Isdation And Characteirzation Of BZIP Transcription Factor, GmbZIP-32, From Soybean (Glycine Max L.)

Soybean is not only a70%source of edible protein in the world, but also anatural resource of vegetable protein, vitamins, minerals and fats et al. However, theadverse environmental factors, such as cold, drought, soil salinization and disease,seriously affect the growth and development of soybean, which bring terrible loss tothe economy. It was reported that bZIP transcription factors played vital roles in plantdefense responses and the processes of adversity stress responses. In this paper, onenew bZIP transcription factor, GmbZIP-32, was cloned from soybean Jilin32. Thestructure and function of GmbZIP-32was analyzed in order to provide theoreticalbases for application of bZIP in the genetic engineering breeding. The experimentresults are following:1. GmbZIP-32was cloned by RT-PCR in soybean Jilin32cDNA. The ORF ofGmbZIP-32is1542bp encoding a polypeptide of513amino acids. The predictedGmbZIP-32protein contained a typical structure of bZIP domain, including one basicdomain, one leucine zipper motif and one glutamine–rich domain. The amino acidsequence alignment and phylogenetic tree analysis indicated that GmbZIP-32wasrelated most closely to the GmbZIP11in soybean and grouped into Class I in bZIPtranscription factors subfamily, implying that they may be diversified from a commonancestor during evolution.2. The yeast effect plasmid pGBK7-GmbZIP-32was constructed, then it wastransformed into the yeast strain AH109. The positive transformants were selectedusing nutrient-deficient medium SD/-Trp/-Ade/-His. The result indicated thatGmbZIP-32had no transcriptional activation ability in yeast.3. The pET28-GmbZIP-32plasmid was constructed, then it was transformed intoE. coli Rosetta (DE3). The recombinant protein of GmbZIP-32was induced in37°Cfor6hours by1.0mM IPTG.4. The expression pattern of GmbZIP-32was analyzed using quantitative real-time PCR. The results indicated that GmbZIP-32had no tissue-specificexpression characteristics which expressed in roots, stems, leaves, flowers andimmature embryos. But its expression was higher in leaves and flowers than in othertissues. GmbZIP-32responded differently to biotic and abiotic stresses such as salt,drought, cold, wounding, ABA, SA, ET and MeJA.5. The coding region of GmbZIP-32was cloned into pCB35SR1R2-GFP. Theconstructed plasmid was introduced into Arabidopsis using Agrobacterium-mediatedtransformation following the floral dip method. The seed germination rate ofGmbZIP-32transgenic Arabidopsis was significantly higher than that of wild typeArabidopsis under the condition of drought and high salt stress; GmbZIP-32transgenic Arabidopsis grew much healthier than wild-type Arabidopsis removingwater for18d. The water loss rates of detached leaves of GmbZIP-32transgenicArabidopsis were lower than that of wild type Arabidopsis. These data indicated thattransgenic Arabidopsis plants constitutively expressing GmbZIP-32showed anincreased tolerance to drought and salt compared to wild-type plants.6. GmbZIP-32transgenic Arabidopsis plants maintained lower level ofmalondialdehyde and higher levels of soluble carbohydrates compared with the levelsin wild-type Arabidopsis plants under the condition of simulated drought and high saltstress. These data indicated that GmbZIP-32improved the resistance of transgenicArabidopsis plants by reducing the accumulation of lipid peroxide decompositionfinal product and increasing the accumulation of osmotic regulation substances.