Map-based Cloning Of EAO1 Regulating Enlarged-aerial-organs In Rice(Oryza Sativa L.)

In recent years,the development of aerial lateral organs in rice has attracted widespread attention from breeders and scientists.They believe that not only reasonable plant types are an important factor for high yield,but it can also open a new path to solve world food security problems.The aerial lateral organs of rice mainly include stems and leaves.The length of stem directly affects the plant height,and stem diameter influences plant lodging resistance,while the size of leaf blades was related to the light-receiving area,all of which directly or indirectly affect single-plant yield.Therefore,it is crucial to understanding the genetic pattern and development mechanism of rice aerial lateral organs.We identified a enlarged aerial organs mutant（eao1）by screening rice EMS-mutational library.This paper performed a serious researches,such as phenotype and histological observations of eao1,gene fine mapping,candidate gene identification,map-based cloning,expression analysis,protein structure analysis etc.The main results are as following:1.The eao1 is a mutant of enlarged aerial organObserving phenotypes at maturity stage,we found mutant was higher than wild type.Further analysis the number and length of internodes revealed that the internode number and length increased.The measurement of internode diameter showed that diameter of the mutant was wider than wild type.Tthe stems are larger and thicker.The leaf length of the mutant was not significantly different from wild type when observed on the leaves,but the width was significantly wider than wild type.The results of agronomic analysis indicated that there are no significant differences in primary branches,but the number of secondary branches significantly increased,the effective spikes,and the seed setting rate decreased.2.Increased number of cells lead to thicker stem and wider leaves of the mutant eao1To clarify the reason for the larged aerial organs,we performed histological analysis of internodes and leaves,which showed that mutant cross section and vascular bundles ares are larger than wild type,the number of vascular bundles and cell layers increased.Besides,all cells in vascular bundles included xylem cells,phloem cells,bundle sheath cells and parenchyma cells were significant increase.3.Genetic analysis and molecule mapping of EAO1Genetic analysis indicated that the larged aerial organs traits of eao1 mutant were controlled by a recessive nuclear gene.Based on the F₂ population derived from a cross between the eao1 and XIDA 1B,EAO1 was fine mapped on chromosome 8between marker F4-4 and F4-4 with the physical distance of 157kb.4.EAO1 candidate gene cloning and transgene plants validationWe screened genes related to the phenotype of mutant and their DNA sequences were sequenced in 26 annotated genes.The result showed a single nucleotide substitution from G to A within gene at the 269_th base of the first exon,caused the encoded glycine to be glutamic.This gene was initially identified as a candidate gene for EAO1.In order to verify that the gene is the mutant gene,we constructed the p CAMBIA1301-EAO1 vector for genome complementation experiments.The phenotype revealed that the related traits such as the plant height and leaves of the complementary transgenic plants were restored to the wild type level.Then the mutation site of the target gene was sequenced and found that was a heterozygous site.The above results indicate that the mutation phenotype of the mutant eao1 is caused by the gene mutation.5.EAO1 is expressed in the cell nucleusThe RT-PCR and QPCR results of wild type and mutant showed that EAO1 was expressed in various tissues in rice plants at heading stage.The entire coding sequence of EAO1 was fused to the N-terminus of green fluorescent protein（GFP）and transformed into tobacco leaves.It was found that EAO1 detected a fluorescent signal in the cell nuclear,indicating that EAO1 is a nuclear protein.6.EAO1 interacts with Os TCP5 and Os TCP12Related experimental results found that the p GBKT7-EAO1,p GADT7-TCP5 and p GADT7-TCP12 recombinant plasmids were all non-toxic and the TPR domain of the EAO1 protein had no self-activating activity.Yeast two-hybrid experiments indicated that p GADT7-TCP5/p BD-EAO1,p GADT7-TCP5/p BD-EAO1,and positive controls grew normally on SD-Trp-His-Ade-Leu nutritional media.It indicated that the TPR domain of EAO1 can interact with Os TCP5 and Os TCP12.The results of Bi FC showed that YFP signal could be detected in the cell nuclear of tobacco leaves.It also indicated that the TPR domain of EAO1 could interact with Os TCP5 and Os TCP12.