Cloning And Functional Analysis Of Stress-related Genes Of Gm/TaAREB Transcription Factors And Promoters In Soybean And Wheat

Abiotic stresses such as drought, high-salt and low temperature severely affect the yield and quality of crops all over the world. Plants can perceive different stress signals from their surroundings, transmit the signals and regulate expression of related genes to respond to adversity. It was reported that transcription factors played vital roles in stress signal transduction, downstream gene expression regulation and improvement of anti-adversity in plant (Liu et al., 2000).Alkalescence leucine zipper proteins (basic leucine zipper, bZIP) are divided into ten subfamilies: A, B, C, D, E, F, G, H, I and S (Jakoby et al., 2002). At present, the studies on the bZIP transcription factors are mostly focused on A subfamily, which contains several important members such as ABF1, ABF2/AREB1, ABF3, ABF4/AREB2, and mainly participate in response to ABA, drought, high salt, heat and oxidation stresses. Furthermore, ABFs-overexpressoin remarkably enhances stress tolerance in transgenic Arabidopsis (Choi et al., 2000).Soybean (Glycine max) with strong salt tolerance and wheat with high drought tolerance were used as materials to isolate genes and promoters of adversity-relevant bZIP transcription factor and further study their functions. The idiographic experiment results are following:1. Gene isolation and sequence blast analysis: By using 5' and 3' RACE technique, a new adversity-relevant transcription factor were cloned from soybean and wheat, respectively, which were nominated as GmAREB (Glycine max ABA Responsive Element Binding Protein) and TaAREB (Triticum aestivum ABA Responsive Element Binding Protein). Homology trees analysis showed that GmAREB and TaAREB had higher homology with Arabidopsis ABF2 and rice OsTRAB1. Phylogenetic analysis indicated that both GmAREB and TaAREB genes had more close relative relationships with Arabidopsis ABF2 and OsTRAB1.2. Analysis of gene expression characteristic and copies: The expression of GmAREB and TaAREB was intensively induced by ABA, drought, high salt and low temperature. GmAREB had one copy in soybean genome while TaAREB had three copies in wheat genome. Tissue-specific expression pattern indicated that GmAREB and TaAREB were induced to express in root, stalk and leaf of soybean and wheat, respectively, and expression quantity in order from high to low was leaf, stalk and root.3. Analysis of gene localization: GmAREB and TaAREB proteins were localized inside nucleus in onion epidermal cells by constructing 163-GFP:GmAREB and 163-GFP:TaAREB fusion expression carriers and transforming by particle bombardment.4. Analysis of DNA-binding specificity and transactivation activity: Electrophoretic mobility shift assays (EMSA) indicated that GmAREB and TaAREB proteins could specially bind with normal ABRE cis-element while they were not able to bind with mutant ABRE cis-element. The above results proved that GmAREB and TaAREB proteins had DNA-binding specificity in vitro. The analysis of transactivation function showed that GmAREB and TaAREB genes had not activation activity in yeast. Further RT-PCR analysis of downstream genes in transgenic Arabidopsis that GmAREB and TaAREB could also activate some downstream stress-related genes expression and have transactivation function.5. Promoter isolation and analysis of transient expression: By TAIL-PCR and adaptor-PCR, part promoter sequence of TaAREB gene and full-length promoter sequence of GmAREB were gained and submitted into promoter database for element forecast analysis. The results showed that responsive elements to ABA, drought, low temperature and salt were discovered. Transient expression carriers with GUS reporter controlled by different promoter fragments of GmAREB gene were constructed, transformed into the mature embryo calli of wheat by particle bombardment, respectively, the calli treated by drought, ABA, salt and low temperature were given transient expression at the different degree, which explained that these promoter fragments contained some stress-responsive elements involved in drought, ABA, salt and low temperature. GUS fluorescence quantitative analysis indicated that there were a drought-responsive element in -400~-696bp section, a low-temperature responsive element, a ABA responsive element and a salt-responsive element in -696~-944bp section.6. Functional analysis of transgenic Arabidopsis for stress tolerance: Transgenic plants were treated under drought stress for 30 days, most wild-type plants were dead after resuming water and survival rate was only 5% (3/60) but that in 29A/35S::GmAREB transgenic plants were 58.3 % (35/60) and 50% (30/60), respectively, and in 29A/35S::TaAREB transgenic plants were 55% (33/60) and 51.7 % (31/60), respectively. Under salt stress treatment for 30 days, all of the wild type were also dead, but transgenic plants grew normally. Survival rates of 29A/35S::GmAREB transgenic plants reached to 53.3% (32/60) and 43.3% (26/60), respectively, and 29A/35S::TaAREB transgenic plants to 51.7% (31/60) and 45% (27/60) , respectively. After treated by low temperature stress, all of control plants were dead too, but part of transgenic plants could still resume growth. The survival rates for 29A/35S::GmAREB transgenic plants were 45% (27/60) and 41.7% (25/60), respectively, and for 29A/35S::TaAREB transgenic plants were 40% (24/60) and 36.7% (22/60), respectively. After oxidative stress, chlorophyl content measurement showed that the leaves of wild type were bleached and chlorophyll content was nearly zero, but part leaves of GmAREB/TaAREB transgenic plants still kept green. The chlorophyl contents in 29A/35S::GmAREB transgenic plants were 10.2 mg/g·FW and 7.3 mg/g·FW, respectively, while they in 29A/35S::TaAREB transgenic plants were 7.5 mg/g·FW and 12.6 mg/g·FW, respectively. The above results proved that GmAREB and TaAREB over-expression increased abiotic stress tolerance of transgenic Arabidopsis.7. Functional identification of transgenic tobacco for stress tolerance: GmAREB and TaAREB transgenic tobacco plants improved the tolerance to drought, salt and ABA on the medium containing 2%PEG, 200 mM NaCl and 100μM ABA by phenotype observation. The results on guard cell stomatic aperture showed that the stomata close speed in GmAREB and TaAREB transgenic plants as obviously quicker than that in wild-type plants (W38) and reduced moisture evaporation, played down transpiration and increased abiostic stress resistance.