The Pharmacological Properties Of Acetylcholinesterase In Pardosa Pseudoannulata, An Important Predatory Enemy Spider Of Insect Pests

Acetylcholinesterase (AChE) is one kind of serine hydrolase and the primary function is suspending the transmission of nerve impulses by hydrolyzing neurotransmitter. acetylcholine (ACh), rapidly at synapses and interrupt the nerve impulses. AChE is the main target of organic phosphorus and carbamate insecticides. The enzyme activity would be passive after the binding of insecticides and enzyme molecular. This binding can cause acetylcholine accumulation at synapses and keep stimulating the receptors. The receptors would be hypererethism for a long time and nerve impulses conduction would be blocked (depolarization blocking). Therefore. the process of insect physiology and biochemistry would be disordered and destroyed, eventually leading to the death of insect. Pardosa pseudoannulata is one of most important predatory enemies against insect pests and play important roles in pest ecological management. So, the selectivity of insecticides between insect pests and P. pseudoannulata is important to obtain the sufficient control by this predatory enemy.In order to understand the insecticide selectivity between predatory enemy spiders and insect pests.2 acetylcholinesterase genes of P. pseudoannulata. AChEl and AChE2. were cloned. The expression patterns in different tissues were also analyzed. In order to study the pharmacological properties of two cloned AChEs. the recombinant proteins were obtained by expressing two genes in insect cells. The results in the present study will provide important evidences in insecticide selectivity between insect pests and predatory enemy spiders, and will also provide useful information to help the molecular design of selective insecticides. 1. Cloning and analysis of two kinds of acetylcholinesterase genes in P. pseudoannulataUsing PCR and RACE techniques, we have cloned 2 full length genes of AChE in P. pseudoannulata, a primary predatory enemy of insect pests. Derived from the first AChE gene, the amino acid sequence contains two conservative regions of insect acetylcholinesterase. which are "WIY(F)GGG" and "TLFGESAE". Derived from the second AChE gene, the amino acid sequence contains a conservative regions of insect acetylcholinesterases, which is "WIY(F)GGG". According to homologous comparison and phylogeny relationships, two new genes were denoted as Pp-acel and Pp-ace2.In order to understand the molecular mechanisms of insecticide selectivity between P. pseudoannulata and insects, the amino acid sequences of insect AChEs and P. pseudoannulata AChEs are compared to find out the key amino acid differences in AChEs between P. pseudoannulata and insects. At the point mutation sites in insect AChEs that are associated with insecticide resistance, several amino acid differences were found between insect AChE2 and P. pseudoannulata AChE2. At the putative important sites for AChE functions, several amino acid differences were also found between insect AChEs and P. pseudoannulata AChEs. These key amino acid differences between insect and P. pseudoannulata AChEs may play important roles in insecticide selectivity between insect pests and predatory enemy spiders, which need further studies to provide direct evidences.2. The expression levels of two AChE genes in different tissues of P. pseudoannulataThe transcript levels of Pp-ace1 and Pp-ace2 in several tissues were determined with real-time PCR techniques. Although two transcripts were identified in most tissues tested, the expression levels of Pp-acel were higher than Pp-ace2 in several tissues, which is consistent with the found in two insect AChE genes. Two AChE genes were highly expressed in P. pseudoannulata legs. The expression level of Pp-acel in legs is 9.8 times of that in abdomen and the time for Pp-ace2 is 2.2 between legs and abdomen. Meanwhile, high expression levels of Pp-ace1 were also found in cephalothorax. digestive tract and heart, and high levels of Pp-ace2 in digestive tract. For both Pp-ace1 and Pp-ace2. no or very slight levels were found in poison gland and fat body. The expression levels in legs differed mostly between Pp-ace1 and Pp-ace2 with the Pp-ace1/Pp-ace2 ratio of 4.5. The Pp-ace1/Pp-ace2 ratio in expression levels is in the range of 1-2.33 in cephalothorax. digestive tract, abdomen, heart and ovarian. 3. The expression of two AChEs in Bac-to-Bac systemsTwo P. pseudoannulata AChEs were successfully expressed in insect sf9 cells using Bac-to-Bac systems. The activities of two recombinant proteins from Pp-acel and-Pp-ace2 reached the highest levels in 72 h after the transference and declined quickly after 96 h. Therefore, we collect the culture medium after 2-3 d when the cells were infested to perform the further experiments. The result indicated that the activity of the recombinant Pp-acel is significantly higher than that of Pp-ace2. The inhibition tests indicated that the expressed Pp-acel is more sensitive to all insecticides tested than Pp-ace2. Although the sensitivity to fenitrothion of the expressed Pp-ace1 is close to that of Pp-ace2, the sensitivity to isoprocarb and fenobucarb of Pp-acel are 31 and 54 times of those of Pp-ace2. Two recombinant proteins are most sensitive to fenobucarb. especially for the recombinant Pp-ace1. The sensitivity of the expressed Pp-acel to fenobucarb is 188 times of that to fenitrothion.