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

[Functional screening of phosphatase-encoding genes from bacterial sources]

Phosphatase [APase] enzymes including phytases have broad applications in diagnostic kits, poultry feeds, biofertilizers and plant nutrition. Because of high levels of sequence diversity among phosphatases, an efficient functional screening method is a crucial requirement for the isolation of the encoding genes. This study reports a functional cloning screening method for the isolation of APase-encoding genes from bacterial genomic libraries in a medium containing a chromogenic substrate. The method was optimized to distinguish the desired signal from the background chromosomal APase activity. This screening method led to the isolation of two novel APase-encoding genes from Pseudomonas putida with no similarities to the known genes in the databases, indicating successful implementation of the developed method

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

Identification and expression analysis of two Arabidopsis LRP-protein encoding genes responsive to some abiotic stresses

Two Arabidopsis thaliana genes, psr9.2 and psr 9.4 appeared to be highly similar to a phosphate-starved induced gene, psr9, isolated from Brassica nigra suspension cells. Sequence analysis classified the encoded polypeptides as members of leucine-rich repeat [LRR] proteins superfamily. The sequence of psr9 proteins comprise a unique N-terminal region encompassing a coiled-coil structure proceeding eleven LRRs along the C-terminal. Expression pattern analysis showed the responsiveness of these genes to various environmental conditions. Although both psr9 genes, psr9.2 and psr9.4, are expressed throughout the plant, the expression of psr9.2 was higher in the root whereas psr9.4 expression was prominent in the shoot. The expression levels were increased proportional to the duration of phosphate deprivation treatment. Plants exposed to cold temperature expressed both genes at high levels in both roots and shoots. In contrast, heat shock increased the expression levels of both genes in the shoots while reducing it in roots. High-salt treatment upregulated the expression of psr9 genes only in the roots. These data may suggest distinct roles for psr9 genes during plant response to various environmental conditions