Effects Of Curcumin On Chitinase Activity And Expression Of Chitinase Genes From The Carmine Spider Mite,Tetranychus Cinnabarinus(Boisduval)

The carmine spider mite, Tetranychus cinnabarinus(Boisduval), is an economically important pest distributed worldwide and infests a large number of host plants. Owing to the characteristics of short maturation period and high reproductive rate, plus long-standing using a large number of chemical acaricides, the carmine spider mite has led to the problem of retard residue, resistance and resurgence. Therefore, it is urgent to find new acaricides with efficiency and low toxicity. Phytogenic acaricides, which is water soluble, and low toxicity to mammals, can produce non-residual effect on environment and can be biodegraded by non-target organisms; hence, these compounds are suitable for applying in integrated mite management. A number of studies have shown that curcumin, the major yellow pigment extracted from Chinese medicine plant Curcuma longa, can be an acaricide. To verify the acaricidal activity of curcumin, Pesticides Labs of natural products have done a lot of bioassay experiments. Early studies found that the mites lost much water and their bodies shrank after exposure to curcumin, and these symptoms are closely related to the integument. Chitin is the principal component of the cuticle, and chitin synthases and chitinolytic enzymes work hand in hand in remodeling chitinous structures. However, most studies of the mechanism of curcumin concentrated on the changes of detoxifying enzymes, and there are few researches focusing on the data of curcumin effects on chitin synthases and chitinolytic enzymes in the mite. Taking chitinase as a starting point, this paper studies the chages of chitinase, the effect on chitin degradation after using curcumin against Tetranychus cinnabarinus, and ascertains the mechanism of curcumin to Tetranychus cinnabarinus from physiological biochemical and molecular level. The main results are as follows:1. Valuated the contact curcumin against female adults of T. cinnabarinus and the various sublethal dosages. A slide-dip method was used to valuate the LC-P equation after 48 h treatment, and the equation was y =-0.474 + 1.234 x, the LC50 values was 2.424 mg/m L,95% confidence intervals was 1.715~3.733 mg/m L. The LC40、LC30、LC20、LC10 values after 48 h treatment were 1.511、0.911、0.504 and 0.222 mg/m L, respectively.T. cinnabarinus were treated with sublethal dosages, the results showed that the egg, larva stage, nymph stage and adult mites period was shortened overall trend compared with the control, and there was no significant difference, but the average life expectancy of adult female mite was significantly shorten.2. Clarified fundamental properties of chitinases from T. cinnabarinus. Improved schales method was used to study the fundamental properties of chitinases from T. cinnabarinus, including the optimum temperature and thermal stability, optimum p H and p H stability, the effets of metal ions on the enzyme activity and kinetic constants Km value. Chitinase of T. cinnabarinus’ s optimal temperature was 35℃, and it was sensitive to high temperature; The chitinase was most active at p H 7.0, and most stable at p H 6.0; enzyme activities were markedly inhibited by the presence of metal ions, but there are a few metal ions activating the enzyme; and hydrolysis of colloidal chitin Km value was 5.15 μmol/L. These findings enrich the study of the biological characteristics of chitinase from mites, and provide a theoretical basis for chitinase-based screening acaricide.Based on the determined properties, the paper further tested chitinase activity of different life stages. The results showed that the chitinase had highly activity at the nymph stage, whereas the activity was lowest in the egg stage. The chitinase specific activity in nymphs was significantly higher than the level in egg. This may be because nymphs of mites was on the molt stage, and it urgently needs chitinase to play a role in the promotion of chitin degradation. With increasing concentrations of curcumin treatment, T.cinnabarinus chitinase activity progressively decreased. After 24 h treatment, there were significant differences in chitinase activity between 2.0, 4.0 mg/m L and the control. After treatment at 4.0 mg/m L, the activity of chitinase was 32% lower than the control. It is inferred that curcumin acaricidal mechanism may be related with chitinase.3. The six c DNA sequences of chitinases of Tetranychus cinnabarinus(Tc CHTs) were cloned and analyzed. Based on the available genome sequence of Tetranychus urticae, used the technology of RT-PCR, Six CHT c DNA sequences were cloned and submitted to Gen Bank(accession numbers: KT956964, KT956965, KT956966, KT956967, KT956968, and KT956969) with names of Tc CHIT1, Tc CHIT2, Tc CHIT3, Tc CHT4, Tc CHT5 and Tc CHT6, respectively. The six CHT c DNA sequences had different domains: Tc CHIT1, Tc CHIT2, Tc CHIT3, and Tc CHT4 were predicted to have one signal peptide and two domains including: chitin-binding domain and catalytic domain, while Tc CHT5 and Tc CHT6 had one transmembrane helice and one catalytic domain. The six CHT c DNA sequences had different domains. These genes had predicted molecular weights of 60.80, 69.03, 104.32, 59.75, 45.63, and 49.05 k Da, respectively, and isoelectric points of 5.44, 8.65, 6.23, 6.07, 6.12, and 8.27, respectively. Phylogenetic analysis showed that chitinase genes from T. cinnabarinus and Tetranychus urticae were clustered together.4. Detected the transcripts of each CHT gene among different developmental stages and the effect of curcumin on the gene expression. Based on the cloning and analysis of the six Tc CHTs, RT-q PCR was used to detect the transcripts of each CHT gene among different developmental stages(egg, larvae, nymph, and adult). Results revealed that CHTs expression levels in larvaes and nymphs were higher than those in eggs and adults, especially Tc CHIT1, Tc CHIT2, Tc CHIT3 and Tc CHT4. The expression of larvae and nymph was nearly hundreds-fold than egg and adult. After the nymph mites were exposed to curcumin for 24 h, the m RNA expression levels of Tc CHTs significantly decreased. It is noteworthy that Tc CHIT1 and Tc CHT5 were more sensitive than others.