Identification And Function Analysis Of Two Novel Genes Involved In Response To Salt Stress

In recent years,the high soil salinization become more and more serious.High soil salinity is a major abiotic factor that limits crop yield.Under the high salt stress,the plant can adjust physiological and developmental situation to survive and reproduce,in which the gene regulation plays a key role.Many scientists have done a lot of researches in the field,but the mechanism of gene expression response to salt stress is still unclear.In the preliminary work our laboratory created a transgenic line sy24(temporarily named sy24)which is transgened by the promoter of salt-induced ERF3 driving LUC(PERF3-LUC).Used as wild type material,sy24 was mutagenized via EMS to screen the mutants with the LUC activity higher than the wild type.In this paper,two of this type of mutants p13h09 and sy33,were studied and the following progress was made.p13h09 using the map-based cloning methd,the mutant was identified to encode a function unknown protein.The gene P13H09 has four exons and 3 introns.In the mutant p13h09,the 263th base(from the initiation codon ATG)in the first exon G is mutated to A,which caused the mutation of the 88th amino acid Gly to Asp in the protein.The mutant p13h09 and its T-DNA insertion mutants were crossed,and FI generation also showed the phenotype LUC activity is similar to mutant p13h09.Further LUC activity of transgenic complementary material ofp13h09 is restored the level of the wild type sy24.These results demonstrated that the alteration of LUC activity in p13h09 is caused by the mutation of P13H09.The mutant p13h09 also showed significantly altered phenotypes on seed germination and root growth when treated with NaCl,sorbitol and ABA.In seed germination,mutant p13h09 is more sensitive to NaCl,sorbitol and ABA,so gene P13H09 may participate in salt stress,osmotic stress and ABA signaling pathways.Gene expression analysis showed that P13H09 express in all tested tissuses,but the highest in the flowers,while the lowest in the leaves.By Agrobacterium infiltration of tobacco,the P13H09-GFP fusion protein is shown to be located in the nucleus,which suggested P13H09 protein plays a role in the nucleus.According to all these results,it is suggested that P13H09 is a novel gene which plays an important role in response to salt stress.By map-based cloning technology,the mutation site of sy33 is narrowed between 1.25M to 3.1 M in 5th chromosome.Aided by the second generation sequencing,the mutant gene in sy33 was identified to encode a function unknown protein(temporarily named S Y33)in the wild type sy24.The gene SY33 has five exons and four introns.In sy33 mutant the 443th nucleotide G(from ATG)is mutated to A,which caused the mutation of the 148th amino acid Gly to Asp in protein.The sy33 seed germination is more tolerant to NaCI than WT.Furthermore,sy33 flowers earlier than sy24.Gene expression analysis showed that SY33 expresses mainly in flowers.According to above results,it is suggested that SY33 plays an important role in plant stress response and the flowering control.In short,the phenotype,gene cloning and the preliminary fiunction analysis ofP13h09 and sy33 are probed in this paper,and our current results provided the next research on the function of P13H09 and SY33 with some important clues.