Reference genes are used as internal controls in gene expression studies, but their expression levels vary according to tissue types and experimental treatments. Quantitative real-time PCR (qPCR) is the most sensitive technique for transcript quantification provided that gene transcription patterns are normalized to an evaluated reference gene. In this study, the suitability of eight commonly used genes (β?-actin, 5.8SrRNA, α?-TUB, GAPDH, RPL13a, RPS18, TBP, SDHA) were cloned and investigated to find the most stable candidates for normalizing real-time PCR data generated from the four different strains (abamectin-resistant, fenpropathrin-resistant, omethoate-resistant, and susceptible strains) and different developmental stages (eggs, protonymphs, nymphs, and adults) of carmine spider mite, Tetranychus cinnabarinus (Boisduval) (Acarina: Tetranychidae). The stability of gene expression was assessed using two different analysis programs, geNorm and NormFinder. Using these analyses, RPS18 and 5.8SrRNA had the most stable expression regardless of the four different strains, whereas RPS18 and α?-TUB were expressed most stably in different developmental stages.
Three heat shock protein 70 (Hsp70) cDNAs were isolated from the carmine spider mite, Tetranychus cinnabarinus. They were tentatively named as TCHsp70-1, TCHsp70-2 and TCHsp70-3. Structural analyses showed that all of the three TCHsp70 cDNAs held the full open reading frame (ORF). Putative protein sequences and a phylogenetic tree suggested that TCHsp70-1 and TCHsp70-3 were cytoplasm HSP70 and TCHsp70-2 was endoplasmic reticulum HSP70. Comparison of deduced amino acid sequences of TCHsp70-1 and TCHsp70-3 showed 84.78% identity, TCHsp70-1 and TCHsp70-2 showed 57.33% identity, TCHsp70-2 and TCHsp70-3 showed 58.26% identity. Real-time comparative quantitative PCR revealed that the relative expression of TCHsp70-2 was lower than TCHsp70-1 and TCHsp70-3 at each temperature tested. TCHsp70-1 and TCHsp70-3 shared a similar expression pattern after cold and heat shock compared with their expression at normal temperature (26 degrees C), but the mRNA expression of TCHsp70-1 was significantly higher and lower than that of TCHsp70-3 at cold and heat shock temperatures (except for 34 degrees C), respectively. This result possibly indicated the expression patterns of TCHsp70 were affected by their location in different cellular compartments. The results also indicated that three TCHsp70s, especially TCHsp70-1 and TCHsp70-3, may play an important role in mediating tolerance to cold, thermal stress for Tetranychus cinnabarinus.
This study aimed to evaluate the acaricidal activity of lettuce (Lactuca sativa) extracts against carmine spider mites (Tetranychus cinnabarinus Boisd.) and isolate the acaricidal components. Acaricidal activities of lettuce extracts isolated from different parts (the leaf, root and seed) using various solvents (petroleum ether, acetone and methanol) were evaluated with slide-dip bioassay and relatively high median lethal concentration (LC50) values were detected. Acetone extracts of lettuce leaves harvested in July and September were fractionated and isolated with silica gel and thin-layer chromatography. Consequently, acetone extracts of lettuce leaves harvested in July exhibited higher acaricidal activity than those harvested in September, with an LC50 value of 0.268 mg ml-1 at 72 h post-treatment. A total of 27 fractions were obtained from the acetone extract of lettuce leaves harvested in July, and mite mortalities with the 11th and 12th fractions were higher than those with the other 25 fractions (LC50: 0.751 and 1.258 mg ml-1 at 48 h post-treatment, respectively). Subsequently, active acaricidal components of the 11th fraction were identified by infrared, nuclear magnetic resonance and liquid chromatography/mass spectrometry. Five components were isolated from the 11th fraction, with components 11-a and 11-b showing relatively high acaricidal activities (LC50: 0.288 and 0.114 mg ml-1 at 48 h post-treatment, respectively). Component 11-a was identified as β-sitosterol. In conclusion, acetone extracts of lettuce leaves harvested in July might be used as a novel phytogenic acaricide to control mites.
The present research envisages the investigation of the optimized semi-solid process parameters of the Al-5Zn-3Mg-2Cu alloy. The effects of remelting temperature and holding time on the microstructure and mechanical properties of modified Al-5Zn-3Mg-2Cu alloy were studied by the recrystallization and partial remelting (RAP) process. The results reveal that with an increase in the semi-solid holding temperature, there was an increase in the average grain size of the Al-5Zn-3Mg-2Cu alloy, but when the temperature was increased to 635°C, the grain roundness decreased owing to the grain coalescence and growth. After holding at 635°C for 32 min, the grains were partially transformed into dendrites. Further, with the prolongation of the holding time, the grain size of alloy gradually grew and the roundness increased, and after holding 32 min at 625°C, the grain roundness began to decrease. The optimum semi-solid process parameters were: 615°C × 32 min and 625°C × 16 min. The Al-5Zn-3Mg-2Cu alloy parts were prepared by thixoforming process. Under the conditions of 615°C × 32 min and 625°C × 16 min, the parts were formed completely and the surface of the parts was smooth and clean. Under the condition of holding 32 min at 615°C, the tensile strength and elongation at the break of the parts correspondingly reached 576 MPa and 9.5% respectively, while they reached 589 MPa and 11%, respectively under the condition of holding 16 min at 625°C.
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