| Rice(Oryza sativa L.)is one of the most important crops and half of the world’s population has rice for main food.Rice tiller number is a very important agronomic trait.Ideally,each tiller can form one panicle.Tillering is the key factor that determines the yield of rice.In this study,the rice reduced tiller number20(rcn20)mutant was used as the experimental material for morphological and cytological observation,map-based gene cloning and functional analysis.The main findings are as follows:1.The recessive mutant rcn20 was isolated from the japonica rice cultivar SD808 by EMS mutagenesis.The rcn20 mutant shows severely decreased tiller number(only 3 to 4 tillers)and slightly reduced plant height,compared with wild type SD808 normally with 15 tillers.2.The paraffin section technique was used to observe the tiller development in the rcn20 mutant.In the rcn20 mutant,the axillary meristem could initiate normally in the seedling stage,and the tiller bud formed but could not elongate in the mature stage.Scanning electron microscopy(SEM)showed that the shape of the epidermal cells of the mutant tiller buds was irregular,whereas the shape of the cells on the surface of the wild-type tiller buds was regular.Immunofluorescence analysis of root tip cells in the meristematic zone and elongation zone revealed that most cells in the rcn20 mutant had altered orientation of the cortical microtubules arrangement during the mitosis interphase.This may result in changes in cell morphology and ultimately suppression in the elongation of the tiller buds.3.Genetic analysis revealed that phenotype of the rcn20 mutant was controlled by a recessive gene.The mutant gene was located in the region of 300 kb on chromosome 3 by map-based cloning.The candidate gene was found to be LOC_Os03g01530(named RCN20,hereafter).The 616 rd nucleotide at the CDS of the gene was changed from G to A,resulting in an amino acid change from alanine to threonine at position 206.The wild-type genomic DNA of this gene was transformed into the rcn20 mutant,which rescued the mutant phenotype.Therefore,LOC_Os03g01530 was confirmed to be the target gene controlling the number of tillers by promoting tiller bud elongation.The RCN20 gene encodes β-tubulin protein.4.Real-time RT-PCR analysis showed that the RCN20 gene had the highest expression in tiller buds,followed by higher expression in leaf sheaths,young panicle and roots.Transgenic rice plants harboring the GUS reporter gene driven by the RCN20 promoter were developed.Histological staining of each tissue confirmed the expression pattern revealed by RT-PCR analysis.5.Os TB1,a TCP family transcription factor,inhibits tiller bud elongation.The loss-of-function tb1 mutant shows increased tiller number.We constructed the double mutant of the rcn20 and tb1 mutant.The tillering phenotype of double mutant is much similar to rcn20,suggesting that the RCN20 gene was located at the downstream of Os TB1.Comparison of the expression level of Os TB1 and RCN20 genes in the tb1 mutant and its wild type Taizhong 65,in the strigolactone receptor mutant d14 and its wild type SD808 revealed that Os TB1 has an inhibitory effect on the expression of RCN20.6.Through the electrophoretic mobility shift assay(EMSA)and chromatin immunoprecipitation assay(Ch IP),the Os TB1 protein was verified to be able to bind to the core sequence of the RCN20 promoter region,indicating that Os TB1 may regulate the RCN20 gene expression directly.7.We further measured the transcriptional activity of Os TB1 through transient expression of the luciferase(LUC)reporter gene in the rice protoplasts.This assay confirmed that the RCN20 gene expression was significantly inhibited by Os TB1.Transgenic rice plants were developed in which expression of the Os TB1 gene can be induced by Dexamethasone(DEX)treatment.Upon DEX treatment of the transgenic seedlings,the expression of Os TB1 was up-regulated whereas the expression of RCN20 was down-regulated.8.The tb1 mutant and its wild type Taizhong 65,the strigolactone receptor mutant d14 and its wild type SD808 were treated with the strigolactone analogue GR24.q RT-PCR analysis revealed that the expression level of Os TB1 was up-regulated whereas that of RCN20 was down-regulated by the strigolactone treatment. |
