Screening And Cloning Of The Mutants Of Salicylic Acid Metabolism And Characterization Of The Function Of A Homologous Gene Of S5H In Arabidopsis

In plants,salicylic acid(SA)acts as an important endogenous hormone to regulate plant disease resistance,stress reponses,growth,and senescnece.The metabolic mechanism is one of the important contents in SA study.In Arabidopsis,SA synthetic and catabolic pathways have not yet been well elucidated.In order to further reveal the molecular mechanism and physiological function of SA metabolism,the s3h*s5h double mutant of Arabidopsis that show dwarf phenotype caused by SA accumulation was mutated by EMS mutagenesis to screen the phenotype-recovery mutants and further were used to clone genes related to SA metabolism.On the other hand,the physiological function of a candidate gene of SA-hydroxylase,At4g10490(DLO2),was studied through bioinformatics,molecular biology,biochemistry,and genetic approaches.The major results are as follows:(1)SA-related mutants were screened using a phenotype-dependent approach.The Arabidopsis double mutant Col-0 s3h*s5h was mutated by EMS,and the phenotypic restoration mutants were screened to establish a phenotypic restoration mutant library.The rosette leaf diameter and SA levels of the selected mutants were counted.In this study,20 mutants related to SA metabolism and leaf senescence have been screened and divided into four categories according to their phenotypes and SA levels:?.The phenotype of the first class of mutants showed larger leaves,delayed leaf senesence,and decreased SA content compared to the s3h*s5h double mutant;?.The phenotype of the second class of mutants showed larger leaf,earlier leaf senescence,similar SA content compared to the s3h*s5h double mutant.?.The phenotype of the third class of mutants showed larger leaf,and later attenuated,but the SA content did not change;?.The phenotype of the fourth class of mutants showed smaller leaves,later senescence,and decreased SA content.(2)The mutants were backcrossed with SA-accumulated Arabidopsis double mutant Col-0 s3h*s5h.F1 seeds were harvested and sowed.After the plants matured,F2 seeds were harvested and continued to be sown to obtain F2 plants.The phenotypic segregation ratio meets 3:1.Selected large F2 plants were used for DNA extraction for DNA re-sequencing.Two mutants have been sequenced in this work.(3)We used the CRISPR/Cas9 technology to create the double mutant s3h'*s5h of the Ws ecotype.The Ws s3h*s5h were crossed with the individual mutants for further map-based cloning.The F2 seeds were harvested and continued to be sowed to obtain F2 plants with a phenotypic segregation ratio of 3:1.A large F2 plant was selected to extract DNA for map-based cloning,and the position of the gene was determined using the diversity of single nucleotide sequences between different ecotypes.(4)The F1 hybrids were obtained using the mutant crossing within 6 Class ? mutants and their phenotypes were observed.F1 hybrids were randomly selected for the analysis of SA metabolism and phenotype.It has been demonstrated that the mutations among the five mutants 108008,109004,113016,116003,24-3 are different.(5)We used bioinformatics methods and public database to analyze the predicted gene expression patterns,and initially obtain the candidate gene At4g10490(DL02)potentially associated with SA hydroxylation.This gene is closely related to the published hydroxylated gene S3H in Arabidopsis.(6)The recombinant protein of DL02 was purified and the enzyme reaction assay was performed using the SA as substrate.The high performance liquid chromatography(HPLC)were used to determine the enzyme reaction product.The results showed that the protein encoded by DL02 gene can catalyze the hydroxylation of salicylic acid to 2,5-DHBA in vitro.(7)The mutants s3h*s5h*dlo2 and s5h*dlo2 were obtained using CRISPR/Cas9 technology.Their leaf phenotype and metabolism were analyzed.In this study,the DL02 gene was knocked out in mutants s3h*s5h and s5h,and the leaf phenotype and metabolism did not change significantly,indicating that the function of DL02 gene in leaf senescence was not obvious.(8)Using Arabidopsis transgenic technology,the pMDC32-DLO2 vector was successfully transferred into SA-accumulated double mutant s3h*s5h to get complementary plants and their leaf phenotype and metabolism were analyzed.The results showed that the phenotype became larger but did not restore WT levels,and its salicylic acid metabolism level had returned to WT metabolism levels.It was demonstrated that DL02 does indeed have the function of hydroxylation of SA in Arabidopsis and has physiological function in plant.The above results indicate we successfully set up an EMS mutant screening system to generate SA related mutants using the Arabidopsis double mutant s3h*s5h;We functionally analysized the protein encoded by At4g 10490 both in vitro and in vivo and proved it is a functional SA 5-hydroxylase.