Resistance Molecular Mechanism Of Tetranychus Urticae (Koch) To Fenpropathrin

Tetranychus urticae (Koch) is the most serious mites on fruit, vegetables and crops, pyrethroids chemical were used control the mites a long time. Pyrethroids chemical has become the main pesticide of mite control on fruit trees in Gansu province, because of the high toxicity of its, and environmental compatibility, and the security of natural enemies. But high-dose and high frequency use of these acaricides resulted in the development of resistance in T.urticae Resistance of T. urticae is caused by the increases in the activity of metabolic detoxification of the insecticide and mutations of target gene. At the molecular level in the three cases can lead to the development of resistance to pesticides, mutation and gene amplification and overexpression. Based on laboratory breed susceptible(S) and fenpropathrin-selected strains (Fe-R) T. urticae, Cloned and compared cDNA of the T. urticae S and Fe-R strains sodium channel gene, esterases gene, cytochrome P450and glutathione s-transferases sequences, in susceptible and fenpropathrin-resistant T. urticae form in molecular level were carried out to clone of sodiun channel gene (the target of pyrethroids) and different detoxification enzyme genes(esterases, cytochrome P450and glutathione s-transferases), to analyze sequences, to relative expression pattern of the different genes, and to reveal the molecular mechanisms of fenpropathrin resistance in T. urticae. For the resistance of the mites management to provide the theory basis.The main results are as follows:1. Cloned two5419bp cDNA fragments (domains I-IV) of the T. urticae para sodium channel gene, encoded1805amino acids (GenBank Accession JN881331and JN881332). Optional exon (SV1) and alternative exon (SV2) were all found in SS and FeR strain VGSC gene.A comparison of the sequences between resistant and susceptible mites identified two changes that were founde only in the resistant strains: a phenylalnine to isoleucine substitution (eqivalent to F15381) in segment6of domain Ⅲ and an alanine to aspartic acid substitution (eqivalent to A1215D) with Ⅱ/Ⅲ intracellular linker region.and was phylogenetically classified within the divergent group of arachida. The T. urticae sodium channel fragement showed highest similarity to other species of arachnida:96.12%with Tetranychus cinnabarinus,51.36%with V. destructo,33.83%with Boophilus microplus.2. The esterase complementary DNAs was identified and cloned from the insecticide-susceptible strain of T. urticae, The complete amino acid sequence of TuCCEl deduced from the cDNA is1869residues with the putative signal peptide consisted of23residues with a predicted molecular weight of88.39kDa and an isoelectric point (pI) of5.7. which was designated as TuCCE1, The cDNA of TuCCE1gene contained an open reading frame (ORF) of1683bp encoding560amino acids (GenBank JN034058), The deduced amino acid sequence of the TuCCE1gene both had the Esterase-lipase and CO esterase.Comparison of the deduced amino acid sequence with the published mite esterase sequence coming from TuCCE1showed highest identity (98%) to the Tetranychus cinnabarimts and Panonychus citri. The TuCCE1had60%amino acid identity to Ixodes scapularis esterase1, and the TuCCE1had32%-36%amino acid identity to the other animal esterase.The predicted amino acid of the esterase gene contained some typical residues of AChE (the three residues that putatively form the catalytic triad, the oxy-anino hole and the sequence GESAG). The esterase gene possess Carboxylesterases type-B signature of F-[GR]-Gx(4)-[LIVM]-x-[LIV]-xGxS-[STAG]-G (S is the active site residue). The corresponding amino acid residue of TuCCE1gene was FGGNPDQVTIFGESAG. And the conserved cysteine that compose disulfiede bridge (EDCLFLNVIVT).Molecular phylogenetic tree of estesrases amino acid sequences from some representative insects and mites were constructed. The results showed that Tetranychus