Functional Analysis Ofrice OsHAD1 In Response To Drought Stress

Rice(Oryza sativa L.)is one of the most important food crops in the world,and its yield and quality are often affected by various adversity conditions.Os SCL30 b is a multistress response gene.Os HAD1 is an enzyme encoding(halogenated acid dehalogenase,HAD)screened by yeast two-hybrid assay using Os SCL30 b as a bait protein.So we cloned the gene Os HAD1 to investigate its role in the regulation of abiotic stress in rice.Preliminary studies on the mechanism of drought tolerance action of Os HAD1 were carried out by constructing p CAMBIA1300 overexpression and antisense expression vectors,35S:Os HAD1-GFP and Os HAD1pro:GUS expression vectors,and the main results were as follows:(1)Gene expression profiling data and q RT-PCR results showed that Os HAD1 was responsive to drought,temperature,ABA and salt.GUS staining of Os HAD1pro:GUS transgenic seedlings showed that Os HAD1 was expressed in stems,internodes,anthers,seed endosperm and roots,while GUS staining in roots was significantly enhanced under various stresses treatments.The gene was subcellularly localized in the nucleus and cell membrane.(2)In the 20% PEG lethality experiment,the antisense-expressed lines exhibited significantly enhanced drought tolerance.The soil culture drought experiment showed similar results.Os HAD1 antisense-expressed lines treated with 10% PEG in moderate simulated drought showed significantly longer root systems than the control,and with increasing PEG concentration,root elongation was significantly promoted.The exact opposite result was exhibited in overexpressed seedlings.(3)The results of DAB and NBT leaf staining under drought stress showed that the reactive oxygen species accumulation was reduced in antisense-expressed lines and the expression of Os Rboh E,a gene regulating reactive oxygen species production,was downregulated.The leaf senescence assay showed that it had higher tolerance to oxidative stress.Meanwhile,superoxide dismutase and peroxidase activities were significantly higher in the antisense-expressed plants,and the expression levels of their corresponding genes were significantly up-regulated.The results were opposite to the previous experiments in the overexpressed lines.(4)After the Os HAD1 transgenic lines were treated with 50 μM ABA solution,the antisense-expressed lines showed lower stomatal opening and slower leaf water loss compared with the wild type.The expression levels of the marker genes Os LEA3,Os SNAC1,and Os RAB21,Os DREB2 B,which are responsive to ABA treatment under drought stress conditions,were significantly higher in the antisense-expressed lines,whereas the expression levels in the overexpressed lines were lower than those in the wild type.These results suggest that Os HAD1 negatively regulates drought stress tolerance through an ABA-dependent pathway.