 19 Mediator subunits（MEDs）were differentially expressed.Among them,the transcripts,including those coding genes of MED4,MED7,MED15,MED36 and other MEDs,were obviously changed in expressed level.The transcripts of MED 7,a key subunit in the central module of the Mediator complex,were significantly differently expression in both ROC22（upregulated 3.28 times）and Badila（upregulated 3.77 times）samples collected under low nitrogen stress for 6 hours,speculating it may play an important role in nitrogen metabolism.Therefore,a further study on this transcript was carried out.A MED7 gene with a full-length cDNA from sugarcane was cloned,which is named as ScMED7（GenBank accession:KP970628）,has a 525 bp open reading frame（ORF）encoding 174 amino acids,and the predicted protein is 19.9 kDa located in the nucleus and cytoplasm,which was confirmed by subcellular localization experiments.ScMED7,containing one typical conserved domain of MED7 proteins,and shares 98%homology with that from Sorghum bicolor（XP₀02447862.1）.ScMED7 was constitutively expressed,yet significantly higher in bud tissues.ScMED7 transcription was obviously induced by low nitrogen,heavy-metal（CdCl2）,low temperature（4℃）and hormone（SA and MeJA）stresses,while inhibited by osmotic stresses of NaCl and PEG.It suggested that ScMED7 plays an essential role in the process of the response to stresses,including the nitrogen stress.