cinnabarinus and Panonychus citri esterase gene belong to the same original group showing the highest identity to the T.urticae was esterase gene.3. The cDNA of cytochrome P450gene contained an open reading frame (ORF) of1533bp encoding510amino acids (GenBank JQ860112), start codon (ATG), stop codon (TAA). A predicted molecular weight of82.98kDa and an isoelectric point (pI) of7.28. Contains the classic hemen-binding sequence motif FSGGKRICPG (446-455), The K helix ESLR (371-374) and a high hydrophobic signal sequence in amino terminus in the CYP3gene. Homologous analysis showed Ixodes scapularis has33%amino acid identity to CYP3gene.4. Two novel GST genes in Omega and Delta families were cloned from T. urticae by using RT-PCR strategy. The two GSTs were designated as TuGSToland TuGSTdl. The TuGSTol cDNA is717bp in length with an open reading frame(ORF) coding for238amino acid, and a predicted molecular weight of38.77kDa and an isoelectric point (pI) of5.89. The TuGSTdl cDNA is678bp in length with an open reading frame(ORF) coding for218amino acid, and a predicted molecular weight of34.23kDa and an isoelectric point (pI) of5.14.The Omega class GSTs have a unique N-terminal extension, ESLVIVNYL and C33of GSH-two features of GSH binding distinguish TuGSTo from typical GSTs. The presence of S11and N49key residues are important for Delta class, and P56-L142-G150-D157may be reflect to key residues for overlap form of protein.Phylogenetic analysis showed that TuGSTol and TuGSTdl genes could be classified into the subfamily of omega and delta class of GSTs. According to the GST sequences of different species could be classified into11clades. The TuGSTol had30%amino acid identity to Bombyx, and the TuGSTdl had63%amino acid identity to Panonychus citri.5. Eight candidate genes has been cloned from T. urticae using RT-PCR technology, and submitted them to the GenBank, GenBank number and length were ELF (JN881325)658bp,5.8SrRNA371bp, GAPDH (JN881330)335bp, RPL13a (JN881328)242bp, TBP (JN881326)365bp, SDHA (JN881329)343bp, a-TUB (JN881325)962bp, β-actin (JN881324)822bp, respectively.The qrtPCR primers were designed and the stability of gene expression was assessed using two different analysis programs, geNorm and NormFinder. In this study, GeNorm recommends that the ranking of gene expression stability value (M) of a-Tublin, SDHA, RPL13a, GAPDH, ELF,5.8s rRNA and TBP were0.381,0.537,0.609,0.625,0.639,0.688,and the ranking of gene expression stability were a-Tublin+SDHA<RPL13a<GAPDH<ELF<5.8srRNA<TBP in different strains, Genes that were most stably expressed were indicated by lower average expression stability values. a-Tublin was the most stable genes for different strains. We also found a good agreement in the reference genes ranking between GeNorm and NormFinder as they both ranked a-Tublin as the best stable reference genes under different strains, this work may act as a starting point for future gene expression study in T.urticae.6. To verify whether sodium channel gene, esterase gene, P450gene and GSTs genes are over-expressed in fenpropathrin-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), fenpropathrin resistant strain (Fe-R). The results showed that the transcript levels of sodium channel and TuCCEl were significantly lower than in susceptible strain, in which sodium channel and TuCCEl had0.301and0.654-fold higher transcript level than that of S strain. Compared with S strain, CYP3and TuGSTol and TuGSTdl genes mRNA expression of the fenpropathrin-resistance(Fe-R) strain were3.296,3.243and6.529, The mRNA expression levels of CYP3D2and TuGSTol and TuGSTdl genes in Fe-R of T. urticae significantly higher than that of S strain.To sum up, Resistance molecular mechanism of T. urticae (Koch) to fenpropathrin, Not only with target-site resistance mutation, but also to the CYP3D2, TuGSTol and TuGSTdl gene expression about the amount of mRNA rise. The results showed that T. urticae was exist in multiple resistance mechanism to fenpropathrin.