Using An F₂ Screen To Estimate The Frequency Of Resistance Alleles To Cry1Ab In Wenzhou Population Of Chilo Suppressalis And Cloning Of An Aminopeptidase N Gene CsAPN3

The rice stem borer Chilo suppressalis (Walker) is one of the most serious pests threatening rice production in China. Bt transgenic rice offers a new effective and safe weapon to control the rice stem borer. Bt rice will be commercialized in China in a few years. Therefore, estimating the frequency of resistance alleles to Bacillus thuringiensis gene CrylAb in field populations of C. suppressalis will be important for establishing the system of early resistance forecasting and resistance manangement strategy. In this study, a modified F2 method was used to estimate the frequency of resistance alleles to B. thuringiensis gene Cry1Ab in a field population of C. suppressalis collected from Wenzhou, Zhejiang Province in 2009. A putative Bt receptor APN gene (CsAPN3) was cloned and sequenced from C. suppressalis.1. Using F2 screen to detect the frequency of resistance alleles to CrylAb in a field population of C. suppressalisAn F2 screen was used to estimate the frequency of resistance alleles to Bacillus thuringiensis Cry1Ab in a field population of C. suppressalis collected from Wenzhou, Zhejiang province in April of 2009. Egg masses of C. suppressalis were collected and every egg mass was used to build one isofemale line, and a total of 201 isofemale lines were established and screened at F2 generation. The frequency of alleles for Cry1Ab was estimated to be around 0.0025 (95% CL:0.0019-0.0031). The results showed that the frequency of resistance alleles to B. thuringiensis gene CrylAb in Wenzhou population of C. suppressalis is low. It is suggested that "high dose/refuge" IRM strategy should be adopted in China for resistance management of Bt rice.2. Laboratory selection for CrylAb resistance in C. suppressalisA strain was estabolished from survivors of 7 candidate isofemale lines of F2 screen. The mixed strain was selected with actived CrylAb for six generations and the susceptibility to Cry1Ab was declined significantly. Resistance level of the selected strain increased to 10-fold compared with the susceptible CN strain. The survival percent under the discriminating dose of CrylAb (2μg/cm2) was increased from 5% before selection to 35% after selection.3. Cloning, sequence analysis and mRNA expression patterns of CsAPN genes from C. suppressalisAn APN gene was cloned by using RT-PCR and RACE and sequenced from C. suppressalis. The complete cDNA sequence of the gene contained 3363 bp and encoded a protein of 1013 amino acids. This gene was designated as CsAPN3 according to its amnio acid similarity to other insect APNs. This APN has the conservative domain GAMEN and HEXXHX18E, and 20 signal peptidase sequences at N-terminal. But there was not a GPI at the C-terminal. The mRNA expression of APN in different tissues (head, mid-gut, fatbody) and developmental stages (larva, pupa, adult) was compared in the susceptible strain with Real-time PCR. The highest expression was detected in the mid-gut tissue and in the larval stage of C. suppressalis.