Resistance Molecular Mechanism Of Tetranych Usurticae Koch To Spiromesifen

Tetranychus urticae Koch is one of the most important worldwide pest mites, it mainly harmed a variety of fruits, vegetables and crops. Because it has variety hosts, strong adjustment, the populations of T urticae have higher intrinsic rate of growth and had developed resistance to various chemical acaricides in different levels. In recent years, it has become the most harmful pest mites in fruits and vegetables, which seriously affects the development of fruit and vegetable industry in Gansu province. Based on molecular level, the sequences of cDNA of the T. urticae SS and Sp-R strains’Acetyl-CoA carboxylase gene, cytochrome P450and glutathione s-transferases were cloned and compared, and the expression pattern of these genes were analysised. All these work were reveal the resistance molecular mechanism of T. urticae to spiromesifen which provide the theory basis to the management of resistance mites.The main results are as follows:1.Through the test of the activities of detoxifying enzymes (CarE, GSTs and MFOs) in susceptible(SS) and Spiromesifen strains (Sp-R).The result showed that the activities of detoxifying enzymes in resistance population were higher than sensitive population.The activities of CarE, GSTs and MFOs in Sp-R were1.83-,2.45-and1.47-folds of the sensitive population separately. That means the resistance of T.urticae Koch to Spiromesifen connect with the activities of detoxifying enzymes.2. The full length of Acetyl-CoA carboxylase gene from T. urticae was cloned by using the RT-PCR(GenBank Accession:JX424763), it contains7068bp encoding2235amino acids,which contain three function domains, the biotin carboxylase(BC),the biotin carboxyl carrier protein (BCCP) and the Carboxyltransferase (CT). The phylogenetic tree of9ACCase amino acid sequences from diffrenet species were constructed by MEGA5.0. The results showed the ACCase of T. urticae have a high identity with Pediculus humamus corporis.3. Two novel GSTs genes in Delta family were cloned from T. urticae by using RT-PCR strategy. The two GSTs genes were named as TuGSTdl and TuGSTd2(GenBank Accession:KC445659and KC445660). Sequence analysis showed that the full length cDNA of two GSTs genes contains648and648bp open reading frame (ORF) encoding215and215amino acids. The putative protein of GSTs gene shows predicted molecular weights of24.57kDa and24.47kDa, with a theoretical pI of6.33and5.49.The presence of S11and N49key residues are important for Delta class, P55-L142-G150-D157may be reflect to key residues for overlap form of protein. Phylogenetic analysis showed a closer relationship of GSTs with delta members of other insects, and with49%amino acid identity to Panonychus citri.4. The cDNA of cytochrome P450gene contained an open reading frame (ORF) of1719bp encoding572amino acids (GenBank Accession:KC445658), start codon (ATG), stop codon (TAA). A predicted molecular weight of65.09kDa and an isoelectric point (pI) of7.61. Contains the classic hemen-binding sequence motif FSAGPRNCIG (498-507). The K helix EGLR (425-428) and a high hydrophobic signal sequence in amino terminus in the CYP4gene. The results showed a high identity of GSTs with Aedes aegypti.5. To verify whether sodium Acetyl-CoA carboxylase gene, GSTs gene, P450genes and esterase genes are over-expressed in Spiromesifen-resistance, real-time PCR with the a-tublin as the reference gene was employed to determine the relative expression quantity of the these genes in different strains of the pesticides, including susceptible strain (S), Spiromesifen resistant strain (Sp-R). The results showed that compared with S strain, Acetyl-CoA carboxylase and TuGSTdl and TuGSTd2genes mRNA expression of the Spiromesifen-resi stance (Sp-R) strain were1.84,3.75and 5.60, The mRNA expression levels of Acetyl-CoA carboxylase and TuGSTdl and TuGSTd2genes in Sp-R of T. urticae were significantly higher than that of S strain. But the CYP4and TuCCEl were lower than that of strain.