Isolation And Fonctional Anaytsls Of A Cytosolic Class Ⅰ Small Heat Shock Protein Gene ZmHSP16.9in Zea Mays

Abiotic stresses such as high temperature, low temperature and drought, influence plantgrowth and development. These stress signals can stimulate plant which can produce a seriesof responsive protein to protect the cell metabolism. Heat Shock Protein (HSP) is one kind ofimportant stress-induced proteins, producing when the plants are in response to externalstresses. Under stress, increasing expression can strengthen the ability of the plants to resistvarious stress factors, which plays a very important role in the long-term co-evolution processof plants and the environment, and has an important significance in taking it as a study ofstress physiology. The small HSP (sHSP) is a kind of heat shock protein family that range insizes from approximately15to30kDa. Many studies in vivo and in vitro indicate that sHSPact as molecular chaperones in cells. As molecular chaperones, sHSP can bind to and facilitatethe synthesizing polypeptides to correct folding; leading new-synthesized polypeptides totransverse the organelle membranes and locating them in different position. Under hightemperature stress, sHSP can prevent the heat-denatured proteins assembling, preventunreversible proteins denaturalization, or be propitious to the renaturation of heat-denaturedproteins. They play very important roles in plant's thermotolerance.In the present study, we isolated and characterized a novel sHSP gene, ZmHSP16.9, frommaize leaves under heat stress. Sequence comparison, expression analysis and furtherfunctional studies on the over-expressing ZmHSP16.9plants were analyzed in this work.These data have the important theoretical and practical significance in further understandingthe functions of sHSP and using genetic engineering to improve corn comprehensiveresistance. The main results are as follows:(1) Molecular cloning and bioinformatics analysis of ZmHSP16.9geneTotal RNA was isolated from maize leaves under heat stress, we isolated the sHSP genesZmHSP16.9by RT-PCR, and the ZmHSP16.9encodes a protein of152amino acids.Comparing the gene with other small heat shock protein homologous sequence alignment, the results showed that the gene and the cytoplasmic class I sHSPs family clustered together.From the amino acid sequence, we can draw the conclusion that the gene with other plantcytosolic class I sHSPs is highly homologous. The amino acid sequence of ZmHSP16.9wasanalyzed in the ProtComp v.9.0database(http://linux1.softberry.com/berry.phtmL?topic=protcomppl&group=programs&subgroup=proloc). ZmHSP16.9was predicted to be localized in the cytosolic. The fusion protein pBI121-ZmHSP16.9-GFP was expressed transiently in onion epidermis, and the GFP greenfluorescence could be detected in the cytoplasm with the fluorescence microscope. The resultsclearly indicate that ZmHSP16.9is mainly localized in the cytoplasm. From these data, wespeculate that the gene belongs to cytosolic class I sHSP genes.(2) Tissue specificity and expression analysis of ZmHSP16.9in Zea maysThe young seedlings of maize were treated by drought, high temperature, lowtemperature, H2O2and ABA, and total RNAs were extracted and Northern blot was carriedout. The results showed that maize leaves under normal growth condition could not detectZmHSP16.9gene transcripts, but ZmHSP16.9mRNA were expressed in the leaves, stems androots of maize under heat-shock treatment, and the level in the leaves was highest. Hightemperature and H2O2treatments led to a significant increase of ZmHSP16.9transcript level.These results suggest that the ZmHSP16.9gene is involved in response to heat temperatureand oxidative stresses.(3) Recombinant of prokaryotic expression vector pET-ZmHSP16.9, antibody productionand Western blotting analysisA recombinant of prokaryotic expression vector pET-ZmHSP16.9was constructed andexpressed in E.Coli. BL21. The strong induced fusion protein was purified and used toimmunize white mice to obtain antiserum. Western hybridization revealed ZmHSP16.9inleaves of maize seeding was induced by heat temperature and oxidative stresses.(4) Identification of the transgenic tobaccoThe coding region of ZmHSP16.9was introduced into the vector pBI121under thecontrol of the CaMV35S promoter and then transformed into WT tobacco (NC89). Thetransgenic lines showed expression of ZmHSP16.9at both mRNA and protein levels byNorthern blot and Western blot. (5) The over-expression of ZmHSP16.9increased seed germination and seeding growthof tobacco under high temperatureCompared to WT plants, the over-expressing ZmHSP16.9transgenic tobacco showed asignificantly higher germination rate, and the seedling growth was better than WT plantsunder high temperature stress.(6) Changes in physiological parameters of tobacco under high temperature stressTo investigate the effect of ZmHSP16.9over-expression in tobacco on the physiologicalresponses to stress, a panel of physiological parameters, including the accumulation of H2O2,O2, antioxidant enzyme activities, antioxidants content, Malondialdehyde (MDA) contentsand relative electrolytic leakage (REL) were examined. All these physiological parametersreflect normally plant responses or tolerance to stress environment, thus serving as the plantphysiological index under stress. As shown in the result, heat stress induced the accumulationof H2O2, O2in the WT and transgenic tobacco, but this accumulation was more in WT.Through maintaining relatively higher antioxidant enzyme activities and antioxidants content,ZmHSP16.9decreased the accumulation of ROS to reduce the damage to cells. After hightemperature treatment, compared with WT plants, the MDA content was significantly lowerin ZmHSP16.9over-expressing plants. Transgenic plants had a lower electrolyte leakage thanWT under high temperature. These results indicate over-expressing ZmHSP16.9tobaccocould enhance the tolerance to the high temperature compared with WT plants.(7) ZmHSP16.9over-expressing tobacco improved tolerance to oxidative stressUnder oxidative stress, ZmHSP16.9over-expressing tobacco showed a significantlyhigher germination rate and the seedling growth was better than WT plants. H2O2in differentconcentration was used to estimate the effects of the oxidative stress on the growth of the WTand transgenic tobacco plants. ZmHSP16.9over-expressing plants showed higher chlorophyllcontent than WT plants under both high and low H2O2concentrations. These data suggest theadvantages of anti-oxidative characterization in the ZmHSP16.9over-expressing tobaccolines.