* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Results
Primary transcript wikipedia , lookup
X-inactivation wikipedia , lookup
Genetically modified crops wikipedia , lookup
RNA interference wikipedia , lookup
Transcription factor wikipedia , lookup
Cancer epigenetics wikipedia , lookup
Oncogenomics wikipedia , lookup
Epigenetics of depression wikipedia , lookup
Short interspersed nuclear elements (SINEs) wikipedia , lookup
Epigenetics of cocaine addiction wikipedia , lookup
Public health genomics wikipedia , lookup
Pathogenomics wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Essential gene wikipedia , lookup
History of genetic engineering wikipedia , lookup
Quantitative trait locus wikipedia , lookup
Microevolution wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Designer baby wikipedia , lookup
Long non-coding RNA wikipedia , lookup
Genome evolution wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Genome (book) wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Genomic imprinting wikipedia , lookup
Gene expression programming wikipedia , lookup
Minimal genome wikipedia , lookup
Biology and consumer behaviour wikipedia , lookup
Ridge (biology) wikipedia , lookup
Transcriptome-wide survey and expression analysis of stress-responsive NAC genes in Chrysanthemum lavandulifolium 汇报人:刘晨 指导老师:寇晓虹 Contents 1 Introduction 2 Materials and methods 3 Results 4 Discussion Introduction • Plants respond to various environmental stress using three primary strategies. • Under high salt conditions, a variety of genes are induced to express. • Genes encoding transcription factors (TFs) are the most highly regulated,which include members of the WRKY,bZIP, MYB (Myeloblastosis), AP2/EREBP (Apetala-2/EREBP) and NAC (NAM, ATAF, and CUC) families . These genes often function together as a large gene family in plants, with differentmembers of the same gene family participating in differentstress responses. Introduction • There is relationship between structure and function of NAC transcription factors • Many members of NAC gene family responded to adversities and hormonal treatments, and some showed tissue-specific expression patterns. • Most of the stress-responsive NACs belong to the ATAF and AtNAC3 subfamilies. • Ten candidate ClNAC genes in C.lavandulifolium responded to multiple-stress and hormoral treatments. Materials and methods seeds germination and plant growth salinity treatment was applied by submerging the roots of the plants with a 200 mM NaCl for 12 h, and the second and third pairs of fully expanded leaves below the terminal bud were collected. RNA-Seq analysis salinity treatment chilling and heat treatment drought stress ABA and SA treatment Collected the second and third pairs of fully expanded leaves below the terminal bud from each treated samples. Cotyledons were collected after the seeds germinated; leaves, stems, and roots were collected from eight-true leaves stage seedlings. C.lavandulifolium specimens with eight- true leaves were transferred into a short-day artificial clima-te chamber for flowering, and then the flower buds, flower petals and seeds were collected, frozen in liquid nitrogen and preserved. RT-PCR analysis RT-PCR adn qRT-PCR analysis Materials and methods Full-length amplification of candidate genes and bioinformatics analysis • Amplifed the candidate genes(ClNAC2, -3, -4, -9, -11, -12, -17, -21, 39, -44) with genes engineering. • The phylogenetic tree was constructed using MEGA4.0 software and the neighbor-joining method [37] with the following parameters: Poisson correction, pairwise deletion, and bootstrap (1000 replicates; random seed). Multiple sequence alignments were performed using the default parameters for ClustalW [38]. The 10 ClNAC genes were classified into the corresponding subfamilies according to Ooka et al. and Fang et al. . We used the TMpred server to predict of the membrane-bound ClNAC members encoded by the 10 candidate genes. Results Salt-induced expression of transcription factors in the leaves of C. lavandulifolium Fig. 1. Distribution of “molecular function” GO)terms of the C. lavandulifolium unigenes in leaves subject to salt between 0 and 12 h. GO annotation categories assigned to fewer than 30 unigenes were not shown. The x-axis refers to number of unigenes differentially expressed in RNA-Seq analysis, and the y-axis represents functional categories (P < 0.01, FDR < 0.05). Results • Activities of transcription regulators were significantly enriched in the leaves of C.lavandulifolium. • A large number of transcription factors in the leaves of C. lavandulifolium responded to the NaCl treatment (see Appendix S4 in Supporting information). Results 44 genes belong to NAC family NaCl treatment 14 genes displayed unchanged expression 6 genes ≥2 28 genes displayed upregulated expression 8 genes ≥4 6 genes ≥8 2 genes displayed downregulate expression 2 genes ≥16 6 genes ≥30 Results Expression pattern of ClNACs in different organs Fig. 2. Heat map representation of tissue-specific expression of 44 ClNACs. Expression patterns of 44 ClNACs genes were analyzed using RT-PCR, and the fluorescence absolute quantification of electrophoretic bands was performed by Image Quant 5.2 software. The colors indicate expression intensity (red, high expression; black, no expression). SE, seeds; CO, cotyledon; LE, leaves; ST, stems; BU, flower buds; FP, flower petals; RO, roots. