ABSTRACT
Bacillus thuringiensis Cry2Ab toxin was a widely used bioinsecticide to control lepidopteran pests all over the world. In the present study, engineering of Bacillus thuringiensis Cry2Ab toxin was performed for improved insecticidal activity using site-specific saturation mutation. Variants L183I were screened with lower LC50 (0.129 µg/cm2) against P. xylostella when compared to wild-type Cry2Ab (0.267 µg/cm2). To investigate the molecular mechanism behind the enhanced activity of variant L183I, the activation, oligomerization and pore-formation activities of L183I were evaluated, using wild-type Cry2Ab as a control. The results demonstrated that the proteolytic activation of L183I was the same as that of wild-type Cry2Ab. However, variant L183I displayed higher oligomerization and pore-formation activities, which was consistence with its increased insecticidal activity. The current study demonstrated that the insecticidal activity of Cry2Ab toxin could be assessed using oligomerization and pore-formation activities, and the screened variant L183I with improved activity might contribute to Cry2Ab toxin's future application.
ABSTRACT
Vegetative insecticidal proteins 3A (Vip3A) were important insecticidal proteins for control of lepidopteran pests. Previous study demonstrated that Vip3Aa and Vip3Ad showed significant difference in insecticidal activities against Spodoptera exigua, while the molecular mechanism remained ambiguous. Here we demonstrated that the difference in insecticidal activities between Vip3Aa and Vip3Ad might be caused by the difference in stability of Vip3Aa and Vip3Ad in S. exigua midgut protease. Vip3Aa was quite stable while Vip3Ad could be further degraded. Molecular dynamics simulation revealed that Vip3Aa was more stable than Vip3Ad, with smaller RMSD and RMSF value. Amino acid sequence alignment indicated that three were three extra prolines (P591, P605 and P779) located on Vip3Aa. We further identified that residue P591 played a crucial role on stability and insecticidal activity of Vip3Aa. Taken together, our study demonstrated that the stability was essential for the insecticidal activity of Vip3A toxins, which might provide new insight into the action mode of Vip3A toxins and contribute to the design Vip3A variants with improved stability and insecticidal activity.
ABSTRACT
A new phosphorescence-labelling reagent (3.5-G-D-P labelling reagent) was developed, based on 3.5-generation polyamidoamine dendrimers (3.5-G-D) as internal acceptor to capture porphyrin (P) molecular. In the disturber of heavy atom, 3.5-G-D-P could emit room temperature phosphorescence (RTP) of 3.5-G-D and P on the surface of polyamide membrane (PAM), respectively. Products (3.5-G-D-P-WGA) of 3.5-G-D-P labelling triticum vulgaris lectin (WGA) could emit strong and stable RTP signal on the surface of PAM, and it also could take specific affinity adsorption reaction (AA) with alkaline phosphatase (ALP). The product of the AA reaction (3.5-G-D-P-WGA-ALP) could keep the RTP characteristics of 3.5-G-D-P very well, and the DeltaI(p) of the system was linear correlation to the content of ALP. The DeltaI(p) of the system with Tween-80 was once for P and twice for 3.5-G-D more than that without Tween-80. Thus, the affinity adsorption solid substrate-room temperature phosphorimetry (AA-SS-RTP) for the determination of trace ALP has been established using Tween-80-3.5-G-D-P to label WGA. The detection limit (LD) of this method was 0.12fgspot(-1) for 3.5-G-D and 0.18fgspot(-1) for P with direct method, 0.14fgspot(-1) for 3.5-G-D and 0.17fgspot(-1) for P with sandwich method, respectively, and the sensitivity was obviously high. This research showed that either using 3.5-G-D or P excitation/emission wavelength to determine the content of ALP in human serum, the results were coincided with ELISA, and the flexibility of AA-SS-RTP was obviously improved and the applicability was wider. Meanwhile, the reaction mechanism of determining ALP by direct method AA-SS-RTP was discussed.