Analysis Of Heat Resistance In Arabidopsis Mutant Athspr And Prokaryotic Expression Of Athspr Gene

A mutant with altered pleiotropic phenotype was isolated using Promoter-Trap Strategy. The functional alterations of the trapped gene caused by the T-DNA insertion made the plant typical of new phenotypes, such as growth retardation, curved leaf, shortened hypocotyls of the whole plant, and reduced stress resistance to harmful enviroment. Cloning and bioinformatics analysis of the related gene showed the novel gene belonged to the HSP100/C1pB family. And the gene was named Athspr (heat shock protein-related in Arabidopsis thaliana). In this study, resistance to heat stress between wild-type and mutant Athspr was compared. The fusion protein incoding by specific sequence of Athspr was expressed and purified by prokaryotic expression technology and Ni affinity chromatography. The main research results are as follows:1. The phenotypes of mutant and wild-type under different heat shock stress were compared. In the short-term heat shock stress, the plants did not show stable differences in heat resistance between wild-type and mutant Athspr. When reduced the preheating time (90min→10min) of the heat shock treatment, the mutant showed higher death rate. The Arabidopsis plants of both wild-type and mutant Athspr had different phenotypic changes under heat shock stress at 30℃. In the MS medium, both wild-type and Athspr mutant plants could not be survived; while in the soil culture, the mutant plants could survive, but not transfferd into reproductive stage. A lower germination rate of the mutant was observed after heat trearment, compared with wild-type;2. ATHSPR protein was extracted by SDS-PAGE gel electrophoresis. Various protein extraction methods were compared in the merits of protein isolation, and it was proved that ammonium sulfate precipitation method could extract and separate ATHSPR protein better. The expression of Athspr gene in the mutant was preliminary analyzed after the heat shock by SDS-PAGE gel electrophoresis. Expression level in the mutant was reduced compared to the wild-type;3. By analysis of bioinformatics,957 base squences at the third exon in Athspr gene was used as foreign gene to construct prokaryotic expression vector. The ATHSPR protein with specific sequence was expressed in the Trans Rosetta (DE3) prokaryotic cells. The protein with high purity had been gained using Ni Sepharose 6 Fast Flow gel, which laid a good foundation for the studying of expression pattern and function of ATHSPR protein.In conclusion, the functional alterations of Athspr gene trapped by the T-DNA insertion affected the heat resistance, and indicated that the Athspr gene may played an important role in the signal pathway under the normal and heat shock conditions.