Characteristic Analysis Of WHSP90 And Functional Identification Of W17 Promoter In Wheat

Plants have evolved defense mechanisms to perceive signals from their surroundings and to appropriately respond to different stresses by modulating the expression of responsive genes. Heat shock protein HSP90 and ERF transcription factors play a central role in abiotic and biotic stress signal transduction to regulate defense gene expression, as well as in improving crop stress resistance. The aim of present study is to investigate the characteristics of the WHSP90 gene and W17 promoter in wheat. This study presents the relational theoretical basis for further exploring stress signal transductions to regulate defense gene expression and improving wheat resistance.1. Analysis of expression pattern: The results of Q-RT-PCR indicated that WHSP90 could be induced by high temperature, drought, high salinity, low temperature, ET, MeJA, ABA and SA. However, the expression pattern was different, WHSP90 respond highly to ET, high temperature and drought. WHSP90 might play different role in different signaling pathway.2. Subcellular localization assay: WHSP90 were fused to the 5′terminus of the coding region of green fluorescence protein (GFP) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Then the fused plasmids were introduced into onion epidermal cells. Transformed cells were incubated for 24 h in the dark and localization of the fusion proteins was then detected using a confocal microscope equipped with appropriate filter. The subcellular localization assay indicated that WHSP90 localized into the cytoplasm and nuclei, suggestings that WHSP90 functions in the cytoplasm and nuclei.3. Prokaryotic expression and polyclonal antibody preparation: WHSP90 gene was inserted into the pET-28a (+) vector and pGEX-4T-1 to get fusion vector His-WHSP90 and GST-WHSP90. The most high expression quantity was induced. The protein of His-WHSP90 was purified using HisTrapTMHP. The purified protein was injected into a rabbit. And the titer of the rabbit's anti-serum was measured by indirect ELISA. The rabbit's antiserum with high titer (﹥125000) was obtained. The polyclonal antibodies can be used for further investigation.4. Acquirement of transgenic Arabidopsis plants: WHSP90 was ligated into the pBI121 vector under control of the 35S promoter. Columbia (Col-0) ecotype Arabidopsis plants were transformed using the vacuum infiltration method. Transgenic Arabidopsis plants of 8 independent lines were used for further analysis.5. Functional Identification of W17 Promoter: By I-PCR, 1859 bp promoter sequence of W17 gene was gained. Transient expression carriers with GUS reporter controlled by different promoter fragments of W17 gene were constructed, and then transformed into the mature embryo calli of wheat by particle bombardment, respectively. The calli treated by drought, ABA, high salinity, SA, GA, MeJA, low temperature and high temperature were given transient expression at the different degree, which explained that this promoter was inducibled by stress, and it contained some stress-responsive elements involved in drought, ABA, high salinity, SA, GA, MeJA, low temperature and high temperature. The expressive level of GUS gene was controlled by the quantity of stress-responsive elements.