Homozygous Identification And Cell Structure Analysis Of Arabidopsis Mutant Athspr As Well As Antibody Preparation Of AtHSPR Protein

An Arabidopsis pletiotropic mutant Athspr was isolated using promoter trapping mediated by T-DNA in our laboratory. The T-DNA insertion site has been determined in the previous study, and the gene related with mutant encordes a new protein associated with heat shock protein by bioinformatics analysis, which has been named Athspr (heat shock protein-related in Arabidopsis thaliana). Athspr mutant showed a dwarf pheno type with curled leaves, shorter petiole and hypocotyl, shorter and thicker siliques, dark green and delayed reproductive growth stage, compared to wild-type. In the present study, the homozygous of Athspr mutant was identified by PCR. Meanwile, the cell structure of leaves, stems, hypocotyls, cotyledons from the mutant Athspr was analysed. Moreover, the polyclonal antibody of AtHSPR was prepared using the specific sequence of Athspr gene.The main research results are as follows:1. The homogeneity of mutant Athspr was determined by PCR, and it was a homozygote.17bp was missed and5bp was mismatched in the RB-side of T-DNA. However, the genome sequence was intact.2. The observation and statistical results from leaf cell sections showed that:(1) The mesophyll cells of midvein, palisade and palisade tissue in mutant Athspr were decreased in size than those in the wild-type, while the epidermic cells showed the same trend;(2) The size and numbers of xylem cells in mutant Athspr were also reduced, compared to wide-type;(3)The chloroplast in mesophyll cell of the mutant Athspr was more than that wide-type;(3) Thylakoid membranes and grana stacks of the chloroplasts in the mutant Athspr was slightly poor developed relative to wild-type the wild-type by the observation of ultrastructure.3. Compared to wild-type, the images of semithin section and SEM from mutant Athspr showed that:(1) The Aspect ratio of S-Cells in stems was decreased;(2) The size of hypocotyl cells and epidermal cells were reduced and the length hypocotyl cells was shortened.4.957bp specific sequences in Athspr gene was used to construct prokaryotic expression vector, pET-30a-At957by bioinformatics analysis. The recombinant protein At957-His6with high purity was gained through IPTG inducing, affinity chromatography and dialysis.5. Polyclonal antibody for AtHSPR was obtained by rabbits which were immunized with the recombinant protein At957-His6. The antibody was purified using the CNBr-activated SepharoseTM4B and can sepecifically combined with AtHSPR protein in Athspr overexpressed Arabidopsis plants.In conclusion, T-DNA insertion indeed caused alteration in function of the protein AtHSPR in the mutant Athspr, and led to the many changes in the cellular architecture and phenotype in the mutant.These data all indicated that Athspr may play a pivotal role in growth and development as well as tolerance of adverse stress.