Cloning And Functional Studies Of Nicotinic Acetylcholine Receptors α7 Subunit In Helicoverpa Armigera(hübner)

The cotton bollworm, Helicoverpa armigera(Hübner)(cotton bollworm), is a major insect pest which can do great damage to many crops like cotton, maize, wheat and flowers and vegetables. The widely planting of Bt cotton has made great success to control the cotton bollworm, as well as increasing selective pressure from the Bt, thus the resistance risk to Bt of cotton bollworm also increased. The expression level of Bt insecticide protein decreased in the later growth stage of Bt cotton, so it still need spray chemical insecticides to control the third and fourth generation of the pest. Though the cotton bollworm has become less resistant to the pesticides because of the planting of Bt cotton, it still remains certain resistance level to traditional pesticides according to the years of field resistance detecting data.Spinetoram, a second-generation product of spinosyns, has a ten times of insecticidal activity more than the spinosad. It has great control effectiveness to the cotton bollworm. With the property of readily availability and the safety as a biological pesticide, it was recommended environmentally friendly and had a low toxicity and no residual. Because of the novel action mechanism and being no cross-resistance with the traditional pesticides, it was greatly accordance with the new pesticides developing trend. It will be helpful to discover new pesticide to study the action mechanism of spinetoram.The transcriptome data in the treatments of spinetoram indicating the m RNA expression level of the n Ach Rs(nicotinic acetylcholine receptor) α7 subunit increased prominently. Hence, we have cloned n Ach Rs α7 subunit, analysized its expression profile and performed preliminary study on function. The full length of n Ach Rs α7 subunit gene in H. armigera was cloned using degenerate primer combined with RACE(rapid amplification of c DNA ends) technology. We found that the full length of the c DNA sequence was 3632 bp(Gen Bank accession number KM884875), the ORF was 1491 bp which encoded a protein of 621 amino acid residues. It had the common character of the Cysteine ring receptor family: the N- and C-terminal ex-cytosolic tail, the four transmembrane domains TM1、TM2、TM3、TM4 and the big cytosolic ring. In the N-terminal, there were ligand binding domain, Cysteine ring composed by two Cysteine residues which were separated by 13 amino acid residues and two closely linked Cysteine residues. The ligand binding domain and transmembrane domains were relatively conserved, while the cytosolic ring varied between different species. The nucleotide sequence and amino acid sequence of n Ach Rs α7 subunit in H. armigera had a very high homology compared with Heliothis virescens, obtaining for 99% and 100% respectively. Phylogenetic tree indicated that the n Ach Rs α5、α6、α7 subunit in H. armigera were closed to the α7 subunit in vertebrate. Multiple alignment of the amino acid the sequences in different insect species showed that the sequential 12 amino acid residues(RSSKSLLANVLD) were the same, this was not occuring in other subunits. The results indicated that there may be some similar functions between these three subunits.Using q RT-PCR to compare the m RNA expression level of n Ach Rs α7 subunit in different development stages and different tissues, as well as the expression change after treated by the spinetoram in H. armigera. We discovered that the expression level in adult was significantly higher than that in egg. The adult had a 7.04 times the amount of the egg. The expression level in the other stages were low with no significant difference. In different part of the larval, the highest expression occured in the head, which had a 5.42 and 6.04 times the amount of the thorax and the abdomen respectively. The expression level increased significantly in the treatment of the LC60 dose of spinetoram, which was 2.01 times more than the control. These suggested that n Ach Rs α7 subunit may be involved in the process of spinetoram killing the cotton bollworm.In the RNi experiment, we injected two si RNA to silence the n Ach Rs α7 subunit gene expression in the 3rd larval of H. armigera and used q RT-PCR to detect the silencing effect. The results showed that the silencing effect was 40.82% and 41.35% for si RNA001 and si RNA002 respectively after injecting 48 h, and that was 73.56% and 66.39% after 72 h. We also detected the susceptibility decreasing of the cotton bollworm after RNAi-mediated silencing to spinetoram. The rate of weight inhibition of larvae injected with si RNA showed no difference compared with that of control larvae injected with RNase-free water in treatments of 1.0 μg/ml of spinetoram. These results suggested that the silencing effect of si RNA001 was better, and n Ach Rs α7 subunit was a potential site of action or the composed subunits of the target receptor.NAch Rs α7 subunit in-vitro expression had been carried out on preliminary exploration. When inject 27.6 ng c RNA of n Ach Rs α7 subunit in H. armigera alone or together with the c RNA of n Ach Rs β2 subunit in mouse with the ratio of 1:1 to the Xenopus oocytes, we recorded changing currents using the two-microelectrode voltage-clamp technique.In conclusion, these results suggested that n Ach Rs α7 subunit may play an important role in the process of spinetoram killing the cotton bollworm. To further explore the interaction between spinetoram and n Ach Rs α7 subunit, and the pharmacological property by constructing the Xenopus oocytes cell expression, and this would provide theoretical basis to study the interaction between the various subunits and insecticidal mechanism of spinetoram.