Working group session:
Functional Genomics
Presentation type:
oral
Authors:
Husnain, Tayyab
Presenter:
Husnain, Tayyab
Correspondent:
Husnain, Tayyab
Abstract:
Epicuticular wax and proline contents were studied in five varieties of Gossypium arboreum, namely FDH-170, FDH-300, FDH-306, FDH-786 and Ravi. Variety FDH-786, being the most drought tolerant, exhibited 9- and 5.75-fold higher levels in wax and proline contents respectively when compared with non-stressed plants. A small alpha-crystalline heat shock protein gene (GHSP26) was isolated and characterized using RACE and genomic DNA PCR. Segments of 1026 bp cDNA sequences and 1108 bp genomic (exon & intron) were obtained. Alignment studies revealed that GHSP26 comprises a single open reading frame of 230 amino acids. Expression studies of the gene in different tissues showed that the gene is expressed mainly in leaves of stressed plants.
We also carried out high throughput analysis of drought tolerant genes from G. arboreum, and developed cDNA libraries from mRNA isolated from leaves and roots of drought stressed plants. Twelve thousand clones were cultured of which 1000 showed novelty in that there was no homology to genes in the NCBI data bank. Selected clones were spotted on the microarray slides and hybridized with probes from control and water stressed plants. Thirty seven ESTs were found up-regulating under drought stressed conditions.
Besides, wax mutants were developed by physical and chemical mutagens and cDNA libraries were constructed from the leaf mRNA to yield clones 78% of which showed no homology with genes in the NCBI databank. Therefore, there is great potential in the search for novel genes from G. arboreum. Selected clones were spotted on the microarray slides and hybridized to yield 40 ESTs involved in wax biosynthesis.
GHSP26 and GUSP genes were cloned in a plant expression vector,pcambia-1301 (CaMv 35S promoter, neomycin phosphotransferase and GUS, a reporter gene). Transformation of Gossypium hirsutum variety CIM 496 with these genes yielded transgenic plants that exhibited 6-fold and 60-fold over expression of GUSP and GHSP26 genes under eight days of drought stress conditions. Experiments are in progress for longer exposure of transgenic plants to drought stresses.
The upstream region of the promoter of stress resistant gene GHSP26 was analyzed. Each deletion construct was analyzed by Agroinfection in Tobacco leaves after treatment of Abscisic acid (ABA), heavy metals and dehydration. Promoter fragment of 716bp showed two-fold or greater induction after each treatment.
Similarly, the promoter of 949bp upstream from translation initiation of the GUSP gene encoding universal stress protein was isolated from genomic DNA of Gossypium arboreum. Deletion analysis revealed that dehydration of abscisic acid and salt responses was affected. A deletion between -208bp and -949bp showed 2-4 fold induction.