cDNA-SRAP and Its Application in Differential Gene Expression Analysis: A Case Study inErianthus arundinaceum

Abstract

<jats:p><jats:italic>Erianthus arundinaceum</jats:italic>is a wild relative species of sugarcane. The aim of this research was to demonstrate the feasibility of cDNA-SRAP for differential gene expression and to explore the molecular mechanism of drought resistance in<jats:italic>E. arundinaceum</jats:italic>. cDNA-SRAP technique, for the first time, was applied in the analysis of differential gene expression in<jats:italic>E. arundinaceum</jats:italic>under drought stress. In total, eight differentially expressed genes with length of 185–427 bp were successfully isolated (GenBank Accession numbers: EU071770, EU071772, EU071774, EU071776, EU071777, EU071779, EU071780, and EU071781). Based on their homologies with genes in GenBank, these genes were assumed to encode ribonuclease III, vacuolar protein, ethylene insensitive protein, aerobactin biosynthesis protein, photosystem II protein, glucose transporter, leucine-rich repeat protein, and ammonia monooxygenase. Real-time PCR analysis on the expression profiling of gene (EU071774) encoding ethylene-insensitive protein and gene (EU071781) encoding ammonia monooxygenase revealed that the expression of these two genes was upregulated both by PEG and ABA treatments, suggesting that they may involve in the drought resistance of<jats:italic>E. arundinaceum</jats:italic>. This study constitutes the first report of genes activated in<jats:italic>E. arundinaceum</jats:italic>by drought stress and opens up the application of cDNA-SRAP in differential gene expression analysis in<jats:italic>E. arundinaceum</jats:italic>under certain stress conditions.</jats:p>

Identification of smut-responsive genes in sugarcane using cDNA-SRAP

“…Previous research has revealed that cDNA-SRAP is a simple, reproducible, and reasonable-throughput approach for the comprehensive analysis of differential gene expression at the cDNA level (Li et al, 2003;Lu and Wu, 2006;Deng et al, 2007;Ma et al, 2008;Que et al, 2012b). In the present study, cDNA-SRAP along with agarose gel electrophoresis, was adopted to analyze how genes in sugarcane function during sugarcane-S. scitamineum interactions.…”

“…The cDNA-SRAP procedure described by Que et al (2012b) was utilized with a minor modification and the primer sequences were selected from previous reports (Table 1) (Li et al, 2003;Deng et al, 2007;Que et al, 2012b). The 50 μL cDNA-SRAP reaction contained 5.0 μL 10X PCR buffer (15 mM MgCl 2 plus), 3.75 µL dNTPs (10 mM), 1.25 U Taq DNA polymerase enzyme, 1.5 μL forward and reverse primers (10 μM) respectively, and 100 ng second strand cDNA as the template.…”

“…The cDNA-SRAP amplified products were concentrated to approximately 10 μL using a centrifugal freeze drying system (Martin Christ, Germany) (Que et al, 2012b). The total volume was loaded onto a 1.6% agarose gel (containing 0.1% Ethidium bromide) for electrophoresis and imaged with a Bio-Rad Gel Imaging System (Hercules, CA, USA).…”

“…The differentially expressed genes obtained were subjected to a BlastX similarity search analysis against the NCBI protein database and annotated according to their homologies with known protein sequences (Que et al, 2012b;Su et al, 2014a).…”

“…It combines simplicity, reproducibility, reasonable throughput ratio, and easy isolation of bands for sequencing. The cDNA-SRAP technique, with cDNA as a template, has been proven to be suitable for analysis of differential gene expression in several plants, including Brassica oleracea (Li et al, 2003;Deng et al, 2007), Spartina angelica (Lu and Wu, 2006), B. napus (Ma et al, 2008) and Erianthus arundinaceum (Que et al, 2012b). Furthermore, the cDNA-SRAP method, together with agarose gel electrophoresis, can overcome the shortcomings of the isotope labeling and silver staining methods by providing a shorter, safer, and less expensive method that requires no special equipment and can be conducted in a typical laboratory (Que et al, 2012b).…”