Map-based Cloning And Preliminary Functional Analysis Of Genes Regulating The Expression Of Heat Shock Proteins In Arabidopsis

Plants are frequentely subjected to heat stress during their growth and developmet.Recently,global warming is generating rapid changes in temperature that seriously affect crop production worldwide.To cope with heat stress,plants synthesize heat shock proteins(HSPs),which are under the control of heat stress transcription factors(HSFs).Howere,many of the key factors mediating the heat response pathways remain unkown.In order to indentify regulators involed in heat stress,we established a forward genetic screening method for mutants with altered HSP expressions,a firefly luciferase reporter gene driven by the HSP18.2 was introduced into Arabidopsis thaliana plants in glabrousl plants(Col-0 background).Seeds from one homozygous line harboring a single copy of HSP18.2:LUC transgene were used as the wild type(P-LUC),and mutagenized with EMS(ethyl methanesulfonate).Then the mutants with altered luminescence expression under heat stress treatment were screened by using a hypersensitive CCD camera.The HL111 mutant with elevated luciferase expression comparing with wild type,and LL807 mutant declined luciferase were obtained in our screening and further studied in this thesis.HL111 mutant displayed the significantly elevated luciferase expression comparing with wild type under the treatment with 38? for 1 hour.In addition,HL111 is hypersensitive to heat stress.Under normal temperature(22?-23?),the growth and development of HL111 are retarded and the plant size reduces.To isolate the HL111 gene,a map-based cloning strategy was used.The mutation was narrowed down to between 11.3M to 11.47M in Chromosome 4.After next-generation sequencing and PCR sequencing,a nucleotide change of G1861A was found in candidate HL111 gene,which caused an early termination of HL111 translation.The bioinformatics analysis showed that HL111 belongs to WD40 repeat-like superfamily and it distributes in both the cytoplasm and nucleus.A T-DNA insertion line of HL111 gene identified from TAIR stocks were crossed with HL111,the seeding of filial generation displayed the significantly elevated luciferase expression,and the T-DNA insertion is also more sensitive to heat stress than the wild type(Col-0),which supports the conclusion that mutation of HL111 gene is indeed responsible for the HL111 phenotype under heat stress.HL111 is expressed in roots,stems,leaves,flowers and siliques,and is the most highly expressed in flowers.In addition,the expression of HL111 is suppressed by high temperature.HL111 can active the expression of gene in yeast,so it may be a transcription factor.In addition,we examined the sudcellular localization of HL111-GFP fusion protein and found that it was entirely localized in the nucleus in wild tobacco.LL807 mutant plants displayed the significantly declined luciferase expression compared with wild type plants under the treatment with 40? for 1 hour.In addition,LL807 is sensitive to heat and salt stresses.The petiole of LL807 mutant is shorter and the length-widenth ratio of the blade is lower than those of wild type respectively,the plant size is also smaller.To isolate the LL807 gene,a map-based cloning strategy was also used.The mutation was narrowed down to between 27.25M to 30.28M in Chromosome 1.After next-generation sequencing and PCR sequencing,five candidate genes within the region were found.However,more works need to be done for determining the identity of LL807.Altogether,we screened two mutants HL111 and LL807 which are hypersensitive to heat stress.HL111 is a repressor,while LL807 is a positive regulator of HSP genes.Both proteins are probably important factors in heat stress-response,and can help us to understand the mechanisms about thermotolerance in plants.