| The carmine spider mite, Tetranychus cinnabarinus, with characteristics of strong reproductive capacity and short generation cycle, has been a serious threat to the production and development of Chinese agroforestry. Although there are many methods to control mite pest, but in the near future period, the control of this mite is still based primarily on the use of synthetic chemical acaricides. Long-term chemical control led to the "3R" has been a serious problem, so the search for a new type of multi target, low resistance acaricidal substances is an important subject to be carried out. Botanical acaricide, safer, environmentally friendlier and more efficient alternatives, has become an important option for screening new acaricide. The study found that scopoletin extracted from Artemisia annua possess a good acaricidal activity, has the potential to be a botanical acaricide. According to the previous studies and the literatures reports, T. cinnabarinus showed obvious nerve symptoms after scopoletin treatment, also suggested that Ca2+-ATPase was one of the major targets of scopoletin.Therefore, based on finding new acaricide to play the acaricidal activity of scopoletin, this study aims to achieve the target location of scopoletin, to clarify its acaricidal mechanism of scopoletin, as well as to lay the foundation for further development and utilization of scopoletin.In this study, molecular biology techniques was used to characterize the biological characteristics of Ca2+-ATPase of carmine spider mite, studied the changes in gene expression levels of T. cinnabarinus Ca2+-ATPase after exposed to scopoletin at various concentrations, cloned the full-length of TcPMCAl gene associated with scopoletin’s targets, and the expression pattern of TcPMCAl was also analysised. With computer-aided technology and structural biology software, the 3-D model of TcPMCAl was builded, determined the binding cavity of the TcPMCAl, eventually deduced the binding sites of scopoletin. Discussed the target sites of scopoletin to T. cinnabarinus Ca2+-ATPase with a molecular level, and laid the foundation for the further development and utilization of scopoletin. The main results are as follows:1. Characterized the biological characteristics of Ca2+-ATPase in T. cinnabarinus, including the optimal temperature, pH and the impact of common enzyme activators, inhibitory effect in vitro of scopoletin on Ca2+-ATPase also was measured.The optimal temperature and pH of T. cinnabarinus Ca2+-ATPase appeared to be 35～38℃ and 6.5 ～1.2, respectively. Initially identified the activation effect of some common enzyme activators Mg2+, Mn2+, Ca2+, Na+, K+, Cu2+, Zn2+, Co2+on Ca2+-ATPase. Scopoletin has a good acaricidal activity, the median lethal concentration (LC50) is 1.142mg/mL against female adult of T. cinnabarinus in 24h. After exposed to scopoletin with different concentrations, Ca2+-ATPase activity of T. cinnabarinus decreased in different extent, which showed that scopoletin has inhibitory effect on Ca2+-ATPase, and the inhibition rate reached a maximum of 80% at scopoletin 31μM. Scopoletin played a considerable inhibitory effect compared with the specific inhibitor vanadate. With the addition of scopoletin and vanadate, the Km and Vm values of Ca2+-ATPase increased to 1.52× and 0.66x in the presence of scopoletin compared with the control.2. Study the gene expression changes of T. cinnabarinus Ca2+-ATPase under scopoletin induced. Induction results of scopoletin indicated that all of the four Ca2+-ATPase genes produced significant stress response, which TcPMCAl, TcPMCA2 and TcSPMCA2 genes were significantly increased, while TcSERCAl gene expression showed a trend first and then decreased as a whole. From time and dose-effect perspective, TcPMCAl gene incense scopoletin most sensitive, performed by the highest relative expression levels reached to 142-fold after 8h exposure to scopoletin at 1.25mg/mL,119-fold after 24h exposure to scopoletin at 0.31 mg/mL. Compared with the control, the relative expression of TcPMCA2, TcSPMCA2 and TcSERCAl reached a maxmine of 73.1-、70-and 140-fold, respectively. Inferred, the acaricidal effect of scopoletin is closely related to TcPMCAl gene.3. Use the transcriptome database and further aligned with the nucleotide sequences from genome datasets of T. cinnabarinus, allowed us to isolate a partial cDNA encoding PMCA1. The remaining 5’and 3’ends were then amplified by developing a RACE/ PCR-based strategy. The full-length cDNA sequence named TcPMCAl and deposited in the GenBank database with accession number KP455490. This cDNA of 4,369 bp contains an open reading frame (ORF) of 3,750 bp. The open reading frame encodes 1249 amino acids residues with a predicted molecular mass of 137.7 kDa and an isoelectric point of 8.10. Analysis of the deduced amino acid sequence revealed the presence of ten membrane-spanning segments (TM) numbered from TM I to TM X. Additionally, the PMCA1 gene from T. cinnabarinus and T. urticae clustered into the PMCA1 family and seemed to share a single clade. This result indicated that similar physiological functions and evolutionary relatedness may exist between the PMCA1 in T. cinnabarinus and T. urticae. This observation is highly supported by the finding that TcPMCAl exhibits 99.7% amino acid sequence identity with T. urticae PMCA1. The results showed that TcPMCAl mRNA was expressed in all stages, suggesting that TcPMCA1 has roles in biological processes throughout all developmental and growth stages. The relative expression levels of TcPMCAl is nymph> adult> larva> egg.4. Construct the three-dimensional structure model of TcPMCAl. Amino acid sequences of TcPMCAl subunit was put in PDB database for a BLASTx search, obtained more than one appropriate template, which Ca2E1P phosphoenzyme intermediate of the in rabbit SERCA Ca2+-ATPase got the highest score,73% sequence similarity. By the conformation analysis of TcPMCAl, the presence of the protein subunit three active binding cavities were found, corresponding protein N-domain, A-domain and P-domain, respectively. Molecular docking was performed using Autodock software, obtained the scopoletin binding conformation in TcPMCA1 activity cavity B which is the active domains (A-domain) of the protein. Carbonyl and the adjacent aromatic ring of scopoletin lead to protein located in the catalytic domain of the active site entrance, and the remainder of this compound is embedded into the channel. The docking results show that, scopoletin mainly interacted with three amino acid residues of TcPMCA1. Among them,11 carbonyl oxygen atoms of scopoletin played a H-bonding with Ala-298 amino acid of TcPMCAl, the binding energy is 2.93; 13 methoxy oxygen atoms of scopoletin played a H-bonding with Lys-301 amino acid of TcPMCA1, the binding energy is 2.99; while the other part of scopoleyin bounded with Cys-299 amino acid residues with π-π conjugated effect.In conclusion, the above research results preliminary cleared the acaricidal mechanism of scopoletin against Tetranychus cinnabarinus. Through hydrogen bonding and π-π conjugated effect, scopoletin bounded to TcPMCA1 subunit, caused active channel blocking, spatial structure destroyed of TcPMCA1, then the substrate can not enter the active center of the enzyme, resulting enzyme activity losed. Finally scopoletin play the role of nerve agent, resulting in the death of mites. This study lays a foundation for the future development and utilization of scopoletin... |
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