One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family, it could be easily used to other prokaryote and eukaryote species
Background Salinity as a most significant environmental challenges affects the growth and productivity of plants worldwide. In this study, the ionic and iso-osmotic effects of salt stress were investigated in Aeluropus littoralis L., a halophyte grass species from Poaceae family, by cDNA-amplified fragment length polymorphism (cDNA-AFLP) technique. To dissect the two different effects (ionic and osmotic) exerted by salt stress, various ionic agents including 200 and 400 mM sodium chloride (NaCl), 200 and 400 mM potassium chloride (KCl) as well as 280 and 406 gl − 1 (− 0.9 and − 1.4 MPa) polyethylene glycol 6000 (PEG) as their iso-osmotic concentrations were applied. Results Application of KCl and PEG significantly reduced the fresh weight (FW) of A. littoralis seedlings compared to control while NaCl treatment markedly enhanced the FW. At the transcriptome level, different observations of changes in gene expression have been made in response of A. littoralis to ionic and osmotic stresses. Out of 69 transcript derived fragments (TDFs), 42 TDFs belong to 9 different groups of genes involved in metabolism (11.6%), transcription (10.2%), ribosomal protein (8.7%), protein binding (8.7%) transporter (5.8%), translation (5.8%), signal transduction (4.3%), nucleosome assembly protein (2.9%) and catabolism (2.9%). The 44 and 28 percent of transcripts were expressed under ionic stress (NaCl-specific and KCl-specific) and osmotic stress (common with NaCl, KCl and PEG), respectively which indicating a greater response of plants to ionic stress than osmotic stress. Expression pattern of eight candidate TDFs including; SYP81 , CAND1 , KATN , ISB1 , SAMDC , GLY1 , HAK18 and ZF30 was evaluated by RT-qPCR at high salinity levels and recovery condition. Conclusion Differential regulation of these TDFs was observed in root and shoot which confirm their role in salt stress tolerance and provide initial insights into the transcriptome of A. littoralis . Expression pattern of ionic and osmotic-related TDFs at A. littoralis can be taken as an indication of their functional relevance at different salt and drought stresses.
Laccases are plant enzymes with essential functions during growth and development. These monophenoloxidases are involved in lignin polymerization, and their expression respond to environmental stress. However, studies of laccases in some plants and fungi have highlighted that many structural and functional aspects of these genes are still unknown. Here, the laccase gene family in Aeluropus littoralis (AlLAC) is described based on sequence structure and expression patterns under abiotic stresses and ABA treatment. Fifteen non-redundant AlLACs were identified from the A. littoralis genome, which showed differences in physicochemical characteristics and gene structure. Based on phylogenetic analysis, AlLACs and their orthologues were classified into five groups. A close evolutionary relationship was observed between LAC gene family members in rice and A. littoralis. According to the interaction network, AlLACs interact more with proteins involved in biological processes such as iron incorporation into the metallo-sulfur cluster, lignin catabolism, regulation of the symbiotic process and plant-type primary cell wall biogenesis. Gene expression analysis of selected AlLACs using real-time RT (reverse transcription)-PCR revealed that AlLACs are induced in response to abiotic stresses such as cold, salt, and osmotic stress, as well as ABA treatment. Moreover, AlLACs showed differential expression patterns in shoot and root tissues. Our findings indicate that AlLACs are preferentially involved in the late response of A. littoralis to abiotic stress.
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