Transcriptome Analysis Of Foxtail Millet(setaria Italic) Under Low Nitrogen Stress And Characteristics And Functional Identification Of SiMYB3

Nitrogen(N)is one of the major nutrients for plant growth and development. Foxtail millet(Setaria italic) has characteristics of tolerance to drought and barren stresses and wide adaptability. It becomes an ideal material for monocotyledon to study mechanism of stresses tolerance. At present, few papers reported genes related to low nitrogen stress response in foxtail millet. Transcription factors play crucial roles in regulating gene expression. Although a few transcription factors related to low nitrogen stress have been identified to date, it is still little known about how MYB like genes involve in low nitrogen response. In this study, we isolated a MYB like transcription factor gene named SiMYB3 and identified its characteristics and functions by transcriptome sequencing of foxtail millet under low nitrogen stress. The major results are as following:1. Transcriptome analysis of foxtail millet under low nitrogen stress. By analyzing the transcriptome of low nitrogen tolerant variety named Zheng 204 under control and stress conditions,we obtained 1889 differential expressed genes. Among those genes 1317 were up-regulated, 572 were down-regulated. They were classified into three groups based on GO functional analysis: the biological process(746), the cellular component(873) and the molecular function(948), and the term enriching more genes was transcription in the group of molecular function. In addition, 1103 genes were annotated into 113 pathways including biosynthesis of secondary metabolites and plant hormone signal transduction. 74 transcription factor genes(up-regulated 58/down-regulated 16) included MYB-like(20 up/5 down)、bZIP-like(7 up/1 down)、WRKY-like(6 up/2 down)、AP2-like(3 up/4 down)families and so on. MYB gene family had the largest numbers comparing with the other families, suggesting that the MYB-like genes play a key role in transcription regulation in response to low nitrogen stress in foxtail millet. Thus we selected the MYB genes for further analysis. In addition, we obtained 13466 and 15095 novel transcripts from control and treatment samples, respectively, which laid the foundation for cloning new genes response to low nitrogen stress in foxtail millet.2. Characteristics of the MYB like genes response to low nitrogen stress in foxtail millet. In 25 members of SiMYB genes, 20 members were up-regulated and 5 members were down-regulated. They were named SiMYB1-25, respectively. Sequence homologous analysis indicated that the length of nucleotide and amino acid sequences of 25 SiMYB genes were diversity. The protein average mass was 32.70 kD and the pI was ranged from 5.06 to 9.42. The phylogenetic tree analysis indicated that the 25 genes were divided into two subfamilies, namely MYB1 R and R2R3-type MYB. The members of R2R3-type MYB subfamily contained two HTH domains and the genes in MYB1 R subfamily had only one HTH domain, and the members in the MYB1 R were further classified into two groups according to the gene structure. In addition, all the 25 genes were localized on the nine different chromosomes, and SiMYB1 and SiMYB7 were near to each other on the second chromosome, suggesting that SiMYB1 and SiMYB7 might be a result of gene duplication in the evolution process.3. Characteristics and function analysis of SiMYB3. Among 25 members of SiMYB family in foxtail millet, the four genes including SiMYB1 to SiMYB4 were highly up-regulated under low nitrogen stress. We found that Si MYB3 was not only induced by low nitrogen(up to 10.2 fold), but also up regulated under low potassium and low phosphorus stresses, up to 4.3 and 6.2 fold, respectively, which indicated that SiMYB3 played an important role in response to nutrition deficiency in foxtail millet. SiMYB3 belongs to R2R3-type MYB subfamily and SiMYB3 protein was localized in the nucleus and mainly expressed in the root. The plants overexpressing SiMYB3 had stronger root system and accumulated more biomass than WT under low nitrogen, low potassium, and low phosphorus stresses. SiMYB3 can promote expression of genes related to transport and utlization of nitrogen, phosphorus, and potassium nitrents, which was a result of transgenic plants enhancing tolerance to low nitrogen, low potassium, and low phosphorus stresses. Besides, SiMYB3 involved in the root development process mediated by low nutrition conditions by enhancing the expression of ANR1 and TAR2, which can provide chance for further analysis of the relationship between low nutrition stress and root development in plants through studying the molecular mechanism of SiMYB3.