Gene Expression Analysis Of Low-nitrogen Responsive MYB Transcription Factor Family Genes And Functional Verification Of ScMYB33-2 In Sugarcane

Sugarcane(Saccharum spp.hybrid)is an important crop for sugar and bioenergy in the world.In China,it is very common for over-fertilization of nitrogen in present sugarcane production,and which increase both the cost and the risk for environmental contamination.Therefore,to improving nitrogen use efficiency(NUE)in sugarcane production needs to be solved urgently.Breeding for nitrogen efficient cultivars become an effective way both to improve the NUE and reduce the nitrogen fertilizer application and the potential for environment contamination.The molecular mechanisms of nitrogen uptake and utilization research are the basis and the key to obtain the cultivars with nitrogen use efficiency.The MYB superfamily is one of the most abundant groups of transcription factors in eukaryotes.Its members play a pivotal role in signal transduction and transcriptional regulation for plant in response to environmental stresses.In recent years,their roles in plant nutrition and productivity have received more and more attention.Some MYBs have been verified their positive role in improving yield and nitrogen use efficiency,while related research in sugarcane has not yet started.Digging and identification of low-nitrogen sensitive response genes,than clarificating their molecular mechanisms of regulation in sugarcane nitrogen uptake and utilization,will provide a beneficial theoretical and practical basis for high NUE sugarcane breeding.In this study,fifty-nine sugarcane MYB gene(ScMYBs)sequences were screened via bioinformatics analysis.Using ROC22,a sugarcane variety with a wide range planting in China,as the materials,the tissue-specific expression profiles in various tissues and the expression patterns of ScMYBs under low nitrogen treatment in sugarcane leaf and root were measured by real-time quantitative polymerase chain reaction(RT-qPCR).Subsequently,the expression characteristics of low-nitrogen-sensitive ScMYBs under different exogenous hormones were analysed.Finally,a low-nitrogen-sensitive sugarcane MYB gene,termed as ScMYB33-2 was isolated and its function in the regulation of nitrogen metabolism was investigated using the tobacco transient expression system.The main results were listed as follows:1.The expression characteristics of ScMYB family genesThe tissue specific gene expression patterns of the 59 ScMYBs were identified in root,bud,leaf,pith and skin of stem tissues,respectively.It was showed that ScMYB2 was a leaf-specific expressed gene,and ScMYB22 was a root-specific expressed gene.ScMYB13 displayed a root/pith-specific expression pattern,while ScMYB60 presented a bud/leaf-specific expression pattern.ScMYB3,ScMYB5,ScMYB21,ScMYB25,ScMYB26,ScMYB32 and ScMYB51 can be detected in 4 tissues.The other 48 ScMYBs were successfully detected in all sugarcane tissues.Among them,ScMYB1,ScMYB21,ScYB39,ScMYB43,ScMYB44 and ScMYB46 showed the highest expression levels in leaf(more than twice as much as it in other tissues),while ScMYB6,ScMYB2 and Sc2RMYB2S1 were the significantly highest in the buds(more than twice as much as it in other tissues).The expression patterns of the 59 ScMYBs both in the leaf and in the root of sugarcane under low nitrogen treatment were investigated in both leaf and root of sugarcane,respectively.Twenty-seven ScMYBs were identified as typical low-nitrogen sensitive response genes in both leaf and root,respectively.Among them,four and fourteen ScMYBs were classified as low-nitrogen sensitive up-regulated type from leaf and root,respectively.Their expression levels were induced by low nitrogen treatment and continuously up-regulated(or generally increase)significantly during the treatment.Twenty three and thirteen ScMYBs were classified as low-nitrogen sensitive down-regulated type from leaf and root,respectively.Their expression levels were suppressed by low nitrogen treatment and continuously down-regulated(or generally decrease)significantly during the treatment.Moreover,among those low-nitrogen sensitive up-regulated type MYB genes,three and thirteen ScMYBs were identified as leaf-specific or root-specific up-regulated genes.Among those low-nitrogen sensitive down-regulated type MYB genes,seventeen and seven ScMYBs were identified as leaf-specific or root-specific down-regulated genes.The results revealed that two regulatory strategies:positive and negative regulation,were developed within ScMYBs in the regulation of nitrogen metabolism in the root of sugarcane.The negative regulation might be the main style of ScMYBs in the regulation of nitrogen metabolism in the leaf.Specifically,ScMYB33-2 is the only one low-nitrogen sensitive up-regulated type gene which was stably up-regulated both in the leaf and in the root during the treatment.ScMYB5,ScMYB20,ScMYB27,ScMYB28,ScMYB41 and ScMYB55 were stably down-regulated in both leaf and root during the treatment.While ScMYB13,ScMYB51,ScMYB54,ScMYB56 and Sc2RMYB2S1 presented an antithetical expression pattern in leaf and root.Moreover,some low-nitrogen sensitive response genes(26)from leaf were selected,and the expression profiles of them under different exogenous plant hormones were investigated.Four of them were induced by ABA and continuously up-regulated(or generally increase)significantly during the treatment,while nine of them showed a reverse expression pattern.Three ScMYBs were identified as ABA insensitive genes.Two of them were induced by MeJA and continuously up-regulated(or generally increase)significantly during the treatment,while ten of them showed a reverse expression pattern.There were four ScMYBs being insensitive to MeJA treatment.Six of them were induced by SA and continuously up-regulated(or generally increase)significantly during the treatment,while seven of them showed a reverse expression pattern.There were four ScMYBs being insensitive to SA treatment.Specifically,the expression of ScMYB33-2 were suppressed by ABA and MeJA,while was induced by SA.2.Cloning and functional verification of ScMYB33-2ScMYB33-2,a typical low-nitrogen sensitive response R2R3-MYB gene was isolated from sugarcane variety ROC22.The full-length cDNA sequence of ScMYB33-2 was 520 bp long,with a 483 bp ORF encoding 160 amino acids.Co-expression network analyses based on tobacco transient expression system indicated that the expression levels of nitrite reductase gene(NbNiR),glutamine synthetase gene(NbGS),glutamate synthase gene(NbGOGAT),aspartate aminotransferase gene(NbASPAT),asparagine synthetase gene(NbAS)were significantly up-regulated,when ScMYB33-2 was over-expressed in N.benthamiana leaf.It was suggested that ScMYB33-2 might play an active role in regulating nitrogen metabolism.The results constitute new interesting MYB candidates for elucidating the molecular mechanisms of nitrogen metabolism regulation in further research,and provide a beneficial theoretical basis for high NUE sugarcane breeding.