The Expression Of Alkaline Phosphatase In Cry1Ac-resistant Helicoverpa Armigera And Synergism Of Its Toxin-binding Region Fragment To Cry1Ac Toxicity

The Cotton bollworm, Helicoverpa armigera (Hübner), is a major and most serious insect pest inthe cotton-growing regions of China. Transgenic cotton that expresses a gene derived from thebacterium Bacillus thuringiensis has been deployed for combating cotton bollworm since1997. Greatsuccuss has been obtained in recent years with the planting of Bt cotton for control of cotton bollwormin China. Because of the Bt gene in plant expression continued in the whole growth period, Cottonbollworm throughout the growth cycle are derived from Bt insecticidal protein selection pressure.Although, so far, no cotton bollworm resistance to a Bt crop has been reported from the field, theselection experiments in the laboratory indicated that at least10species of insects are at the potentialrisk of being able to resist Cry1Ac toxin, and there also are some reports about H. armigera. Thisindicates that the potential crisis is the resistant evolution of Helicoverpa armigera.In order to effectively delay the Bt resistance generation, a lot of research on Bt mechanism hasbeen done at home and abroad. At present, a number of insect midgut proteins have been identified asputative Cry1A toxin-binding receptors on midgut epithelial cells in many lepidopteran species,including cadherin, aminopeptidase-N(APN), and alkaline phosphatase (ALP). Binding of the Crytoxins to specific receptors of the larval midgut is required for toxicity. Alterations of toxin-bindingreceptors are reported as main mechanisms of Bt resistance. Therefore the expression and enzymeactivity of the receptor protein were associated with the generation of Bt resistance. In order to increasethe insecticidal activity of Cry toxins and ensure its sustainability, the use of synergistic factors forenhancing the insecticidal efficacy and delaying the resistance are reported in many papers. The reportsshowing that the toxin-binding region fragment of receptor could significantly synergist the toxicity ofCry1A toxins, such as the toxin-binding region fragment of Cad and APN. Whereas, the studies of ALPfragments effect on Cry1A toxicity has not been reported. The results as followed:（1） The relative expression of alkaline phosphatase mRNA of Cry1Ac-susceptible strain96S andfour different Cry-resistance strains were determined using real-time qPCR. The results showedthat the relative expression levels of ALP of the four Cry1Ac-resistant strains were significantlydecreased, further validation by the enzyme activity analysis, the results showed that theenzyme activity of ALP of the four Cry1Ac-resistant strain were also significantly decreased,and with the increase of resistance ratio, the expression quantity and enzyme activity of ALPshowed a trend of gradual decline.（2） Using PCR amplification, the915bp cDNA fragment of H. armigera ALP (GenBank accessionno. EU729322) encoding the Cry1Ac toxin-binding region was cloned, encoding a predictedprotein of305amino acid residues, with predicted molecular weight of34.63kDa andisoelectric point of6.92. This fragment was expressed in E.coli cells, and formed inclusionbodies during expression. After solubilization and purification, the resultant peptide had theexpected size of37kDa on SDS-PAGE). The Western blot analysis suggested that the fragmentwas expressed and Oligomers were observed. （3） Bioassay analysis of Cry1Ac toxicity with additional ALP fragment, bioassays were performedagainst Cry1Ac-susceptible strain96S using sublethal doses of Cry1Ac toxin, the resultsindicated that ALP fragment synergisted Cry1Ac toxicity to H. armigera larvae significantly.Functional analysis of ALP fragments，a ligand blot analysis indicated that the ALP fragmentcould bind to activated Cry1Ac toxin. whereas, Western blot analysis suggested that it could notbind to BBMV and no significant Cry1Ac oligomerization was observed with the addition ofALP fragment in the BBMV.In conclusion, these results indicated that down-regulation of the ALP was associated with theCry1Ac toxin resistance in H.armigera, and with the increase of the resistance ratio, the expressionquantity and the enzyme activity of ALP showed a trend of gradual decline. This suggests that the ALPplays an important role in the evolution of resistance and is a key factor for the development ofresistance. Therefore, this result helpe exploiting this target for monitoring resistance gene frequency infield populations, and may develop an appropriate molecular-based resistance screen method. Thetoxin-binding region fragment of receptor could significantly synergist the toxicity of Cry1A toxins, sothis research can provide a method to cope with the resistance of H. armigera to transgenic cotton.