Isolation and Functional Analysis of the bZIP Transcription Factor Gene TaABP1 from a Chinese Wheat Landrace

In plants, basic leucine zipper （bZIP） transcription factors play important roles in regulatory processes, including stress response, pathogenic defense and light response as well as organ and tissue differentiation. Chinese wheat landrace Pingyaoxiaobaimai （PYXBM）, an original parent of drought tolerant wheat varieties grown in northern China, is significantly tolerant to abiotic stresses such as drought, cold and nutrient deficiencies. In order to isolate key stress-responsive genes and then improve stress tolerances of conventional varieties, a bZIP transcription factor gene was isolated from a cDNA library of drought-treated PYXBM using the in situ plaque hybridization method, and was designated as Triticum aestivum L. abscisic acid （ABA）-responsive element binding protein 1 （TaABP1）. It encodes 372 amino acids, and contains three conserved domains （C1-C3） in the N terminal and a bZIP domain in the C terminal which is a typical protein structure for the group member of bZIP family. Transcriptional activation analysis showed that TaABP1 activated the expression of downstream reporter genes in yeast without ABA application. TaABP1 protein fused with green fluorescent protein （GFP） demonstrated that the localization of TaABP 1 protein is in the nucleus. Expression pattern assays indicated that TaABP1 was strongly induced by ABA, high salt, low temperature and drought, and its expression was stronger in stems and leaves than in the roots of wheat. Furthermore, overexpression of TaABP1 in tobacco showed significant improvement of drought tolerance. Data suggested that TaABP1 may be a good candidate gene for improving stress tolerance of wheat by genetic transformation and elucidation of the role of this gene will be useful for understanding the mechanism underlying drought tolerance of Chinese wheat landrace PYXBM.

Construction of Agropyrum intermedium 2Ai-2 Chromosome DNA Library and Cloning of Species-Specific DNA Sequences

The univalent from the meiosis-metaphase spreads of F1 (Z2× wheat variety Wan7107) wasidentified to be Agropyrum intermedium 2Ai-2 chromosome by GISH. The 2Ai-2 chromosomes weremicroisolated and collected. After two rounds of PCR amplification, the PCR products wereranged from 150-3 000 bp,with predominant fragments at about 200-2 000 bp. Using Ag.intermedium genomic DNA as a probe, Southern blotting analysis confirmed the products originatedfrom Ag. intermedium genome. The products were purified, ligated to pUC18 and then transformedinto competence E.coli DH5αto produce a 2Ai-2 chromosome DNA library. The microcloningexperiments produced approximately 5 ×105 clones, the size range of the cloned inserts was 200-1 500 bp, with an average of 580 bp. Using Ag.intermedium genomic DNA as a probe, dot blottingresults showed that 56% clones are unique/low copy sequences, 44% are repetitive sequences inthe library. Four Ag. intermedium clones were screened from the library by RFLP, and threeclones(Mag065, Mag088, Mag139)belong to low/single sequences, one clone(Mag104)was repetitivesequence, and GISH results indicated that Mag104 was Ag.intermedium species-specific repetitiveDNA sequence.

Transformation of Coat Protein Encoding Gene from Soil-Borne Mosaic Virus into Wheat

CWMV-CP1 target gene and bar selection gene were co-transferred into commercial wheat variety of Yangmai158 by particle bombardment. In total, 145 resistant plants to 3 -5 mg L-1 Bialaphos were obtained, 21 plants were identified to be positive in T0 generation by PCR-Southern test, and the transformation frequency had 0.99%. T1 plants were further tested by PCR and Southern hybridization. Results demonstrated that the alien resistance gene had been integrated into the wheat genome. The segregation ratio of CP1+ to CP1- in T1 generation was 1.0 to 1. 3, and didn't agree with Mendelian rule. RT-PCR result from T2 plants showed that the alien gene CWMV-CP1 had stable expression in wheat genetic background.