Molecular Characterization And Function Analysis Of Aminopeptidase N-Bt Toxin Receptor From Chilo Suppressalis Midgut

The striped stem borer (SSB), Chilo suppressalis (Walker), is an important rice pest, widelydistributed in the main rice-growing areas of China. An alternatively attractive strategy for control ofSSB is to produce insecticidal proteins by introducing the corresponding insecticidal genes into rice.However, potential resistance risk of target pest has been cited as the main threat to the utility of Bt rice.Therefore, the key to successfully sustainable application of Bt rice is to delay resistance evolution andimplement resistance management strategies. Moreover, the illustration of insect molecular resistantmechanism to Cry toxin is the base to implement effective insect resistance managementstrategy.Aminopeptidase N (APN) protein located in the midgut epithelium of lepidopteran species hasbeen implicated as a Cry1A receptor and reported to be associated with insect resistance. In this paper,full lengths of APNs cDNA sequences were amplified and molecular characterization and function ofAPNs were analyzed further. The main results are as follows:1.cDNAs of four APN isoforms were identified and sequenced by degenerative primer PCR andRACE techniques. The full-length sequences of four APN cDNAs in SSB were named as CsAPN2(Gene bank access: JQ088281), CsAPN3(Gene bank access: JF519739), CsAPN4(Gene bank access:HQ901596), CsAPN5(Gene bank access: JQ088280). Based on these results, the APN sequencefeatures were derived from deduced amino acid sequences of APN cDNAs. The molecular weights ofAPN proteins encoded by the sequences were109.613,115.207,109.205,114.724kDa, respectively.2.The mRNA transcript levels were assessed for four genes in three different gut tissues anddifferent developmental stages of larvae using real-time quantitative PCR. The results showed that therewere obviously spatio-temporal profiles for four CsAPNs, that is, CsAPNs mained expressed in larvalmidgut, followed by hindgut and foregut. Moreover, CsAPNs had high expressing levels in third instarlarvae. Also, APN emzyme activilites changed consistently with the transcript levels of CsAPNs.3.Binding of Cry1Ab with purified BBMV from midgut of the3rdinstar SSB was detected withligand blot and molecular size of bound protein is about120kDa. Bound protein band was cut from thegel and used for its identification with HPLC-MS.120kDa SSB BBMV bound protein was identified asAPN, which showed that CsAPN was probable Bt toxin receptors..4.Whether the APN was the receptor of Cry1A toxin was confirmed via the antibody blockingtechnology. After the preparation of polyclone antibody, the antiserum and the Cry1A toxin were mixtedat a certain proportion and bioassay for SSB neonate was conducted. Larval survival anddevelopmental rates of antiserum treatment were significantly higher than that of control treatment.Reduced Cry1A toxicity in bioassays with anti-APN sera suggests that SSB midgut epithelial APN arefunctional receptor of the Cry1A toxin.5.To verify the involvement of APNs in Cry1A toxicity and SSB resistance, RNA interference(RNAi) was employed to knockdown APN1, APN2and APN3, which were the candidate genesassociated with insect resistance, by siRNA micro-injection technique. The mRNA transcript level,protein expression and SSB neonate susceptibility to Cry1A toxin were detected with qPCR and RT-PCR, Western-blot and biosaasy, respectively, after siRNA injection of24h,48h and72h. We findthat the transcript levels of APNs were different in time and efficiency. Western-blot detection showedthat the protein expressions of APNs were the lowest after silented for48h and72h.. Further, bioassayindicated that SSB larval mortality of RNAi treatment was significantly reduced compared with controltreatment. It deduces that the down-regulated expression of APNs is functionally associated with thedecrease of SSB susceptibility to Cry1A toxin.Based on these researches, molecular characterization and function of CsAPNs are clearly clarified.This study is very meaningful in elucidating the molecular mechanism of C. suppressalis resistance toBt toxin, establishing and improving the monitoring and forecasting system for resistance to Bt rice,implementing proactive resistance management strategy, and realizing sustainable utilization andhealthy development of Bt rice.