Functional Analysis Of Heat Shock Proteins And Large DNA Fragment From The Resurrection Plant Boea Hygrometrica

Boea hygrometrica is a resurrection plant that can survive dry and alkaline andcalcium-rich soil on Karst rocks. In order to identify novel genes and genetic elements thatimprove stress-tolerance, transgenic Arabidopsis plant populations of BIBAC genomic libraryand EST library from B. hygrometrica were screened under various stress conditions and thefunction of the identified genes were analyzed.From BIBAC library, two clones were found capable of enhancing stress tolerance intransgenic plants. Bh1-78can improve transgenic Arabidopsis the resistance of alkalinitystress, and Bh3-39can improve transgenic Arabidopsis the resistance of osmotic, calcium andalkaline stresses. Transgenic plants grew well under control condition, showing no obviousdifference from the wild type. Under stress conditions, in contrast to the poor growth and lowsurvival rates of the wildtype, transgenic plants exhibited better growth, longer primary rootsand higher survival rates under the stress. Besides, transgenic plants harboring L1-4and itssubclone S21also have the ability to adapt to soil drying. These data indicate the presence ofresistant genes or genetic elements in these clones.To characterize the genes encoding heat shock proteins (HSPs) identified from B.hygrometrica EST library, the gene expression pattern and the phenotypes of theoverexpression transgenic plants were examined under various stress conditions. The resultsshowed that the genes coding for small heat shock protein (BhsHSP)(Bh16.6CI and Bh35.9),HSP70(BhHSP70-1) and HSP40(BhDNAJC1and BhDNAJC9) were induced by CaCl2,NaCl, alkali, MV and ABA treatments. Besides, another HSP40gene BhDNAJC8was verylow under alkali treatment, but up-regulated in the other four treatments. The expression ofthese six genes increased expression over time under all tested conditions with the exceptionof MV treatment. The transgenic lines overexpressing these genes showed stress-resistancephenotypes under most of the tested stress conditions. These results suggest that BhHSPs mayplay protective roles in plant stress tolerance.In desiccated leaves of B. hygrometrica, an HSP-coding gene was cloned and designatedBhDNAJC2. Sequences analysis revealed that BhDNAJC2belonged to HSP40family and contained a J domain, which is likely a region for interaction with HSP70proteins. It represents a novel member of the family according to the sequence comparison with knownHSP40s and the structure of the phylogenetic tree. Real-time PCR data showed thatBhDNAJC2is induced by dehydration, rehydration, ABA, oxidative stress and cold stresses.Overexpression of BhDNAJC2in Arabidopsis confered plant tolerance to20%PEG,20μMABA, pH9.0,150mM NaCl,60mM CaCl2stresses, as indicated by the stable levels ofFV/FM and low relative electrolytic leakage value. Furthermore, confocal microscopicobservation showed that BhDNAJC2-YFP fusion protein was mainly visible in the cytoplasmand the nucleus. Taking together, our data have indicated a potential role of BhDNAJC2inmaintaining the stability of cytosol and nuclear proteins, in an HSP70dependent manner,under dehydration and rehydration process, saline-alkali stress and cold stresses.Together, our results have demonstrated large DNA transformation approach fordissection of genetic elements have a significant advantage.The HSP genes from EST librarymay involved in the protective reactions in B. hygrometrica under stress conditions. Both methods can beused for improving stress tolerance of crops.