Bt Resistance Selection, Receptor Gene Cloning And Synergism Of PTD To Cry1Ac Toxin In Plutella Xylostella

The environmentally friendly insecticide crystal proteins produced by Bacillus thuringiensis are of great scientific interest because of their potency and specificity to a wide range of insect pests. Because of their highly specific toxicity, Bt toxins offer tremendous benefits for insect pest management The laboratory selection experiments show that many pests can also adapt to Bt toxins. So far, documented cases of resistance to Bt in open-field populations of pests are limited to one insect species, the diamondback moth (Plutella xylostella). However, greatly increased use of Bt toxins, whether delivered by conventional sprays or by genetically engineered crops, raises the likelihood that pests will adapt. So growing public concern surrounding Bt use has sparked worldwide debate over current policies.A new and efficient delivery technology termed protein transduction has been proved to cargo exogenous macromolecules or charged compounds into living cells. Small sequences in some proteins, especially virus protein, have been reported as protein transduction domain (PTD). Which can deliver various molecules such as protein, DNA, liposomes, even 45nm dextran particles encapsulating magnetic beads into cells. In these research two kinds of PTDs - Tat and Rev were used to explore their interaction between PTD and Bt insecticidal protein. The main result of this paper were as follows:Two resistant diamondback moth populations with resistance ratios of 24.36 folds to Cry1Ac and 38.16 folds to Bt were achieved by the selection with CrylAc toxin and Bt. Their developmental and reproductive characteristics were studied, which showed the resistant populations possessed fitness disadvantages, including lower egg number and hatch rate, prolonged larval period and lower sex rate() compared with the susceptible one.By designing the degenerate primers and using RT PCR and RACE methods, five genes encoding APN proteins and one gene encoding partial cadherin-like protein, two kinds of Bt receptor proteins were cloned and sequenced in Plutella xylostella, The amino acid sequences of these APNs possessed the common character of APN family. Homology analysis showed those APN genes belonged to three types. In comparison with APN2 in susceptible population, eight amino acid differences were observed in the APN2 of resistant population to Bt insecticide and fifteen amino acid difference were observed in resistant population to CrylAc toxin. One fragment of 427bp cDNA sequence of the cadherin-like protein gene in the fourth instar larvae of susceptible population of diamondback moth was amplified, cloned, sequenced and analyzed using RT-PCR method. Homologous analysis of cloned cDNA amino acid sequences of cadherin-like protein in diamondback moth and corresponding sequence of other source of amino acid sequences of cadherin-like protein revealed that there is high degree of amino acid sequence homology between the diamondback moth and other insects.Double-stranded oligomeric nucleotide encoding 11 amino acid Tat and 17 amino acid Rev protein transduction domain were synthesized, and fusion genes- pET21b-Tat-Cry1Ac, pET21b-Rev-Cry1Ac and pET21b -CrylAc with 6His recombinant were constructed and expressed in E.coli. The highlyexpressed Tat-CrylAc-6His , Rev-Cry1Ac-6His and Cry1Ac-6His fusion protein were purified by Ni2+-NTA-agarose. The LC50 of the three purified fusion proteins to resistant and susceptible diamondback moth were determined. Which showed Tat and Rev synergized Bt 4.84 and 1.29 fold to susceptible population,and 9.69 and 4.99 fold to resistant population,respectively. The synergistic effect of PTD to Bt as well as its potential to overcome Bt resistance was first identified. We also first used the PTD in the field of insecticidal protein and found a mew way to overcome insect resistance by using the carrier ability of PTD.