| The analysis of evolution, cloning and expression pattern of plant resistance genes isan important part of interaction between microorganisms and plants. Nucleotide-bindingsite (NBS) disease resistance genes are prevalent in plant and play an integral role indefending plants from a range of pathogens, including bacteria, fungi, oomycetes, viruses,and nematodes. Therefore, it is important to investigate the NBS disease resistance genesin maize. In the present study, by using bioinformatics approach, a full set of diseaseresistance (R) candidate genes encoding NBS from the complete genome of maize wasidentified and characterized based on the analysis of gene structural diversity,chromosomal locations, conserved protein motifs, gene duplications, and phylogeneticrelationships. In addition, gene expression patterns of NBS-encoding genes1) obtained byEST database;2) induced by Bipolaris maydis (Nisikado) Shoemaker and maize sugarcanemosaic virus were also studied. Accordingly, the selected target genes were cloned. Ourresults are showed as follows:1. One hundred and seven NBS-encoding genes were classified into four types basedon the structure of N-terminal motifs and leucine-rich repeat motifs (LRRs): NBS (N,7),NBS-LRR (NL,31), CC-NBS (CN,11) and CC-NBS-LRR (CNL,58). These genes wereseparately located on each chromosome individually or in clusters, with non-randomdistributions. The numbers of NBS-encoding gene on each chromosome were different,among which chromosome10contained the maximum number (28) of NBS-encodinggenes.2. Fifteen putative conserved motifs were found in the four types of NBS-encodinggenes by MEME analysis. In the NL and CNL types, four conserved regions in the NBSdomain were revealed, including the P-loop, Kinase2, GLPL and MHD. However, in the Nand CN types, either one or three of the four conserved regions could be detected. Nodistinguish differences were found among the conserved motifs of different NBS-encodinggenes types, and Kinase2motif was the most conserved that could be found in each types.3. Compared the maize NBS-encoding genes with those of monocotyledon rice,sorghum and dicotyledon Arabidopsis, Lotus japonicus, we found that firstly, the numberof NBS-encoding genes of maize was least, and secondly, the NBS-encoding genes ofmaize lacking of TIR motifs, which were similar to that of monocot plant rice and sorghum.Moreover, the analysis of the duplications of NBS-encoding genes showed a lower level in maize than those in rice and Arabidopsis. The percentage of NBS-encoding genes inmulti-gene families (two or more members per family) of maize (38.3%) was significantlylower than those in Arabidopsis and rice (46.6%and53.7%, respectively) genomes.Furthermore, the number of gene clusters in maize (22) was lower than that in Arabidopsisand rice (39and104, respectively). Based on phylogenetic analysis of NBS-encodinggenes of maize and other three plants, we found that the phylogenetic relationships ofNBS-encoding genes in maize was closest to sorghum and farest to Arabidopsis.4. The amino acid sequences of the NBS-encoding genes combined with thepublished NBS-encoding genes from several plants were aligned to construct thephylogenetic tree. The results showed that ZmNBS3had high similarity and formed agroup with the maize rust disease (Puccinia sorghi) resistance gene Rp1-D, and ZmNBS42were highly related to the maize resistance gene analog sequence RGA4. On the other hand,ZmNBS55showed high similarity to three paralogs of sorghum root rot (Periconiacircinata) resistance gene Pc, and ZmNBS56were closely related to wheat leaf-rustdisease (Puccinia triiticina) resistance gene Lr10. The above analysis provided apreliminary basis for cloning NBS disease resistance genes in maize.5. The predicted NBS-encoding genes and their gene expression profiles showeddifferent expression levels under plant exposure to the pathogens and exogenous SAstimulation. Semi-quantitative RT-PCR analysis on the RNA isolated from maize leaves,resulted in47of58chosen NBS-encoding genes exhibiting transcriptional activities. In thetreatment of Bipolaris maydis (Nisikado) Shoemaker and SA,73%of these geneexpression levels were upregulated induced by Bipolaris maydis (Nisikado) Shoemaker.ZmNBS7,34,49,62,69,74,85,86,91,102and103presneted higher expression levels inthe induction of Bipolaris maydis (Nisikado) Shoemaker and SA, while the expressionlevels of ZmNBS56,55,51,48,42and27were only upregulated by the application ofBipolaris maydis (Nisikado) Shoemaker. Induced by maize sugarcane mosaic virus, on theother hand, the gene expression levels of ZmNBS39,49,56,61,62,65,74,77,91and103increased, among which ZmNBS39,56,61,62and91showed strong sensitivity of theinduction. ZmNBS49,56,62,74and91showed increased gene expression levels in bothof the two pathogens induction, while ZmNBS42,55and ZmNBS39,77showed specificupregulated expression levels in the two pathogens induction respectively. Furthermore,maize NBS-encoding gene expression patterns were studied using corresponding ESTdatabase, resulted in the alignment of ZmNBS to six groups on the basis of tissue andorgan types, including shoot, root, tassel, ear, endosperm and multiple tissues, we found77 out of107ZmNBS were supported by expression evidence with diverse expressionpatterns.6. According to the phylogenetic analysis of maize NBS-encoding genes and differentexpression patterns of pathogen induction, ZmNBS3,39,42,55,56and77were indicatedthat could be used as important candidate genes. After comprehesive consideration,ZmNBS56was finally selected and cloned from maize B73(ZmNBS56B). The cDNAsequences of ZmNBS56B was1494bp, encoding497amino acids. The analysis of proteinsecondary structure showed that ZmNBS56B was hydrophobin with membrane spaningdomain. The function of this gene should be further investigated. |
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