Repressed Expression Of LeHSP84 In Tomatoes

HSP90(heat shock protein 90) is a molecular chaperone and is one of the most abundant proteins in unstressed cells. It is a ubiquitous molecular chaperone found in eubacteria and all branches of eukarya, but it is apparently absent in archaea. Whereas cytoplasmic HSP90 is essential for viability under all conditions in eukaryotes, the bacterial homologue HTPG is dispensable under non-heat stress conditions. In vivo HSP90 is a dimeric protein constituted by three regions: the N-terminal domain which binds the ATP, the central domain involved in the ATPase activity and in the recognition of client proteins and the C-terminal domain responsible for the dimerisation and binding of several co-chaperon. HSP90 has no activity before its dimerisation.HSP90 plays important roles in animal cells, but the function of HSP90 in plant cells has been less researched. In the Arabidopsis thaliana, this family includes 7 members. The AtHSP90-1 through AtHSP90-4 proteins constitute the cytoplasmic family, whereas the AtHSP90-5,AtHSP90-6 and AtHSP90-7 proteins are predicted to be within the chloroplastidial, mitochondrial and endoplasmic reticulum compartments. Recentaly study shows HSP90 chaperone complex is important in plant development and responsiveness to external stimuli. In particular, HSP90 is crucial for R-protein-mediated defense against pathogens.A cDNA of 2588bp encoding a full-length LeHSP84 had been cloned from tomato, which is a chromogene. The result of sequence analysis showed that the HSP84, locating in cytoplasm, was a member of HSP90 family. In this study, an antisense LeHSP84 cDNA fragment was introduced into tomato by Agrobacterium-mediated transformation. The results showed that the resistant ability to heat and salt stress of tansgenic plants was similar to that of control plants. The results were shown as follows:1) The generation of tomatoes with repressed LeHSP84: The result of PCR and Southern blot showed that the tomato plants transformed the antisense LeHSP84 cDNA fragment had been amplified DNA segments with the NPTII primers, while the control plants haven't, which indicate that the antisense LeHSP84 cDNA fragment had been transformed into the tomatoes. The Northern blot showed that the expression of LeHSP84 in transformed tomato was lower than that in the control plants. It indicated that the LeHSP84 gene in transformed tomato plants had been in repressed expression.2) Thermotolerance of the transgenic plants: The control plants and the transgenic plants were exposed to high temperature of 40℃,42℃,44℃and 46℃for 2h, and then were transferred to normal conditions for recovery for 4 weeks. The results showed that during the recovery phase the growth state of the transgenic plants had no obvious difference from the controls. But after 46℃, The results of Fv/Fm decrease slower in transgenic plants than the controls.3)Salt stress of transgenic tomato: The transgenic plants and controls were treated by 200 mmol/L NaCl. The plant growth was obviously repressed after salt treatment for 20 days, and partial leaves scorched. The growth state of the transgenic plants had no obvious difference from the controls. A set of parameters(relative water content, Na+ , K+ content, proline content, MDA content, SOD enzyme activity)were measured in transgenic plants and the controls after the treatment. The result was that these parameters had no obvious difference between the transgenic plants and the controls.