Map-based Cloning And Preliminary Functional Analysis Of A Negative Regulator SAPH Repressing The Heat Shock Proteins In Arabidopsis

Heat stress is usually defined as a condition in which temperatures are sufficiently high for enough time to irreversibly damage plant function or development.So plants use a variety of metabolic pathways and molecular mechanisms to resist heat stress.Nowadays,many factors associated with heat stress have been found in research,such as heat shock proteins(HSPs)and heat stress transcription factors(HSFs).However,many of the key factors mediating the heat response pathways remain unkown.To study the mechanism of inhibition of heat shock genes,a forward genetic screening method was established previously for mutants with altered HSP expressions.In the screening,transgenic plants containing a firefly luciferase reporter gene(LUC driven by the HSP18.2 was used as the wild type(P-LUC)and mutagenized with EMS.Then the mutants with altered luminescence expression under heat stress were screened by using a hypersensitive CCD camera.HL1401 is one the mutants with increased LUC expression under heat stress and studied in this thesis.HL1401 mutant displayed the significantly elevated luciferase expression compared with the wild type under 38? for 1 hour.In addition,HL1401 plants are more tolerance to heat stress than the wild type.Furthermore HL1401 seeds are more insensitive.to ABA treatment in germination.Under normal growth conditions,HL1401 plants were greatly reduced in size and flowered earlier than WTs.To identify the gene associated with the phenotypes observed in hl1401,map-based cloning was used for searching the mutational gene in HL1401 mutant and the mutation was narrowed down to between 11.47M to 11.54M in Chromosome 4.A mutation of G to A in the exon of a gene encoding a spliceosome-associated protein was detected in this region of HL1401.The gene was designated SAPH(Spliceosome-Associated Protein in Heat).This single-nucleotide change results in an amino acid substitution of Gly to Arg.The bioinformatics analysis showed that SAPH probably participates in pre-mRNA splicing and localizes in the nucleus.In TAIR website,the SAPH gene is predicted to have two isoforms of mRNA,one containing 15 exons,the other having 16 exons.Our experimental analysis showed that the latter isoform predicted by TAIR is not correct.The real isoform contains a mini-exon which exists in the fourth intron of the first type of splicing.Including of this mini-exon introduced a pre-mature stop codon TAA in the mRNA.Several T-DNA iso-alleles from TAIR also showed reduced size early flowering compared to the wild type plants.Furthermore these alleles were able to complement the LUC phenotype when crossed with HL1401.Hence the above phenotypes observed in HL1401 plants are indeed resulted from the mutation of SAPH.SAPH gene expresses in all tested tissues including roots,stems,leaves,flowers and siliques.Imaging of the GFP-SAPH fusion protein showed that SAPH is localized in the nucleus.The expression of SAPH was not induced or inhibited by high temperature treatment.However the ratio of two splicing isoforms were reallocated under heat shock conditions.In summary,SAPH protein was a negative regulator of heat tolerance and may play important roles in pre-mRNA splicing.Our results are helpful for understanding the molecular mechanisms of heat response and tolerance.