Identification And Function Of An Abiotic Stress Resistant-associated Gene COO91₀4051 From The Terrestrial Cyanobacterium Nostoc Flagelliforme

Nostoc flagelliforme,known as "facai" in Chinese,is a terrestrial nitrogen-fixation cyanobacterium that distributes in arid and semi-arid desert regions of China.N.flagelliforme can withstand extreme environmental stresses,such as desiccation,low and high temperatures,and intense solar radiation.Identification of abiotic stress resistant genes from N.flagelliforme not only aids to understand the adaptation mechanisms of this speices in harsh environments,but also helps to obtain functional genes for future use in genetically modified crops.Our group has previously performed the screening of candidate stress resistance-associated genes from N.flagelliforme.In this subject,we will focus on one of these candidate genes,Nfl:COO91₀4051(NCBI),to elucidate its roles in stress resistance.Our previous screening found that the COO91₀4051 gene was up-regulated by drought,ultraviolet and cold stresses,but no further functional clues have been reported about it yet.In this study,low temperature stress experiments showed that COO91₀4051-overexpressing Anabaena sp.PCC 7120 showed obviously low temperature resistance as compared to the control strain(the empty vector transformed strain).The contents of phycobilimin and carotenoides of the transgenic COO91₀4051 strain were obviously higher than those of the control strain.The total content of unsaturated fatty acids in the former was also obviously higher than that of the latter,but their contents in malondialdehyde(MDA)was not mcuh different.These results suggested that COO91₀4051 at least enhanced the cold resistance of the transgenic strain through increased protective pigments and ratios of unsaturated fatty acids.Further,we performed transcriptome sequencing and performed functional classification of differentially expressed genes,including genes involved in photosynthesis,respiratory,genes involved in redox reactions,and genes involved in synthesis of sugar,lipids,proteins,vitamins and metabolism of secondary metabolites,etc.Some photosynthesis and glycosyltransferase-related genes were up-regulated,while some genes involved in respiration and ribosomal submits encoding were down-regulated.The transcriptomic data implied that transgenic strains might also increase photosynthesis and reduce respiration and protein synthesis to save more energy as well as increase the synthesis of polysaccharides to resist the cold stress.In addition,we also conducted other abiotic stress experiments.The transgnic COO91₀4051 strain were more resistant to mannitol(Man)and methyl viologen(MV)stresses than the control strain,while both strains showed no obvious difference under high temperature and ultraviolet stresses.Finally,we found that the nitrogen addition or not also affected the stress adaptation capability of the transgenic COO91₀4051 strain.In nitrogen-free media(BG110),the transgenic strain grew realtively faster than the control strain.Both strains were able to complete the differentiation of heterocysts and showed no difference in heterocysts frequency after 24-h cuture,but quantitative analyses showed that the expression levels of genes with regard to heterocyst differentiation and nitrogen fixation in the transgenic strain was significantly higher than those in the control strain.These results implied that cell differentiation were more rapid in the transgenic strain.Taken together,we found that the COO91₀4051 gene from N.flagelliforme can confer resistance to multiple abiotic stresses,being particularly prominent in cold resistance.