Cloning And Analysis Of Elements Of Vernalization-related Genes And Studies On A Wheat Dwarf Mutant

I. Cloning and analysis of the full-cDNA and promoter sequences of vernalization-related genesThe full-length cDNA sequence of VER2 gene, 1195 bp, was screened out from wheat (Triticum aestivum. L. cv Jingdong No.l) vernalized apex cDNA library. The ORF of VER2 gene encodes 300 AA. Analysis of the VER2 protein structure showed that VER2 has plant disease_resp (disease-resistance response protein) and Jacalin (jasmonate-induced lectin) domains, suggesting that stress response signalings are involved in vernalization. There are a nuclear-localized signal peptide and many kinds of phosphorylation sites in the VER2 protein.TaSPY, M21 (TaRan) and M10 had all obtained. The TaSPY consists of 11 tetratricopeptide (TPRs) and the C-terminal half is likely to be the catalytic domain. The genes TaRan and M10 encode two kinds of small GTP-binding proteins respectively.Using an improved pool-PCR and high-density hybridization screening approachs, a TAC clone with 41.7 kb insert containing VER2 gene was isolated from a transformation-competent artificial chromosome (TAC) library of wheat (Triticum aestivum-Haynaldia villosa). Sequence determination and analysis showed that 11 genes were involved in the TAC. Four exons of gene 3 were completely corresponding to the cDNA sequence of VER2 gene. Genomic sequence of VER2 has three introns: 292 bp (+361~+652), 149 bp (+843~-+991), and 121 bp (+1197~+1317). Analysis of the transcription initiation site (TATA-box in -30 upstream of 5'-franking sequence region) and the polyA signals site (AATAAA) showed that the previously cloned cDNA sequence of gene VER2 was full-sequence.The upstream sequence from transcription start site of VER2 displayed two kinds of repeat regions: large-fragment repeat sequence (L-repeat, 2161 bp) and small-fragment repeat sequence (S-repeat, 482 bp). The response elements were involved in the promoter region, such as abscisic acid response elements (ABRE), MeJA-response elements (CGTCA-motif, TGACG-motif), endosperm expression elements, MYB binding sites, similar elements to GA response element (GARE), and so on. To further investigate the function of the promoter, A 6 kb promter fragment was deleted and led to 10 fragments. Their expression vectors were constructed for transient expression or transgenic plant with GFP and GUS report genes, respectively. The largest fragment (full region of the promoter, -5895 to +73) driving GFP (pGFP201-5990) was bombarded into vernalized or non-vernalized immature wheat leaves. The vernalized immature leaves showed bright green fluorescence after incubation for 24 hours,however no green fluorescence was observed in non-vernaliztion leaves under the same conditions. Suggested that vernalization was essential for the function of VER2 promoter in the immature leaves of winter wheat. The sequence which was an vernalization induced promoter has not been reported yet.II. Studies on a wheat dwarf mutantA wheat dwarf mutant line was obtained in 1997 in our lab. The dwarf wheat was identified as a GA_insensitive dwarf mutant (gaid) for its semi-dominant blocking GA signaling pathway. Physiological analysis showed that the gaid mutant is associated with hypersentivity to high [ABA]. The germination and growth of gaid mutant was completely inhibited at the [ABA] of 10-6 M, whereas the same inhibitive effect on the wild type was achieved at the [ABA] of 10-5 M. The roots of gaid mutant grew curly and short in solidified medium (low oxygen) with high-irradiance white light (63.5 Es-1m-2). However, the abnormal phenotypes of gaid roots were weaker or dispeared in low-irradiance white light or along the surface of culture media. The curly and short growth of gaid roots can be overcome by low concentration (10-8M) of ABA. However, application of IAA, TIBA (an auxin transport inhibitor), ACC or AOA (ethylene biosynthesis inhibitor) was unable to recover the phenotype similar to the root growth in wild type plants.Based on effect of ethylene on plant growth, we proposed that GA may regulate plant growth by two different pathwa...