Construction of suppression subtractive hybridization libraries and identification of brown planthopper-induced genes

Abstract

A suppression subtractive hybridization technique was used to screen for brown planthopper (BPH)-inducible genes in rice (Oryza sativa). cDNAs from a BPH-resistant rice line (B5) infested by BPH were used as the tester population, and mixed cDNAs from a BPH-sensitive line (MH63) and a control (uninfested B5) as the driver population. After hybridizing and cloning, forward and reverse subtraction cDNA libraries were obtained, containing 5700 clones. These clones were further analyzed by differential gene expression screening, and 154 clones that were clearly induced by BPH were identified. Sequencing analysis and homology searching showed that these clones represent 136 single genes, which were assigned to functional categories, including 10 putative cellular functions, according to categories established for Arabidopsis. The 136 genes include 21 known to be related to disease, wound and other stresses, most of which were found to be up-regulated in BPH feeding responses. In addition, an Oryza cysteine inhibitor and a beta-glucosidase belonging to the 21 genes group were found in the rice response to BPH feeding, these two genes have previously been shown to be induced in plant responses to chewing insects. Our results not only confirm that several identical genes are activated in defense mechanisms against both sucking and chewing insects, but also show that genes have overlapping functions in both pathogen and insect resistance.