Map-based Gene Cloning And Functional Analysis Of Rice Moderately Increased Tiller Mutant Mit1

Rice(Oryza sativa)tillering is an important agronomic trait that is one of the key factors of rice yield.Previous studies have confirmed that the outgrowth of tiller buds of rice dwarf and high-tillering mutants is regulated by strigolactones,product of the MAX/RMS/D(more axillary branching)pathway.Strigolactones derive from carotenoids and inhibit axillary bud outgrowth.In this study,we used a rice moderately increased tiller(mil1)mutant as the experimental material.Using the map-based cloning approach,we identified the mutated gene.The functional verification and analysis of the mutant gene were carried out,which proved that the gene is a new gene in the pathway of SLs.These results lay the foundation for further revealing the mechanism of SLs regulation of rice tillering and plant branching.The main results are as follows:1.The mutant mit1 exhibited reduced plant height and moderately increased tiller number.Each internode of mit1 showed shortening at the similar proportion and the plant height was about 80%of that of the WT at the mature stage.Tiller number of mit1 increased,about 2 times of the WT.mitl could produce seeds normally and number of grains per panicle is about 70%of that of the WT,but total grain number and yield per plant were increased compared with the WT.2.Through the map-based cloning approach,the candidate gene was mapped within a 2 Mb interval within the centromere region of chromosome 12.Sequencing analysis of the candidate genes in this region found the LOC_Os12g21710 was the mutant gene,which was named MIT1 and the mutation caused a 10 bp deletion.According to the Rice Genome Annotation Project,MIT1 encodes an isomerase in plant carotenoid biosynthesis.The putative enzyme was termed 15-cis-?-carotene isomerase(Z-ISO).Genetic complementation completely rescued the abnormal phenotype of the mitl mutant.Thus we concluded that LOC_Os12g21710 corresponds to MIT1 gene.3.Sequence alignment showed that the similarity of MIT1,ZmZ-ISO and AtZ-ISO proteins in Rice,Maize and Arabidopsis were higher.Rice Genome Annotation Project Website(http://rice.plantbiology.msu.edu/)predicts that MTI1 is composed of 368 amino acids,and has a N-terminal chloroplast transit peptide according to the ChloroP 1.1 Predi-ction Server prediction.In addition,MTI1 is predicted by the TMHMM Server version 2.0 to have five transmembrane regions,and according to HMMPfam prediction,there is a NnrU domain.Phylogenetic analysis shows that homologous genes of Z-ISO are present in higher plants and bacteria and they all have a common origin,the NnrU gene.4.Real-time quantitative PCR analysis revealed that the MIT1 expression level is high in panicles,culms and leaf sheaths,and low in axillary buds,leaf blades,flowers and roots.The spatial distribution of MIT1 gene expression was further examined by using the MIT1 promoter driven GUS(?-glucuronidase)gene as a reporter.The results of this experiment are consistent with those of RT-PCR.To determine the subcellular localization of the MIT1 protein,we performed a transient expression experiment of MIT1 in rice leaf protoplasts.We found that MIT1-GFP was localized only in the chloroplasts.5.By measuring the indoor light and dark grown rice seedlings 9,15,9'-tri-cis-?-carotene and 9,9'-di-cis-?-carotene content,chlorophyll content determination,deetiolation experiment,and so on,we confirmed mit1 is a Z-ISO mutants and MIT1 encodes Z-ISO.6.The measurement of SLs produced in the root exudates showed that wild type accumulated markedly higher levels of 2'-epi-5-deoxystrigol(epi-5DS)than the mit1 mutant.Further,exogenous application of a synthetic SL analogue,GR24,effectively inhibited the outgrowth axillary buds of mitl and d17(a SL biosynthesis mutant),but not d3(a SL signaling mutant).These results indicate that mitl is a mutant of the SLs biosynthesis pathway.7.In order to further understand the position of the mutation gene MIT1 in SLs synthesis pathway,we generated mitl d17 and mitl d3 double mutants.The results showed that MIT1 is located at the upstream of D17 in SLs synthesis pathway,and does not belong to the SLs signalling pathway.In addition,according to the function of the 27,MIT1 is also located in the upstream of D27.