application of a non-radioactive DD-AFLP method for profiling of aeluropus lagopoides differentially expressed transcripts under salinity or drought conditions

Aeluropus lagopoides is a salt and drought tolerant grass from Poaceae family, distributed widely in arid regions. There is almost no information about the genetics or genome of this close relative of wheat that stands harsh conditions of deserts. The main aim of this research was to isolation and characterization of salt and drought inducible genes from A. lagopoides by Differential Display Amplified fragment length polymorphism [DD-AFLP] method. In this research A. lagopoides was grown under salt or drought conditions and after modifying the DD-AFLP method several fragments were isolated and after nomination their induction was studied by reverse northern blotting. DD-AFLP led to the improvement of a non-radioactive method for which many parameters were optimized. Having screened approximately 1600 transcript-derived fragments, 1.4 percent of them showed varied expression levels in response to high salt or drought treatments. The relative abundance of twenty one selected differentially expressed fragments was inspected by reverse northern blotting that affirmed the potential of this applied method. Sequence comparisons revealed that some of the isolated genes are involved in osmotic adjustment, regulation of transcription, cation transportation and stress responses. These data clearly show that the modified DD-AFLP method was a successful and reliable approach for the isolation of differentially expressed genes

Differential expression of Arabidopsis thaliana acid phosphatases in response to abiotic stresses

The objective of this research is to identify Arabidopsis thaliana genes encoding acid phosphatases induced by phosphate starvation. Multiple alignments of eukaryotic acid phosphatase amino acid sequences led to the classification of these proteins into four groups including purple acid phosphatases [PAPs]. Specific primers were degenerated and designed based on conserved sequences of PAPs isolated from plants, fungi and animals. RNA profiles of Pi-fed and Pi-starved A. thaliana roots were compared with two methods established for gene-family-directed differential display of pap genes. Having analyzed the differentially displayed fragments, seven pap encoding cDNA clones were isolated. One of the clones was a transsplicing product of two genes that encodes an acid phosphatase carrying a zinc-finger domain. Six other clones were predicted to encode secretory phosphatases. Reverse-northern blotting and semi-quantitative RT-PCR revealed distinct expression patterns for each gene under diverse environmental conditions such as Pi starvation, high-salt concentration, cold shock, nitrogen and sulfur deprivation. The presented data can provide some clues for dissecting the possible roles of PAPs in Pi acquisition. Keywords: Acid phosphatase; Purple acid phosphatase; differential display; gene expression; Arabidopsis thaliana; Abiotic stress

Hyoscyamine 6 - Beta - hydroxylase gene isolation from in vitro cultured roots of hyoscyamus niger l. and hyoscyamus tenuicaulis schonbeck - temesy

The H6H gene for hyoscyamine 6b-hydroxylase, which converts hyoscyamine to scopolamine, was isolated from H. niger and H. tenuicaulis. The roots of 14 days sterile seedlings were transferred to a modified liquid B5 medium containing 1mM indolebutyric acid, and after appearance of the lateral roots, subcultured in a free hormone medium. Following a week, the total cellular RNA of the roots was extracted. cDNA of H6H gene was synthetized by RT-PCR, favored gene was amplified by the PCR