Presentation on theme: "Acknowledgments We appreciate Sand West and Lily Luo for their laboratory work in this work. Abstract The sugarcane borer, Diatraea saccharalis, is a major."— Presentation transcript:
Acknowledgments We appreciate Sand West and Lily Luo for their laboratory work in this work. Abstract The sugarcane borer, Diatraea saccharalis, is a major corn pest and a primary target of Bt corn in the mid-south region of United States. The Bt-resistant allele to Cry1Ab protein in Bt corn was identified from D. saccharalis by Huang et al. (2007). To better understand the molecular mechanisms of Bt resistance, the Cry1Ab-susceptible (Cry1Ab-SS) and –resistant (Cry1ab-RR) strains of D. saccharalis were subjected to a microarray analysis. Results showed that the expression levels of many genes were substantially different between the Cry1Ab-SS and – RR strains. After microarray analysis and real-time PCR verification, we found that the gene transcripts coding for a chymotrypsin-like protease, a glutathione S-transferase omega 1, and a lipase were significantly up-regulated in Cry1Ab–resistant strain. This cDNA microarray gene expression information could be used to identify new genes that are associated with Bt resistance development in the sugarcane borer. Materials and Methods Insect strains A Cry1Ab-SS and a Cry1Ab-RR strain of D. saccharalis were provided by Huang et al. (2007a, 2007b) of Louisiana State University. Microarray analysis A total of 35,262 NimbleGen chip probes (5 probes per sequence) were synthesized using the sequences of contigs and singletons obtained from the above cDNA library. Total RNA was prepared from the 3 rd instar larvae using TriZol (Invitrogen, Carlsbad, CA). DscDNAs were synthesized by using the SuperScript Double-Stranded cDNA Synthesis Kit (Invitrogen, CA) according to the manufacturer’s protocol. cDNA samples were labeled with One-color DNA Labeling Kit and hybridized to the microarray chips, and data were Results Microarray analysis of gene expression in the Cry1Ab-SS and –RR strains of the sugarcane borer Total RNA was prepared from the 3 rd instars of the Cry1Ab-SS and –RR strains of D. saccharalis. After over 7,146 genes screening, we found that the gene transcripts coding for a chymotrypsin-like protease, a glutathione S-transferase omega 1, and a lipase were significantly up-regulated in Cry1Ab–resistant strain (Table 1). Three criteria for selection of microarray between Cry1Ab-RR and Cry1Ab-SS were referenced to Vawter et al. (2004). cDNA Microarray Analysis of Gene Expression in Bacillus thuringinsis- susceptible and -resistant Sugarcane borer, Diatraea saccharalis (Fabricius) Zibiao Guo a, Yu Cheng Zhu a, Fangneng Huang b, and Randall Luttrell a a SIMRU, ARS-USDA, Stoneville, MS 38776, b Department of Entomology, Louisiana State University, Baton Rouge, LA Literature cited Bautista, M.A.M., Tadashi, M., Miura, K., Tanaka, T., RNA interference-mediated knockdown of a cytochrome P450, CYP6BG1, from the diamondback moth, Plutella xylostella, reduces larval resistance to permethrin. Insect Biochem. Mol. Biol. 39, 38e46. Castro, B.A., Riley, T.J., Leonard, B.R., Baldwin, J., Borers galore: emerging pest in Louisiana corn, grain sorghum and rice. LA Agric. 47, 4e6. Huang, F., Leonard, B.R., Gable, R.H., Comparative susceptibility of European corn borer, southwestern corn borer, and sugarcane borer (Lepidoptera: Crambidae) to Cry1ab protein in a commercial Bt-corn hybrid. J. Econ. Entomol. 99, 194e202. Huang, F., Leonard, B.R., Andow, D.A., 2007a. Sugarcane borer (Lepidoptera: Crambidae) resistance to transgenic Bacillus thuringiensis maize. J. Econ. Entomol. 