Map-based Cloning And Functional Analysis Of Two Rice Mutant Genes Controlling Plant Type

Improving rice plant type is a key research field for rice breeding to increase rice yield in recent years.More and more researches suggested that it is a reliable method to optimize plant type and increase production using the genes of regulating plant type in rice.However,as a complex trait,plant type was controlled by multiply genetic factors and the regulating mechanisms of plant type were large unknown.For example,as the key plant hormone of controlling rice plant height and tillering,the responsive genes in the downstream of strigolactone?SL?signal were mostly unknown,and transcriptional and post translational modifications of key members in SL synthesis and signal transduction pathways have not been studied.Furthermore,some genes not only control the plant type traits in vegetative growth stage,but also regulate the traits that formed in reproductive growth stage.Although people have realized those phenomenons,the mechanisms were not yet clear.In order to study the regulation mechanism of plant type function genes in rice,we isolated an indica high-tillering dwarf mutant 4?htd4?,a spontaneous mutant of rice,from the restorer line Gui99.Moreover,a rice mutant,dwarf and deformed flower3?ddf3?,was isolated from T-DNA mutants with Oryza sativa cv.Dong-jing as the receptor.In this paper,we study the map-based cloning and their functions of these two mutant genes,and the main results are as follows.?1?Compared with wild type,the main morphological characteristics of mutant htd4 were dwarf and high-tillering.Mapping and sequencing analysis showed that htd4 was a novel allelic mutant of D14,in which a single base substitution forms a premature termination codon.RT-qPCR analyses revealed that expression levels of the SL genes D10,D17,D27,D3 and D14 increased significantly,while expression of D53 decreased in htd4,compared with the wild type.A subcellular localization assay showed that the mutant of D14 in htd4did not disturb the normal localization of D14 proteins.However,a BiFC assay suggested that the mutant-type D14 could not interact with D3.Those results indicated that the signal transduction pathway of SL was interrupted,which lead to the high-tillering dwarf phenotype of htd4 mutant.Furthermore,compared with other D14 allelic mutants,htd4 was the first mutant of D14 discovered in indica,and probably have the fertility restorer genes,which may be of great value in breeding applications.Our results enriched the genetic regulation mechanism of SL controlling rice tiller formation and provided important material resources.?2?Compared with wild type,the ddf3 mutant exhibited severe dwarfism,a greater number of tillers and significantly decreased fertility.All internodes of ddf3 were shorter than wild type,and histological analysis revealed that internode cell elongation was significantly inhibited in ddf3.In the ddf3 mutant,pollen activity was significantly decreased,and the development of stigma was abnormal.Genetic analysis indicated that the ddf3 mutant phenotypes were controlled by a single recessive nuclear gene.Using an F₂ mapping population generated from a cross between ddf3 and Yangdao 6?9311?,the DDF3 gene was mapped to a 45.21-kb region between InDel markers M15 and M16 on the long arm of chromosome 7.Sequencing revealed a 13.98 kb-deletion in this region in the ddf3 mutant genome that resulted in the complete or partial deletion of ZF?DHHC type zinc fnger protein?,EP?expressed protein?and FH2?Actin-binding FH2 domain-containing protein?genes.Knockout results showed that only FH2 knockout,the plants showed severe dwarfing.ZF and EP did not show obvious plant type change after knockout.Therefore,the deletion of FH2 gene was the reason why mutant ddf3 had the severe dwarf phenotype.RT-qPCR analyses revealed that in wild type the transcriptlevels of FH2 were almost the same in all organs,while ZF was mainly expressed in the panicle,and no expression of EP was detected in any organ.Our works laid a foundation for further analysis of the functions of these candidate genes and enriched the research contents of rice plant type regulation mechanism.