Mechanism Of Nicotinic Acetycholine Receptor Pathway By Periplaneta Americana Deoxypodophyllotoxin

Deoxypodophyllotoxin is a derivant of podophyllotoxin which belongs to lignan. It was found that deoxypodophyllotoxin could showed an effect, such as antifeedant, growth inhibition and toxic on the insects. Study on the mechanism of the deoxypopophyllotoxin as drug indicated that it had an affect on ion channel, cytoactive and cytokines, and produced antineoplastic, antiviral and antiinflammatory function. However, no specification study on the mechanism of intracellular so far. Based on the previous results, we study the mechanism of "nicotinic action" symptoms. We gene cloned partial sequence of nicotinic acetylcholine Receptor α6, calmodulin and Ca2+/calmodulin-dependent kinase Ⅱ and studied the expression level of m RNA from Peripleneta americanas treated with deoxypodophyllotoxin, and the research is to prepare for the study on protein fuction change.Part One Insecticidal activity of deoxypodophyllotoxin and the symptoms of Periplaneta americana by deoxypodophyllotoxin[Objective] To observe the symptoms of Periplaneta americana treated with the deoxypodophyllotoxin in microsyringe, and collect the death data of insect. [Method] 3μL deoxypodophyllotoxin of each solution was injected through 3th and 4th segmacoria with microsyringe. The solution was dissolved in DMSO and diluted to a series concentration before it was applied to insect. Observe the symptoms and collect the death data each 24 hours. [Result] The data was imported to professional software DPS,and the software would work out at LD50. It was 0.66 mg each insect at the 2nd day, and decreased to 0.4mg each insect at the 8th day. After 2d, the trend turned to slow down the following days. After treated with deoxypodophyllotoxin, some insect showed overexiciting, stagnancy, paralysis and death, others could be stagnancy at beginning and return to normal at last or some would be no change. [Conclusion] Insecticidal activity on periplaneta americana is time dependance. Deoxypodophyllotoxin can be cumulative action, which depends on the concentration of drug treated to insect. The "nicotinic action" showed that the drug may have affect on insect just like imidacloprid, however insects treated with imidacloprid can return to normal even if it was paralysis before, this phenomenon is different from those treated with deoxypodophyllotoxin. It means that mechanism of deoxypodophyllotoxin is different from imidacloprid.Part Two Gene cloning of three signal molecules n ACh R, Ca M and Ca MKⅡ[Objective] To study the expression level of critical signal molecules on nicotinic acetycholine receptor pathway after deoxypodophyllotoxin was applied, partial m RNA of n ACh R, Ca M and Ca MKⅡwas cloned and sequenced. [Method] Total RNA was isolated from head of P. americana, and then total RNA was reverse transcriptased to c DNA. c DNA was applificated, purified with degenerate primers and semi-nested PCR techonology before it was cloned to E.coli and sequenced. The three sequences were analysed by NCBI Blastn with m RNA of other insect in Gen Bank. [Result] Sequenced m RNA of n ACh R was subunit α6, the length of sequence is 539bp; length of Ca M m RNA sequence is 435bp; length of Ca MKⅡ m RNA sequence is 513 bp. The accession number of the three sequence in Gen Bank is KC473398,KC473397 and KC473399 respectively. Identity of n ACh R alpha6 between P. americana and Tribolium castaneum is 84%, which is the highest of all; identity of Ca M between P. americana and Graphocephala atropunctata is 83%, which is also the highest; and highest identity of Ca MKⅡ was between P. americana and T. castaneum, that is 79%. The identity can somehow showed on the gene phylogenetic tree of the three molecules. [Conclusion]Three sequences can be identified. And three sequenced m RNA make it possible to explore the change of expression level with Real Time PCR teconology.Part Three Expression level change of n ACh R, Ca M and Ca MKⅡ after the insect was treated with deoxypodophyllotoxin[Objective]Explore the expression level change of n ACh R, Ca M and Ca MKⅡ after the insect was treated with deoxypodophyllotoxin. [Method] Total RNA was isolated after periplaneta americana was treated with deoxypodophyllotoxin.Expression level of male and female periplaneta americana which were collected at different time, was analysed by real time PCR teconology with special primers of each sequence. [Result]Expression level of five experimental groups(10-150μg/μL) increase significantly when the insect injected with low concentration of deoxypodophyllotoxin, and decrease while injected with high concentration of deoxypodophyllotoxin. [Conclusion] n ACh R pathway may involved in DOP toxic mechanisms to the P. americana.