Functional Study Of OsTOD1 Gene In Rice Reproduction

Rice is a model monocot plant system for plant growth and development studies.Meanwhile,it is also an important cereal crop,and the rice yield affects the world’s food security.With the rapid expansion of population,a series of ecological problems have been followed.Human beings can never get rid of the haze of food security.Therefore,it is very important to increase rice yield to solve the growing food demand problem.Fertility plays a key role in the production of rice yield.In recent years,molecular mechanism of rice fertility regulation has become a research hotspot in the field of plant developmental biology,which has also promoted the application of rice in genetic improvement.In addition,the key step to determine normal fertility is the successful fertilization.Therefore,researches of rice reproduction have important theory and practice significances and obtaining more sterile mutants is the basis of the researches of rice reproductive development.In the model dicot plant Arabidopsis thaliana,the researches of the molecular mechanism of plant reproduction are more in-depth.In previous work of our laboratory,a fertility related gene AtTOD1(TurgOr regulation Defect 1),which is specifically expressed in pollens and silique guard cells,is cloned from Arabidopsis thaliana.It encodes an alkaline ceramidase which is located in Golgi.AtTODl regulates pollen tube growth by regulating the turgor pressure.The Arabidopsis todl mutant shows defects in pollen tube growth and penetration efficiency,which results in reduced fertility and decreased seedset.The current thesis is based on the study of AtTOD1,and it has found Os01g11220,the orthologous gene of AtTODl in rice by bioinformatic analysis.It is renamed as OsTOD1.This project takes the gene as the starting point to study its function in rice reproduction,in order to further understand the mechanism of pollen tube growth regulation in monocots,and to provide new germplasms for rice breeding.The total length of the OsTOD1 gene is 2122 bp,and it contains 4 exons and 3 introns.OsTOD1 and AtTOD1 genes have conserved collinearity,which are closely linked with KAT1(POTASSIUM CHANNEL IN ARABIDOPSIS THALIANA 1)gene.OsTOD1 gene is highly expressed in the reproductive tissues of rice,especially in pollen grains and pollen tubes,but with low expression in vegetative tissues.Agrobacterium mediated tobacco leaf transformation experiment shows that OsTOD1 is colocolized with endoplasmic reticulum marker.OsTOD1jg,OsTOD1jc and OsTOD1ig,three different forms of the OsTOD1 gene,are transformed into the Arabidopsis todl mutant respectively.The result shows that they are able to partially rescue the Arabidopsis todl mutant phenotype.Three lines of rice todl mutant of homozygous transgenic plants are obtained by genome editing.Compared with the wild type,it is found that there are no significant difference in the vegetative growth stage.At the stage of reproductive growth,there is no obvious abnormality in the development of the reproductive tissues.The external and internal forms of the flowers are normal,and the vitality of the pollen is normal.The pollen tube is germinated and cultured in vitro,which shows that the germination of pollen tubes are also normal.In order to further study the phenotype of ostodl mutant,using the wild type as a control and staining pollen tubes by aniline blue in 6 hours after pollination,which shows that the pollen tube growth retardation of the mutant ostodl.These results indicate that the OsTOD1 gene may regulate rice pollen tube growth.Furthermore,using KAT1 as the marker gene,it is demonstrated that AtTOD1 has collinearity and synteny with TOD1 genes in many other angiosperm species,from basal angiosperms such as water lily(Nymphaea colorata),to monocots such as banana(Musa acuminate),pineapple(Ananas comosus),and rice(Oryza sativa),and eudicots such as tomato(Lycopersicon esculentum).However,it is not collinear with papaya(Carica papaya)and gymnosperm species,such as ginkgo(Ginkgo biloba)and loblolly pine(Pinus taeda).TOD1 from water lily can also partially rescue the Arabidopsis todl mutant phenotype,which indicates that TOD1 is functionally conserved in the basal angiosperm water lily.However,TOD1 from papaya and ginkgo can not rescue the Arabidopsis tod1 mutant phenotype,which indicates that TOD1 is not functionally conserved in the two species.