| Glycine-rich proteins (GRPs) of plants are widely spread in a variety of monocotyledons and dicotyledons, implying their diverse and essential roles. Study of gene expression regulation of GRPs by promoter analysis can help to unravel their possible biological functions. A rice GRP gene, Osgrp-2, has been previously isolated and sequenced (GenBank U40708). Its expression is virus-inducible as well as regulated developmentally and induced by mechanical injury. The 5' deletion analysis of the Osgrp-2 promoter in tobacco transient expression systems revealed the presence of a positive regulatory region (-2290 to -1406) and two negative regulatory regions (-2401/-2291 and -1405/-1022) for the promoter activity. A 1023 bp fragment of Osgrp-2 promoter (-1021 to +2) fused with GUS was transformed into tobacco and proved to be capable of conferring vascular-specific expression.In this study, a VS99 fragment of Osgrp-2 promoter had been strictly tested for its function acquired. Directed repeat the VS99 fragment and then fused it to the upstream of the 35S-89 minimal promoter. The fused sequence was cloned to HindIII- EcoRI digested expression vector pBI121. Transform tobaccos with the recombined plasmid, and GUS activity was assayed in various tissues. The experiments had revealed that the VS99 fragment can confer vascular-specific expression. To identify the cis-elements, Nucleoproteins of stem and leaf of six-week tobaccos had been extracted and electrophoretic mobility shift assay (EMSA) had been preceded with the VS99 fragment. The specific blot suggested specific binding sequence in the VS99 fragment. Aligning the VS99 fragment with the five existed promoters of vascular-specific expression limited 13-bp high-specific sequence. Yeast one-hybrid would be proceded between directed three copies of 13-bp fragment and cDNA library of rice for screening trans-acting factors interacting with the 13-bp fragment. At present, 45 clones had been obtained and 23 of them have been correctly amplificated by PCR. The PCR products will be sequenced and then aligned in NCBI. Graduate student: Chen Wanli Major: Crop Culture and Farming System Supervisor: Associate Prof. Li Wenhua Prof. Fang Rongxiang... |
