CDNA-AFLP Analysis Reveals Differential Gene Expression Under Drought Stress In Agropyron Mongolicum Keng

Drought is one of the most serious abiotic stresses with significantly negative impacts on plant growth and crop yield. Agropyron mongolicum Keng is an important genetic resource and has excellent ecological values in forge improvements, especially for drought resistance. This study used the cDNA-AFLP technology to isolate and characterize differential genes expressed under drought stress and further investigated the molecular and regulation mechanism on drought resistance of A. mongolicum.1. Establishment and optimization of cDNA-AFLP system in A. mongolicum. Double-strand cDNAs were reversely transcripted from 2μg total RNAs. In 20μL restriction endonuclease and ligation reaction, 5μL cDNA, 2μL NEB bufferⅡ, 3 U each for PstⅠand MseⅠ(NEB company), 0.2μL 100×BSA, 75 U T4 DNA ligase, 2μL 10×T4 DNA ligase buffer, adaptors were added, and then incubated at 37℃for 3 h and 16℃overnight. The digested and ligated products were diluted 10×as pre-amplification primary template; pre-amplification products were diluted 15×as selective amplification primary template; selective amplification products were added into 10μL loading buffer and denatured for 10 min at 98℃, and then put in an iced bath immediately. The selective amplification products were analyzed on 6% denatured acrylamide/bisacrylimade gel and then stained by silver.2.Total 25 pairs of primer combinations were selected and used for cDNA-AFLP analysis. Total 143 AFLP bands were scored, and 15 differential transcript-derived fragments (TDFs) were sequenced and their bioinformatics information was primarily analyzed.Our NCBI Blastn search showed that eight TDFs had significant homologous nucleotide sequence with Hordeum vulgare, Triticum aestivum, Oryza sativa and Zea mays, two TDFs had significant homologous nucleotide sequence with Psendomonas, one TDF had significant homologous nucleotide sequence with Homo sapiens, and the others could not find any significant homologous nucleotide sequence. Our Blastx search in NCBI non-redundant protein database showed that only ten TDFs had significant homologous nucleotide sequence with H. vulgare, Z. mays, T. aestivum and some microorganic proteins, others five TDFs, accounting to 30% total TDFs, could not find any significant homologous nucleotide sequence,. The functions of these TDFs genes could be grouped into different categories, such as transcriptional regulator and regulation, calcium binding protein, ribosomal protein, active oxygen scavenging protein, membrane protein. Some unknown genes and new genes expressed under the drought stress were also found in this study. Our results have revealed some important pathways of drought-resistance in A. mongolicum, which will be very valuable for us to further study on drought-resistance mechanism in A. mongolicum and also further explore the potential utilizations of more excellent genetic resources in A. mongolicum.