Font Size: a A A

Identification Of Native Receptors Of Cry1Ac Toxin In The Midgut Of Chilo Suppressalis (Walker)

Posted on:2014-04-09Degree:MasterType:Thesis
Country:ChinaCandidate:Y M ZhuFull Text:PDF
GTID:2283330482962454Subject:Agricultural Entomology and Pest Control
Abstract/Summary:PDF Full Text Request
The rice stem borer, Chilo suppressalis (Walker) is one of the most serious pests threatening rice production in China. Transgenic Bt rice can effectively control the damage of rice stem borer and other lepidopterous pests. The toxicity of transgenic Bt rice depends on the binding between CrylA toxins expressed in transgenic Bt rice and the receptor proteins in the midgut brush border membrane vesicles (BBMV). The Cry1Ac binding proteins and their functional role as native receptors in the midgut of Chilo suppressalis were studied with pull-down assays and RNA interference (RNAi). The results will provide theoretical foundation for understanding the mode of action of Bt CrylA toxins to Chilo suppressalis.1. Separation of CrylAc binding proteins of BBMV in C. suppressalisCry1Ac toxin binding proteins in BBMV prepared from midguts of C. suppressalis were captured under non-denaturing conditions using modified Pull-down method and bio-tinylated CrylAc toxin. These binding proteins were separated by SDS-PAGE and detected by western-blot analysis. They were detected in the 10 gel bands with molecular weight of 350,270,200,160,140,70,60,48,40 and 37.5 kDa, respectively.2. Identification of CrylAc binding proteins of BBMV in C. suppressalisThe proteins from each of the 10 gel bands were separated and identified by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Three categories of protein were detected. Some proteins were indentified as Cry1Ac toxin candidate receptors which have been reported to be functional receptors in other insect speicies, including cadherin from the 60 kDa band, aminopeptidase N3 (APN3) and aminopeptidase N (APN) from the 200,160 and 140 kDa bands, and aminopeptidase N4 (APN4) from the 45 kDa band. Some proteins were identified as the Cry1Ac-binding proteins previously reported in other insects, such as V-ATPase subunit and heat shock protein (Hsp). In addition, a large amount of identified proteins were related to metabolism and cell signals, but their involvement in toxin binding and mode of action was not clear.3. Function verification of binding protein APN3 and APN4 as Cry1Ac receptors in C. suppressalisRNAi-mediated gene silencing was used to further verify if the identified Cry1Ac-binding protein APN3 and APN4 are functional receptors. The results showed that the expression of apn3 and apn4 in the larvae fed with dsapn3 and dsapn4 were reduced by 50% and 46% compared respectively to that fed on dsGFP. The larvae fed with dsapn4 had significantly lower mortality than those fed with dsGFP (a positive control) when treated with 0.8μg Cry1Ac/100 μl. However, the larvae fed with dsapn3 or dsGFP showed no significant difference in mortality. It suggests that APN4 may act as a functional receptor of Bt toxin Cry1Ac in BBMV of C. suppressalis.
Keywords/Search Tags:Chilo suppressalis (Walker), Cry1Ac, Binding proteins, Receptor, Aminopeptidase
PDF Full Text Request
Related items