Map-based Cloning And Functional Analysis Of A Salt Tolerance Gene OsTUB1 In Rice(Oryza Sativa L.)

Salt stress is one of the main environmental factors that restrict plants growth and development.It can not only lead to crop yield reduction,but also make crops wither and die.According to the incomplete statistics of UNESCO and FAO,the area of saline alkali land in the world is 95,438 billion hectares,of which the total area of China is about 99.13million hectares.Because no suitable crops can be found to plant in saline alkali land,saline alkali land as a very valuable land resource is wasted.Rice is one of the major crops and sensitive to salt stress.The salinization of soil seriously restricts the yield and quality of rice.Therefore,it is necessary to excavate salt tolerant rice germplasm resources,analyze the genetic and molecular mechanism of rice salt tolerance,and lay a good theoretical foundation for the cultivation of new salt tolerant rice varieties.At the same time,it is of great significance to maximize the use of saline soil resources,improve land use efficiency and the ecological environment.Microtubules are one of the basic components of the cytoskeleton.Tubulin is the basic component of the microtubule organization.It regulates the organization stability and function of cells in a variety of ways.Previous studies have reported that the stability of microtubule organization is related to salt stress in Arabidopsis.However,whether microtubules regulate ion homeostasis under salt stress and its molecular mechanism remain unclear.This study identified a salt hypersensitive mutant shs1（salt hyper-sensitive 1）.The regulation molecular mechanism of microtubules in ion balance under salt stress was analyzed through genetics,molecular biology,functional genomics and cell biology.Further research found that this gene encodes aβ-tubulin,named OsTUB1.Further analysis revealed that the mutation of OsTUB1 resulted in the disorder of rice microtubule organization under salt stress.At the same time,it was found that OsTUB1 and SRS3interacted to regulate the stability of microtubule organization under salt stress and the subcellular localization of Na⁺transporter SKC1 on the membrane was changed,thus affecting the ion balance between inside and outside the cell and reduces the ability of plants to resist salt stress.The related results are as follows:1.In this study,a new rice salt sensitive mutant shs1 was isolated and identified by screening the survival rate of W017 EMS mutant under salt stress.Under salt stress,we found that the mutant showed salt sensitive,and its survival rate was significantly lower than that of wild-type.Further analysis of the content of Na⁺,K⁺and Na⁺/K⁺ratio between wild-type and shs1 after salt treatment showed that shs1 accumulated more Na⁺in the shoot,which restricted the growth of shs1 and caused the death.These results indicated that SHS1plays an important role in rice under salt stress.2.Genetic mapping of the OsTUB1 gene was performed using the F₂ populations from a cross between shs1 and cultivar N22.We mapped the shs1 gene to a 180 kb genomic region on chromosome 1 between the SSR makers hly8 and C12 using～4500 F₂ mutant plants.Detailed genomic sequence analysis identified a non-synonymous single base change（G to A）at position 523-bp from the start codon of the second ORF（Os01g0282800）,which encodes aβ-tubulin 1 protein OsTUB1.The SNP caused the change of valine to isoleucine located at the conservative region of its tubulin domain.Furthermore,through gene knockout,transgenic complementation and overexpression experiments,it is confirmed that the mutation of Os01g0282800 is the reason of salt sensitive phenotype of shs1.Os01g0282800 encodes aβ-tubulin containing 447 amino acids.Subcellular localization showed that it is located in the cytoplasm microtubules in tobacco and rice protoplasts,but the mutant protein cannot co-locate with microtubules.The expression of OsTUB1 was induced by salt stress.In wild-type,the regularly organization of the microtubules in the cortical cells was disrupted with less microtubule arrays formed under salt stress,indicating that salt stress disrupts the formal organization of microtubules.By contrast,more distorted and sparse microtubule arrays were observed at the elongation zone of shs1 mutant as compared to wild-type.Histochemical analysis showed that OsTUB1 was mainly expressed in roots and stems.Further analysis showed that the mutation of OsTUB1 affects the transcription levels of ion-balance related genes,especially the sodium transporter SKC1.These results suggested that the organization stability of microtubules regulated by OsTUB1 is necessary for salt tolerance and ion balance in rice.3.SRS3 encodes Oskinesin-13A protein,which is essential for the regulation of microtubule organization and affects plant growth.Bi FC and Pull down experiments proved that SRS3 interacts with OsTUB1,and the mutant protein cannot interact with SRS3.Further immunofluorescence experiments and phenotypic analysis showed that the microtubules of srs3 mutants were also significantly damaged under salt stress,and the mutants were also showed salt sensitive under salt stress.Overexpression of SRS3 increased salt tolerance in rice.These results indicated that the interaction between SRS3 and OsTUB1is important in regulating microtubule organization and ion balance under salt stress.4.Our studies found that the membrane localization of sodium transporter SKC1 was altered in shs1 and srs3 mutants.Protein level analysis showed that the protein level of SKC1 was significantly reduced in mutants under salt stress.At the same time,we treated wild-type protoplasts with Oryzalin,a microtubule depolymerization drug,and found that the destruction of microtubule organization affected the subcellular localization of SKC1protein.Furthermore,we separated the membrane and cytoplasm of wild-type and mutant protoplast by gradient centrifugation,and western blot detected SKC1 protein distribution in plasma membrane and cytoplasm.The results showed that the localization of SKC1 on the membrane in mutant was reduced.These results suggested that the stability of microtubule organization regulated by SRS3 and OsTUB1 is crucial for the correct localization of SKC1.5.We analyzed the phenotype of OsTUB1 overexpression transgenic lines in seedling stage（120 m M Na Cl～7‰salt concentration）and field（～5‰salt concentration）.And then,we compared the plant height ratio（PHR）,effective panicle number ratio（EPNR）,1000grain weight ratio and yield per plant ratio（YPPR）between normal field and saline field.Furthermore,we analyzed the survival rate and sodium potassium content in seedling stage.These results showed that OsTUB1 overexpression lines were more salt tolerant compared with wild-type.Meanwhile,we compared the ion contents in mature seeds of wild type and overexpression lines under normal and salt field.Na⁺content decreased in OE lines grown in saline fields compared to wild type.In normal or saline fields,OsTUB1 overexpression improved K⁺content,and decreased the Na⁺/K⁺ratio.Therefore,OsTUB1 enhances salt tolerance and stabilizes yield of rice under salt stress,providing new strategies and material basis for salt tolerance breeding.