Cloning And Functional Analysis Of The Pollen Wall Development Gene OsLTPG47 In Rice(Oryza Sativa L.)

Rice(Oryza sativa L.)is one of the most important staple food crops in the world,and an excellent model plant for developmental biology studies in monocots.With the increasing population and the reducing available land for cultivation,food security has been facing a huge challenge.The utilization of male sterility in rice has played an important role in improving grain yield.Anther development is the basis of the male reproductive development in rice,thus studies on the genetic and molecular mechanisms for anther development are important for both basic and applied sciences.This study isolated rice male sterile mutan,and did researches on phenotype,gene-cloning and gene function to reveal the mechanism of the target gene.The main results are as follows:1.A new male sterile mutant was isolated from a rice mutant library created by?-ray mutagenesis of a japonica rice cultivar Nipponbare.The mutant exhibits normal vegetative development and female organ formation,but it is completely male sterile and has shrunken anthers and aborted pollen grains.2.Scanning electron microscopy assays showed that the three-dimensional spaghetti-like cutin layers and ubisch bodies were stick together in mutant anther,the bud apertures were inane and their epidermal surfaces were covered with randomly distributed cutin materials in mutant pollen grains.Observation of anther transverse sections showed that tapetal degeneration was delayed in the mutant.The inner and outer layers of exine were swollen and the bacula was irregularly shaped,which can not form a typical microspore exine in mutant.3.Genetic analysis suggested that the mutant is a genetically sporophytic male-sterility mutant,which is controlled by a single recessive locus.Using an F₂ segregation population,this locus was mapped to a 32-kb region on chromosome 1.Sequencing analysis uncovered a 1-bp deletion in the frist exon of a gene LOC?Os01g42210,which resulted in a premature stop codon.4.Genetic complementation and gene knock-out test confirmed that the sterile mutant was conferred by LOC?Os01g42210.This is a newly evolved gene in the poaceae family encoding a lipid transfer preotien(LTP),thus this gene is named Os LTPG47.5.Expression analysis by q RT-PCR showed that Os LTPG47 expression was constitutive at low leveles.To understand the role of Os LTPG47 in the regulatory network of tapetal development,the expression levels of 9 known anther functional genes were examined by q RT–PCR.The result showed that,in the osltpg47 anthers,expression of API5,CYP703A3 and C YP704B2 were significantly down-regulated,which suggested that the mutation of Os LTPG47 interrupted the genetic networks of rice anther development.6.Subcellular localization assays indicated that Os LTPG47 was mainly localized to the plasma membrane,this localization requires the N-terminal signal peptide and C-terminal TM domain in this protein.The observation of GFP signal in the GFP-Os LTPG47 transgene plants also confirmed that Os LTPG47 was mainly located in plasma membrane.7.Yeast two-hybrid assays showed that Os LTPG47 formed homologous d immers through the 143-197 amino acid region of the C-terminal.Os LTPG47 also weakly interacted with a GPI transamidase Os PIG-K.8.Chemical analysis of anther cutin was showed that the contents ofcutin monomers(C16:0?-HFA acid,C18:1?-HFA acid,C18:2?-HFA acid,C18:2 FA,and C18:3-HA acid)were significantly decreased compared with the WT.On the contrary,wax analysis showed an increase of alkanes was detected in the mutant anthers.In this study,a male sterile mutant osltpg47 was isolated.Using map-based cloning,genetic complementation,gene knockout and molecular function assays,the molecular mechanism of Os LTPG47 involved in the regulation of rice pollen wall development was elucidated.