Proteogenomic Analysis Of The Midgut Of Near Isogenic Plutella Xylostella (Lepidoptera:Plutellidae) Strain Resistant To Cry1Ac Toxin

Bt crystalline toxins, produced by the bacteria Bacillus thuringiensis, are the most widely used biological insecticide in pest management. Genes encoding Bt insecticidal proteins are commonly incorporated into transgenic crops to protect plants from insect feeding damage. The widespread use of Bt foliar sprays and transgenic Bt crops provide the opportunity for the evolution of Bt resistance to arise in populations of targeted insects. Understanding resistance mechanisms will provide new insights into the mode of action of Bt toxins to develop the effective management of resistance, which is still controversial. Plutella xylostella (L.) was the first of six lepidopteran species to develop field resistance to insecticidal Bt toxins. Different populations selected for Bt resistance often show diverse responses in terms related to resistance mechanism.In order to elucidate the mechanism of resistance to Cry1Ac, the near-isogenic P.xylostella with>5,000-fold resistance were constructed. And the biology characterization of the near-isogenic resistant strain was also studied. In addition, we carried out an in-depth proteogenomic analysis using shotgun HPLC-ESI-MS/MS approach to identify genes and gene networks putatively involved in various physiological and toxicological processes in the P. xylostella larval midgut. And then iTRAQ and Multiple Reaction Monitoring (MRM) were used to discover different expression proteins between the two strains. Many different expression proteins and candidate receptors were discover. The results were as follows:1. Construction of near isogenic P.xylostella strains resistant to CrylAc toxinTo elucidate the mechanism of resistance to Bt CrylAc toxin, we developed near-isogenic line (NIL) strains with>5,000-fold resistance. Two pairs of NILs were generated using either backcross or recombinant inbred line methodologies and then evaluated for near isogenicity using ISSR markers. One backcross line, BC7F3(renamed NIL-R), showed98.24%similarity to the susceptible parental strain, DBM1Ac-S, suggesting its optimal use for resistance characterization studies.2. Characterization of near isogenic P.xylostella strain resistant to CrylAc toxinThe cross-resistance spectrum in the NIL-R strain suggested high levels of cross-resistance to Cry1Ab and Cry1Ah but no cross-resistance to CrylCa or Cry1Ie. The interpretation of the overall data seems to suggest the involvement of an alteration in the binding of CrylA toxins to a common receptor, not recognized by CrylCa or Cry1Ie toxins. Three times of life tables and comparisons of the biological parameters were investigated to minimize the effects of environmental factors and personal error to obtain more real and accuracy date on the fitness of resistance. There were no significantly difference in the development of egg, larvae, the longevity of adult and fecundity of every female adult between the near isogenic resistant strain NIL-R and susceptible strain with DBM1Ac-S, which indicated the Cry1Ac-resistant P.xylostella strain did not lead to developmental asynchrony in our stains. And then the net reproductive rate (R0), the gross reproduction rate (GRR), the finite rate of increase (λ) and the mean generation time (T) of the two strains were similar. The relative fitness of P.xylostella, measured as a ratio of Ro (the net reproductive rate) of resistant strain divided by Ro of the susceptible strain, were ratios of1.02,0.94and0.93respectively in repeats of life table study, which reflected the lack-of-fitness costs of Cry1Ac resistance in NIL-R strain. Single pair genetic cross analyses between the two strains revealed that Cry1Ac-resistance in DBM was controlled by a single, autosomal, recessive locus. The experiment of inheritance of resistance based on the log dose-probit line indicated that resistance might be controlled by a single locus and autosome and be incompletely recessive with degree of dominance of F1and F1’ progeny were-0.74and-0.71, respectively. These informations provide reference in the study of resistance to Cry1Ac proteins in P. xylostella.3. Proteogenomic analysis of the larval midgut by shotgun ESI-MS approachWe carried out an in-depth proteogenomic analysis using shotgun HPLC-ESI-MS/MS approach to identify genes and gene networks in the P. xylostella larval midgut. A total of876,341tandem mass spectra were searched against a database of predicted P.xylostella protein sequences generated from public databases, and a whole-genome six-frame translation. This search identified15,887distinct peptide sequences, total5270proteins (including58containment protein), and a comparison with the publicly available proteome from the published P.xylostella genome identified1,568new peptides suggesting corrections or additions to the current annotations. In-depth analysis identified proteins putatively involved in nutrient digestion and insecticide resistance. Presence and expression at high levels of numerous enzymes of absorption and transport of fatty acids and lipids and glycolysis pathway indicate that active metabolism processes of carbohydrates, and lipids occurred in the larval midgut of P. xylostella. 4. Comparative Proteomics Analysis on the near isogenic P.xylostella resistant to Cry1Ac toxinThe iTRAQ and multiple reaction monitoring (MRM) were used to discover different expression proteins between the near-isogenic resistant strain and susceptible strain. Based on iTRAQ,128different expression proteins were obtained. Notably, one aminopeptidase N and one ATP-binding cassette were found to express lower in resistant strain than susceptible strain, which were reported as Bt receptor before. This may make some evidences that aminopeptidase N and ATP-binding cassette are the receptors to CrylAc in P.xylostella. And then one glucosyltransferase and two enzymes related to medication (methyltransferases and phosphatase) were also found to express higher. There were also two detoxifying enzymes expressing in higher level. The results evaluating by MRM agreed with these from iTRAQ. In addition, some candidate Bt receptors reported in other species were analyzed in these two P.xylostella strains. Finally, all the candidate receptors including aminopeptidase N, ATP-binding cassette, Alkaline Phosphatase, cadherin and beta-1,4-Ga1NAc transferase bre were found had low expression in resistant strains, which may indicated that these receptors may all play some role in the process Cry1Ac resistance in P. xylostella.