C2H2 zinc finger protein (ZFP) genes have been extensively studied in many organisms and can function as transcription factors and be involved in many biological processes including plant growth and development and stress responses. In the current study, a comprehensive genomics analysis of the C2H2-ZFP genes in B . rapa was performed. A total of 301 B . rapa putative C2H2-ZFP (BrC2H2-ZFP) genes were identified from the available Brassica genome databases, and further characterized through analysis of conserved amino acid residues in C2H2-ZF domains and their organization, subcellular localization, phylogeny, additional domain, chromosomal location, synteny relationship, Ka/Ks ratio, and expression pattern. We also analyzed the expression patterns of eight B . rapa C2H2-ZFP genes under salt and drought stress conditions by using qRT-PCR technique. Our results showed that about one-third of these B . rapa C2H2-ZFP genes were originated from segmental duplication caused by the WGT around 13 to 17 MYA, one-third of them were highly and consecutively expressed in all tested tissues, and 92% of them were located in nucleus by prediction supporting then their functional roles as transcription factors, of which some may play important roles in plant growth and development. The Ka/Ks ratios of 264 orthologous C2H2-ZFP gene pairs between A . thaliana and B . rapa were all, except two, inferior to 1 (varied from 0.0116 to 1.4919, with an average value of 0.3082), implying that these genes had mainly experienced purifying selection during species evolution. The estimated divergence times of the same set of gene pairs ranged from 6.23 to 38.60 MY, with an average value of 18.29 MY, indicating that these gene members have undergone different selective pressures resulting in different evolutionary rates during species evolution. In addition, a few of these B . rapa C2H2-ZFPs were shown to be involved in stress responses in a similar way as their orthologs in A . thaliana . Comparison between A . thaliana and B . rapa orthologous C2H2-ZFP genes showed that the majority of these C2H2-ZFP gene members encodes proteins with conserved subcellular localization and functional domains between the two species but differed in their expression patterns in five tissues or organs. Thus, our study provides valuable information for further functional determination of each C2H2-ZFP gene across the Brassica species, and may help to select the appropriate gene targets for further in-depth studies, and genetic engineering and improvement of ...
The key step for the toxicity of Bacillus thuringiensis subsp. israelensis (Bti) is the interaction between toxins and putative receptors; thus, many studies focus on identification of new toxin receptors and engineering of toxins with higher affinity/specificity for receptors. In the larvae of Aedes aegypti, galectin-14 was one of the genes upregulated by Bti treatment. RNAi knockdown expression of galectin-14 and feeding recombinant galectin-14 -thioredoxin fusion protein significantly affected survival of Ae. aegypti larvae treated with Bti toxins. Recombinant galectin-14 protein bound to brush border membrane vesicles (BBMVs) of Ae. aegypti larvae, ALP1 and APN2, and galectin-14 and Cry11Aa bound to BBMVs with a similarly high affinity. Competitive binding results showed that galectin-14 competed with Cry11Aa for binding to BBMVs and ALP1 to prevent effective binding of toxin to receptors. These novel findings demonstrated that midgut proteins other than receptors play an important role in modulating the toxicity of Cry toxins.
Cry11Aa displays high toxicity to the larvae of several mosquito species, including Aedes, Culex, and Anopheles. To study its binding characterization against Culex quinquefasciatus, Cry11Aa was purified and western blot results showed that Cry11Aa could bind successfully to the brush border membrane vesicles. To identify Cry11Aa-binding proteins in C. quinquefasciatus, a biotin-based protein pull-down experiment was performed and seven Cry11Aa-binding proteins were isolated from the midgut of C. quinquefasciatus larvae. Analysis of liquid chromatography-tandem mass spectrometry showed that one of the Cry11Aa-binding proteins is the ATP-binding domain 1 family member B. To investigate its binding property and effect on the toxicity of Cry11Aa, western blot, far-western blot, enzyme-linked immunosorbent assay, and bioassays of Cry11Aa in the presence and absence of the recombinant ATP-binding protein were performed. Our results showed that the ATP-binding protein interacted with Cry11Aa and increased the toxicity of Cry11Aa against C. quinquefasciatus. Our study suggests that midgut proteins other than the toxin receptors may modulate the toxicity of Cry toxins against mosquitoes.
Aedes aegypti is a crucial vector for human diseases, such as yellow fever, dengue, chikungunya, and Zika viruses. Today, a major challenge throughout the globe is the insufficient availability of antiviral drugs and vaccines against arboviruses, and toxins produced by Bacillus thuringiensis (Bt) are still used as biological agents for mosquito control. The use of Cry toxins to kill insects mainly depends on the interaction between Cry toxins and important toxin receptors, such as alkaline phosphatase (ALP). In this study, we investigated the function of A. aegypti C-type lectin-20 (CTL-20) in the tolerance of Cry toxins. We showed that recombinant CTL-20 protein interacted with both Cry11Aa and ALP1 by the Far-Western blot and ELISA methods, and CTL-20 bound to A. aegypti larval brush border membrane vesicles (BBMVs). Binding affinity of CTL-20 to ALP1 was higher than that of Cry11Aa to ALP1. Furthermore, the survival rate of A. aegypti larvae fed with Cry11Aa toxin mixed with recombinant CTL-20 fusion protein was significantly increased compared with that of the control larvae fed with Cry11Aa mixed with thioredoxin. Our novel results suggest that midgut proteins like CTLs may interfere with interactions between Cry toxins and toxin receptors by binding to both Cry toxins and receptors to alter Cry toxicity.
The mosquito Aedes aegypti is associated with the spread of many viral diseases in humans, including Dengue virus (DENVs), Yellow fever virus (YFV), Zika virus (ZIKV), and Chikungunya virus (CHIKV). Bacillus thuringiensis (Bt) is widely used as a biopesticide, which produces Cry toxins for mosquito control. The Cry toxins bind mainly to important receptors, including alkaline phosphatase (ALP) and aminopeptidase-N (APN). This work investigated the function of a C-type lectin, CTLGA9, in A. aegypti in response to Cry toxins. Our results showed by far-western blot and ELISA methods that the CTLTGA9 protein interacted with brush border membrane vesicles (BBMVs) of A. aegypti larvae and with ALP1, APN, and Cry11Aa proteins. Furthermore, molecular docking showed overlapping binding sites in ALP1 and APN for binding to Cry11Aa and CTLGA9. The toxicity assays further demonstrated that CTLGA9 inhibited the larvicidal activity of Cry toxins. According to the results of molecular docking, CTLGA9 may compete with Cry11Aa for binding to ALP1 and APN receptors and thus decreases the mosquitocidal toxicity of Cry11Aa. Our results provide further insights into better understanding the mechanism of Cry toxins and help improve the Cry toxicity for mosquito control.
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