Study On Function Of Arabidopsis Salicylate Hydroxylase DLO2 And Generation Of Low Salicylic Acid Genetic Materia

Salicylic acid?SA?is one of the nine plant hormones,which plays an important role in plant disease resistance,seed germination,stomatal opening and closing,root nodule formation,DNA damage repair and leaf aging.Salicylic acid 3-hydroxylase?S3H?and salicylic acid 5-hydroxylase?S5H?are key enzymes in the metabolism of Arabidopsis thaliana SA.They can catalyze the hydroxylation of SA to 2,3-dihydroxybenzoic acid?2,3-DHBA?and 2,5-dihydroxybenzoic acid?2,5-DHBA?respectively,and then regulate the level of endogenous SA.In our early research,through sequence alignment and phylogenetic tree analysis,we found a homologous gene DLO2?At4g10490?of S5H gene.In vitro enzyme activity analysis showed that DLO2 can catalyze SA to generate2,5-DHBA.However,the biochemical and biological functions of DLO2 are not clear and need to be further elucidated.In this study,the DLO2 function of SA hydroxylase gene was further explored by means of molecular biology and physiological and biochemical techniques.At the same time,the genetic material with low SA content was created by using 35S promoter and S5Hpromoter to drive the overexpression of S3H in wild type.The research results are as follows:?1?Purification and enzyme activity detection of DLO2 recombinant protein.The recombinant DLO2 protein was purified by affinity chromatography with a size of 75 kD.The purified DLO2 was enzymatically reacted with SA as substrate.The results showed that,similar to S5H,DLO2 can catalyze the formation of 2,5-DHBA with SA as the substrate;the optimal pH and temperature of DLO2 enzyme activity were 7.0 and 20?,respectively;the V_maxax and K_m of DLO2 were 65.6±4.0 nmol mg^-11 H^-11 and 31.4±4.3?m respectively.DLO2 expression pattern analysis.The expression of DLO2 and S5H genes in different developmental stages and tissues was detected by fluorescence quantitative PCR.?2?The results showed that DLO2 and S5H were highly expressed in senescent leaves,but low in non senescent leaves,and the trend was basically the same;in different tissues,DLO2 and S5H genes were the highest in stems,followed by flowers,and the lowest in silique.In addition,the expression of DLO2 gene in different periods and tissues was significantly lower than that of S5H.Under the conditions of exogenous SA treatment,pathogen Pst DC3000 infection,cold stress and so on,the expression of S5H was up-regulated to a certain extent,but DLO2 and S5H genes were different,and there was no significant change before and after treatment.The results showed that the expression of S5H was induced by SA concentration,while DLO2 was not induced by SA concentration.?3?Phenotype and metabolism of dlo2 mutants in different background.However,in the background of Col-0 and s3hs5h mutants,the phenotype did not change significantly.In the background of s3hs5h double mutant,DLO2 gene was overexpressed,and the diameter of rosette leaf was significantly larger than that of s3hs5h double mutant,while SA content was lower than that of s3hs5h double mutant.?4?Preliminary analysis of phenotype and metabolism of Arabidopsis thaliana with low SA.Compared with wild type Col-0,the diameter of rosette leaf of 35S-S3H and S5Hpro-S3H super expressed transgenic plants increased significantly.SA detection showed that the content of free SA and total SA in transgenic plants decreased significantly.Compared with NahG transgenic plants,there was no significant change in the diameter of rosette leaf and SA in 35S-S3H transgenic plants,while the diameter of rosette leaf increased and SA content decreased significantly in S5Hpro-S3H transgenic plants compared with NahG transgenic plants,in which the diameter of rosette leaf in single copy plants increased by about 10%,but free SA and total SA in single copy plants decreased slightly.?5?Senescence phenotype analysis of single copy genetic material with low SA content.S5Hpro-S3H showed a late senescence phenotype,and its chlorophyll content and Fv/Fm were measured.The results showed that the chlorophyll content of S5Hpro-S3H was higher than that of Col-0,and there was no significant difference between S5Hpro-S3H and NahG.?6?Susceptible phenotype analysis of mutants with low SA content in single copy.NahG and S5Hpro-S3H are more susceptible than Col-0.The number of spores of NahG and S5Hpro-S3H is not significantly different from Col-0,but they are significantly higher than Col-0.In addition,the free SA content of S5Hpro-S3H single copy transgenic material was about 40-50%lower than that of NahG.In a word,DLO2 has the ability of catalyzing SA to produce 2,5-DHBA.The double mutant of s5hdlo2 has the obvious phenotype of lotus leaves becoming smaller,while the overexpression of DLO2 in s3hs5h also recovers,which indicates that DLO2 has certain biological functions.In addition,the genetic material with low SA content created by plant endogenous gene S3H is a good genetic material for SA research,which can be used in the field of SA metabolism.