Extraction of differential expressing aphid-resistance genes of sorghum (Sorghum bicolor L. Monech) and construction of suppression subtractive hybridization (SSH) library

Abstract

Experiments were conducted for the extraction of differential expressing aphid-resistance genes of sorghum (Sorghum bicolor L. Monech) in the experimental laboratories and fields of Hebei Agricultural University, China and Shenyang Agricultural University, Liaoning Province, China, during 2010 to 2011 and suppression subtractive hybridization (SSH) library was constructed. The seeds of two sorghum varieties (Henong-16 and Qian-3) were grown and aphids were infested through natural and artificial way on sorghum seedlings (10-day old) with a paint brush. Total mRNA was isolated from fresh leaves samples using Trizol reagent and plant RNA mate (TAKARA). Integrity of RNA was confirmed by 1.2% agarose gel electrophoresis. SSH was performed using PCR-Select cDNA subtraction kit user manual according to the manufacturer’s instruction (Clontech Laboratories, Inc, USA). cDNA that contained specific (differentially expressed) transcripts were denoted as tester and the reference cDNA as driver. Tester and driver cDNAs were hybridized after two rounds of subtractive suppression PCR and the pMD18-T vector (TAKARA, Dalian, China). After preliminary screening by subtractive hybridization, plasmid restriction enzyme digestion, colony PCR for 100 forward and 100 reverse clones were sequenced by two-way hybridization using Mega BACE1000 to obtain better quality of 200 expressed sequence tag (EST) sequences. Cross-match software and ClustalW2 were used to obtain vector sequence shielding and multiple comparisons. Using BLAST at NCBI database for homology comparisons, it was concluded that a number of EST sequences which had different degrees of homology with known proteins or genes and another six EST sequences did not have any significant homology in the database. These sequences might have representation for new and unknown genes, or higher variability of non-coding region cDNA sequences.

Key words: Extraction, sorghum, SSH, aphid-resistance genes.