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.) Expression pattern of ClNACs under abiotic stresses and hormone treatments Fig. 3. Heat map representation of the expression data of 44 ClNACs in response to diverse environmental stress stimuli and hormonal treat-ments. Expression data for the ClNAC genes were calculated based on RT-PCR expression values in the leaves of treated samples at different time points. Fig. 4. Expression patterns for the ClNAC genes in response to multiple environmental stimuli and hormonal treatments were extracted from RT-PCR. Expression patterns of ClNAC genes under multiple-treatments are presented with white squares, plus or minus signs. Results • we compared the results of RNA-Seq and RT-PCR after 12-h salt treatment. Among the 44 ClNACs, only ClNAC11 (whose expression was down-regulated in RNA-Seq, but unchanged in RT-PCR), ClNAC14 (whose expressi-on was upregulated in RNA-Seq, but unchanged in RT-PCR) and ClNAC15 (whose expression was unchanged in RNA-Seq, but up-regulated in RT-PCR) showed different expression patterns between the two technologies, suggesting the RNA-Seq was a useful and powerful tool to monitor gene expression. • Ten ClNACs(ClNAC2,-3,-17,-4,-9,-11,-21,-12,-39 and -44) were chosen as candidate genes to perform further study. Results Phylogenetic analysis of candidate NAC genes Fig. 5. Phylogenetic relationship among candidate ClNAC genes and other plant NAC homologous genes. Phylogenetic tree of NAC domain-containing proteins from C. lavandulifolium, Arabidopsis and Oryza sativa. Amino acid sequences were aligned using ClustalW and a neighbor-joining tree was constructed with a 1000-bootstrap replication support. The subfamilies within the NAC family, as designated by Ooka et al. [8] were grouped as indicated. Abbreviations for the name of the subfamilies are as follows: ATAF Arabidopsis transcription factor-like family, NAP NAC-like activated by APETALA3/PISTILLATA family, SENU5 tomato senescence up-regulated 5-like family, NAC2 Arabidopsis thaliana NAC protein 2-like family, NAM no apical meristem transcription factor-like family, NAC1 Arabidopsis thaliana NAC protein 1-like family, ANAC001 Arabidopsis NAC protein 001 transcription factorlike family, TERN tobacco elicitor-responsive NAC protein-like family, TIP turnip crinkle virus interacting protein-like family, ONAC001 Oryza sativa NAC protein 001 transcription factor-like family, ONAC002 Oryza sativa NAC protein 002 transcription factor-like family, ONAC022 Oryza sativa NAC protein 022 transcription factor-like family. Accession numbers of NAC sequences from other plant species used in the analysis are in listed in Supplement 5. Results Results Discussion Transcription factors in C. lavandulifolium responded to salt stress • In C. lavandulifolium, except for four gene families(MYB (Myeloblastosis), HSF (Heat-Shock Factor), AP2/EREBP (Apetala-2/EREBP) and WRKY (named after the WRKY amino acid motif)), we found that 28 ClNACs were up-regulated in C. lavandulifolium, making it the most highly induced TF family in this species. Discussion • Members of the plant NAC gene family display tissue-specific expression and are involved in various processes of plant growth and development. Gene Express tissue Biological process 22 ClNAC(Group C-F) genes buds and petals flowering 5 ClNAC(Group D) genes cotyledons seed gernination 9 ClNAC genes roots later root formation and development Discussion Mechanisms of C. lavandulifolium NAC genes in response to abiotic stresses Expression analyses showed that there were15 NAC genes in C. lavandulifolium that were up-regulated by ABA (see Groups III and V in Figs. 3 and 4), suggesting the existence of other ABA-dependent stress resistance pathways in plants that may be regulated by NAC TFs. ClNACs in Groups I, II and VI responded to abiotic stresses but were not regulated by ABA Discussion Candidate stress-responsive NAC genes in C. lavandulifolium Subfamily ATAF Members functions ClNAC3, ClNAC17, OsNAC5 and 6 stress-responsive genes ClNAC2, ANAC019, ANAC055 and ANAC072/RD26 in A. thaliana stress-responsive genes NAP ClNAC12, -39 and -44 stress-responsive genes SENU5 ClNAC9, CarNAC1 in chickpea functions in the process of plants’ response to aging ONAC022 ClNAC11, LOV1 and ONAC063 in rice NAC 1 ClNAC21, ANAC021/22, GmNAC11 and GmNAC20 SNAC AtNAC3 increase the number of lateral roots of transgenic Summary • ATAF-type gene ClNAC17 and the NAC 1-type gene ClNAC21 can respond to six kinds of treatments. • Further studies of these 2 genes are needed in order to provide tress-responsive genetic resources for the Chrysanthemum abiotic resistant transgenic breeding.