Map-based Cloning And Preliminary Functional Analysis Of SAPH2,a Gene Involved In Regulation Of Heat Shock Proteins In Arabidopsis

Along with the global climate warming,the emergence of extreme high temperature weather is more and more frequent.Plants have evolved a variety of regulatory mechanisms to deal with the high temperature stress,such as heat shock response.The plant exposed to high temperature will produce heat shock response,including the induction of heat shock proteins(HSPs).It is of importance to study the mechanism of heat shock response because not only is it the basis of understanding the thermo tolerance difference among different plants,also can provide scientific basis and practical experiences for the breeding high temperature resistant transgenic crops.Although many plant biologists tried their best to probe the mechanisms on heat shock response and thermotolerance of plants,at present how HSPs are transcriptionally regulated is unclear yet.In order to indentify the regulators involved in regulating the expression of HSPs,our lab established a forward screening method for mutants with altered expression of HSP18.2.In brief,an chimeric gene of HSP18.2 promoter and luciferase gene(LUC)was introduced into Arabidopsis thaliana through flower-dipping method to obtain the transgenic plant(designated P-LUC).Then the P-LUC seeds were mutagenized by EMS and the mutants with altered LUC activity compared with the wild type(P-LUC)under high temperature stress were screened using a hypersensitive CCD camera.The mutant hl761,the object of this research,is one of mutants with higher brightness than the wild type obtained through the above method.Preliminary genetic and phenotypic analyses showed that hl761 is a recessive mutant with higher LUC chemiluminescence level than the wild type when treated with 38?temperature.To isolate the hl761 gene,a map-based cloning strategy was used.The mutation was finally narrowed down to between 5.7M to 6.1M in Chromosome 1.Through genomic sequencing of hl761 combined with specific PCR product sequencing,a nucleotide change of G656A was found in candidate hl761 gene,which caused a change from Gly to Glu in the coded peptide.This altered amino acid is located in the G-patch structure domain of the protein.This gene does not have an intron and encodes a spliceosome-associated protein(temporarily designated Spliceosome-Associated Protein 2,SAPH2).The mutation of SAPH2 in hl761 may affect the function of the protein.Further phenotype analysis of mutant hl761 showed that hl761 was high temperature stress sensitive.When treated under 38? for 18 h,mutant hl761 died whereas the wild-type P-LUC could survive the stress and restored normal growth after transferred to normal temperature condition(22?).Additionally hl761 also showed increased sensitivity to cold stress and the leaf morphology also alters under normal growth conditions.In order to verify the result of map-based cloning,we tried two types of complementary analyses of the hl761.(1)A T-DNA insertion line of hl761 gene identified from TAIR stocks was crossed with hl761 to check the LUC activity of the filial generation and the result showed that F1 seedlings displayed the significantly elevated luciferase expression compared with the P-LUC,consistent with the phenotype of hl761.(2)The wild type SAPH2 gene was transferred into hl761 to check the phenotypes of the transgenic plants,and the results showed that the transgenic plants displayed the similar luciferase expression and thermotolerance to P-LUC.These results demonstrated that the phenotype of mutant hl761 is indeed resulted from the mutation of SAPH2.The expression analysis of SAPH2 show that SAPH2 was expressed in root,stem,leaf,flower and silique,and the highest expression was in flowers.Furthermore,the high temperature suppresses the expression of SAPH2 slightly but significantly.Comparative transcription analysis between wild type P-LUC and mutant hl761 showed that the transcriptional levels of most HSFs and HSPs in mutant hl761 under heat stress were significantly lower than wild type.These results could partially explain the high temperature sensitivity of hl761 and also hinted an important role of SAPH2 in heat shock response and thermotolerance in plants.Whether SAPH2 mutation in hl761 causes a pre-mRNA splicing anomalies remains to be further studied.The present results provide the clues for further probing the function and mechanism of SAPH2 in regulation of heat shock gene expression and the heat-resistance in plants.