Isolation of a cotton reversibly glycosylated polypeptide (GhRGP1) promoter and its expression activity in transgenic tobacco.
Reversibly glycosylated polypeptides (RGPs) are thought to be involved in polysaccharide metabolism. A cDNA of the cotton (Gossypium hirsutum) RGP gene, designated GhRGP1, has previously been characterized, and is preferentially expressed in fiber cells. In order to investigate its temporal and spatial control, we isolated a 624bp fragment upstream of the GhRGP1 coding sequence using a polymerase chain reaction (PCR)-based genomic walking method, transcriptionally fused the 624bp promoter sequence to the beta-glucuronidase (GUS) gene, and analyzed the stable gene expression in tobacco (Nicotiana tabacum). In 4-week-old transgenic tobacco plants, the highest expression level was observed in roots, and the GUS activity was 1.13- and 6.65-fold higher than that in stems and leaves, respectively. In the reproductive growth stage, the GUS expression level was highest in the pistils and the GUS activity in the stigmas and styles were 17.6-fold higher than that in the ovaries. High GUS activity was also detected in the anthers. In addition, histochemical staining for GUS activity on transgenic tobacco plants further indicated a higher expression in the trichomes, seeds and vascular tissues of stems. Abiotic stress treatments on transgenic tobacco plants showed that wounding and dehydration induced GUS expression. These results demonstrated the spatial and temporal regulation of a cotton RGP promoter in a model plant, and provided an important insight into the factors that control the fiber development and stress responses of the gene.
Wu
Ai-Min
AM
Laboratory of Molecular Biology and MOE Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.
Ling
Chen
C
Liu
Jin-Yuan
JY
D001483:Base Sequence; D049108:Cell Enlargement; D016386:Chromosome Walking; D035264:Flowers / Q000033:anatomy & histology / Q000254:growth & development / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D003368:Gossypium / Q000235:genetics*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D018517:Plant Roots / Q000033:anatomy & histology / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014026:Nicotiana / Q000033:anatomy & histology / Q000235:genetics* / Q000378:metabolism; D014867:Water / Q000378:metabolism
D006023:Glycoproteins; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014867:Water