100, 164e171. Huang, F., Leonard, B.R., Wu, X., 2007b. Resistance of sugarcane borer to Bacillus thuringiensis Cry1Ab toxin. Entomol. Exp. Appl. 124, 117e123. Vawter, M., Ferran, E., Galke B., Cooper, K., Bunney, W., Byerley W., Microarray screening of lymphocyte gene expression differences in a multiplex schizophrenia pedigree. Schizophrenia Res 67, Validation of microarray data using quantitative RT-PCR To confirm the differential gene expression determined using the microarray, quantitative RT-PCR was performed on a subset of differentially expressed cDNAs. The RT-PCR analysis was conducted to examine the expressions of three up-regulated genes, e.g., a chymotrypsin-like protease, a glutathione S- transferase omega 1, and a lipase in the Cry1Ab-SS and –RR strains of D. saccharalis (Fig. 1). Fig. 1. Gene expression levels of three genes in the 3 rd instars of the Cry1Ab-SS and –RR of D. saccharalis using real-time PCR. Absolute transcript abundance of (A) glutathione S-transferase omega 1(GST), (B) lipase (LP) and (C) chymotrypsin-like protease (CHY) was determined using qRT-PCR and SYBR green. Bars represent the means and standard errors of 4 total RNA samples that each contains a pool of total RNAs from 3 larvae. Significant differences between Cry1Ab-SS and –RR (Student’s t-test, p<0.05). A. Introduction The sugarcane borer, Diatraea saccharalis, is a major corn pest and a primary target of Bt corn in the mid-south region of United States (Castro et al., 2004; Huang et al., 2006). However, molecular resistance mechanisms of Bt resistance in corn stalk boring pests are poorly understood because of the lack of a highly resistant insect strain for study. Recently, Huang et al. (2007a) identified a resistance allele to Cry1Ab protein in Bt corn from D. saccharalis. The Cry1Ab-resistant strain of D. saccharalis provides a probability to explore the mechanisms of Bt resistance in corn borer species. This study was to identify new genes that are associated with Bt resistance development in the sugarcane borer. To achieve this goal, microarray was performed to compare gene expression levels between Bt-susceptible and -resistant strains of D. saccharalis. acquired according to NimbleScan v.25 User’s Guide through FSU Microarray processing facility. Data were statistically analyzed by using SAS and presented as the mean of fold change. The fold change was calculated by dividing the gene expression level of the Cry1Ab-RR by that Cry1Ab- SS. Confirmation of Quantitative real-time PCR (qRT– PCR) Total RNAs of 3rd instars from Cry1Ab-SS and -RR strains of D. saccharalis were extracted by using TriZol reagent. Concentrations of total RNAs were measured with a NanoDrop spectrophotometer. The primers were designed by RealTime PCR SciTool (Coralville, IA). Each RNA sample was diluted to10ng/μl. The qRT–PCR assays were performed using iScript™ One-step RT-PCR Kit with SYBR® Green (Bio-Rad, Hercules, CA) in a Thermal Cycler PTC200 with Chrome4 detector attached (Bio-Rad). The following thermal cycling profile was used: 50 ⁰ C for 10 min (cDNA synthesis), 95 ⁰ C for 5 min (iScript reverse transcriptase inactivation), followed by 40 cycles of 95 ⁰ C for 10 s and 59 ⁰ C for 30 s. A melting curve was established to check amplification specificity. The cycle threshold (Ct) value was used to calculate the transcript quantity of the target genes based on standard curve (Bautista et al., 2009). Data was analyzed with Student’s t-test, and significance was defined at p<0.05. B. C. IDGene Fold change (Cry1Ab-R/Cry1Ab-SS) SW2037 glutathione S- transferase omega 12.44** SG215 chymotrypsin-like protease1.85* RG825lipase2.56** Table1. A selection of genes up-regulated in the Cry1ab-RR and Cry1Ab-SS. Gene profiling was performed using microarray analysis. Expression level is presented as the mean of fold change (n=4). Significant gene expression difference between Cry1ab- RR and Cry1Ab-SS for each gene is *p<0.05 and **p<0.01 Summary In this study, increased gene expressions of at least 3 candidate genes in Bt-resistant strain were confirmed by using microarray global gene expression analysis and real-time PCR validation. These genes encode a chymotrypsin-like protease, a glutathione S-transferase omega 1, and a lipase. Comparisons of expression profiles of these candidate genes between Cry1Ab-susceptible and - resistant strains of D. saccharalis by RT-PCR showed apparently increased expressions in the resistant strain as compared with the susceptible strain. Such differential expressions of the candidate genes may suggest their involvement in Cry1Ab resistance. Indeed, certain glutathione S-transferase, chymotrypsin-like proteases and lipase have previously been found to be associated with activation or degradation of Bt protoxins and toxins.