Growth And Transcriptomic Study Of Nostoc Flagelliforme Under Low-nitrogen Cultivation And Osmotic Stress

N.flagelliforme is a hairlike terrestrial nitrogen-fixing cyanobacterium species distributed on arid and semi-arid steppes of the northwestern regions of China.It regularly encounter adverse environmental conditions,deficiency of water and nitrogen source often appear in its growth and development.In this study,aquatic-living N.flagelliforme in response to 15 days low-nitrogen cultivation and 24 h osmotic stress was researched in different levels through ultrastructural observation,physiological test and high-throughput sequencing.The results are as follows:1)After 15 days low-nitrogen cultivation and 24 h osmotic stress,the structure of filaments,division cells and vegetative cells remained unchanged under observation of transmission electron microcopy(TEM).2)Low-nitrogen cultivation might inhibit the effects of photosynthesis,damage to cell membrane integrity and break the osmotic balance,increase the level of celluar redox and oxygen free radicals.Cell water content had no significant difference in response to 15 days low-nitrogen cultivation;chlorophyll a,trehalose and proline were significantly lower in low-nitrogen cultivation compared to control group;Ralative electrical conductivity ratio,cell activity,MDA and SOD were significantly higher in low-nitrogen cultivation.Osmotic stress would cause loss of cell water and inhibit the effects of photosynthesis,cause damage to protein structure damage to cell membrane integrity and break the osmotic balance,increase the level of celluar redox and oxygen free radicals.Cell water content and chlorophyll a were significantly lower in 24 h osmotic stress compared to control group;Trehalose,proline,ralative electrical conductivity ratio,cell activity,MDA and SOD were significantly higher in osmotic stress.3)A high-quality genome scanning database of N.flagelliforme was constructed successfully.By the Illumina high-throughput genome sequencing scanning technique,in total,67.49 million pieces of short sequences(Raw Reads)were generated.Among all the raw reads,the overall sequencing outputs more than 95.1% Phred-like quality scores at Q20 level(an error probability of 1%).A 8,756,642 bp genome with 41.5% GC content were obtained after de novo assembly.9,131 Unigenes with average length of 965 bp were obtained,and the length of 44,908 pieces(44.39%)is more than 500 bp.Compared to published DNA sequence of Nostoc Anabaena sp.PCC 7120 and Nostoc punctiforme ATCC 29133,number of genes in N.flagelliforme was more than other two species,their genome size and GC content were similar.4)We sequenced transcriptome of N.flagelliforme by using the Illumina high-throughput transcriptome sequencing technique.After 15 days low-nitrogen cultivation,many genes involved nitrogen metabolism were significantly up-regulated in response to low-nitrogen condition,however,genes involved in photosynthesis were significantly down-regulated.15.70 million pieces of short sequences(Clean Reads)were generated.Among all the clean reads,the overall sequencing outputs more than 99.17% Phred-like quality scores at Q20 level(an error probability of 1%).Mapping rate was 76.57% with 43.67% GC content were obtained after de novo assembly.Compared to control group,348 genes were differentially expressed for N.flagelliforme grown in low-nitrogen condition,151 genes were found to be differentially up-regulated,197 genes differentially down-regulated.Which indicate that aquatic-living N.flagelliforme could fix nitrogen in atmosphere under nitrogen cultivation and enhanced nitrogen metabolism to biosynthesis crucial amino acids and proteins,but its photosynthesis was inhibited.We sequenced transcriptome of N.flagelliforme in response to 24 h osmotic stress by using the Illumina high-throughput transcriptome sequencing technique.14.47 million pieces of short sequences(Clean Reads)were generated.Among all the clean reads,the overall sequencing outputs more than 99.11% Phred-like quality scores at Q20 level(an error probability of 1%).Mapping rate was 84.75% with 44.13% GC content were obtained after de novo assembly.Compared to control group,289 genes were differentially expressed for N.flagelliforme grown in osmotic stress,88 genes were found to be differentially up-regulated,201 genes differentially down-regulated.Many genes involved nitrogen metabolism,RNA degradation and sulfur metabolism were significantly up-regulated in response to osmotic stress,however,genes involved in starch and sucrose metabolism,galactose metabolism and fatty acid metabolism were significantly down-regulated.N.flagelliforme enhanced nitrogen metabolism to biosynthesis crucial amino acids and proteins in response to osmotic stress.Starch and sucrose metabolism,galactose metabolism and fatty acid metabolism were all restrained under osmotic stress.But RNA degradation and sulfur metabolism had been enhanced to overcome the osmotic stress.In short,low-nitrogen cultivation and short-term osmotic stress had no significant change in cell structure in N.flagelliforme.Unique physiological response to adversity stress had an important role for N.flagelliforme.Enhanced nitrogen metabolism could be a unique way for N.flagelliforme to adapt low-nitrogen stress and osmotic stress.The unique physiological characteristics and molecular regulation mechanism in N.flagelliforme has an important value and significance to improving the desert environment.Our research provides important information to artificial culture and molecular regulation mechanism in abiotic stress of N.flagelliforme.