The Cloning And Function Study Of Key Gene Function In High Osmotic Stress "sensing" Signaling Pathway In Arabidopsis

Water,has an important effect on the growth and development of plants.A deficiency of water can cause drought,which is one of the major factors restricting crop production.Therefore,it is important to study the mechanism of plant sensing and responding to the environment stimuli,e.g.drought,to improving agricultural productivity.Calcium is a second messenger which can convert the physical,chemical,and biological signals in external environment to the internal biological signals in plants.When plants are exposed to external stresses,intracellular calcium ions will undergo a series of changes,inducing the downstream defense response in plants.However,only a few plant environment sensors have been found due to the complexity of plant regulatory networks and deficient mutants.Our laboratory is screening for mutants that are not sensitive to penetration based on calcium ion imaging technology.OSCA1 is identified as a previously unknown plasma membrane protein and forms hyperosmolality-gated calcium-permeable channels,revealing that OSCA1 is an osmosensor.Applying the same method,forward genetic screening,we expect to obtain new components of osmotic stress signal in plants.Plants expressing aequorin were treated with 500mM Sorbitolto to detect the changes of intracellular calcium concentration.We rescreened mutants by calcium signal under hyperosmotic stress.10 candidate mutants screened from EMS mutagenesis library were further identified for their physiological and developmental responses to osmotic stress,specific calcium signal analysis and the candidate mutant recessive analysis,and mutant osca6 was selected for the further research according to its stable recessive mutant phenotype with specific response to hyperosmosis stress.A map-based cloning population of osca6 was constructed,and three rounds of screening were carried out by using Sorbitol solution as hypertonic stress.Finally,23F2 lines with stable phenotype were selected from 606 individual lines,and samples were pooled together and the whole genome was sequenced.The target gene was preliminarily determined to be located on the lower arm of chromosome 3 according to the mutation rate line chart of SNP,and the range of mutation sites was further narrowed down by fine positioning.The SNP and related genes within the range were analyzed,and finally three candidate genes were found.The candidate gene was confirmed by complementarity analysis.The mutant osca6 was insensitive to hypertonic stress treatment compared with the wild type on different concentrations of Sorbitol medium,and the seedlings grew better than wild-type.Compared with the wild type,osca6 was insensitive to drought in soil under drought,and its water content was higher than wild type.In addition,functional complementary was performed on the mutant osca9 by the identified candidate gene.The results showed that the complement of the target gene could restore the calcium signaling phenotype of the mutant osca9 under hypertonic stress.To determine if the mutant osca9 is defectively specific to hyperosmolality over other stimuli,we analysed[Ca²⁺]i elevation in response to KCl,NaCl,CaCl₂ and H₂O₂ and observed no difference between wild type and osca9.These results demonstrated that[Ca²⁺]i increases induced specifically by hyperosmolality are impaired in osca9.On the solid stress medium,osca9 was much more sensitive to ionic and non-ionic hyperosmotic stress than wild-type,e.g.KCl,NaCl,CaCl₂ and Sorbitol.These results indicate that mutagenic gene of osca9 might be another major component of the hyperosmolality-induced[Ca²⁺]i increase(OICI)except OSCA1.In conclusion,this study screened an important factor that may be involved in the osmotic stress perception signaling pathway and cloned the target gene.It provides some theoretical basis and potential molecular genetic targets for engineering drought-resistant crops.