Bt杀虫最重要的2个环节是原毒素在昆虫中肠的有效酶解和酶解后活性片段与中肠受体的特异性结合.在前期工作中已发现部分Bt毒素可被茶小绿叶蝉肠液有效酶解成相应活性片段.为进一步了解茶小绿叶蝉是否存在相应受体或受体是否能与毒素结合,从受体的角度展开研究.首先,分析前期获得的茶小绿叶蝉转录组数据,发现该虫存在大量ALP、APN和Cadherin潜在受体基因,筛选部分候选基因,通过RT-qPCR验证表达量差异,结果显示其与转录组数据一致;其次,通过表达分析数据中的FPKM值,筛选出这3类潜在受体基因中表达量高且在肠内外表达差异显著的基因,有8个ALP、7个APN、8个Cadherin;再次,通过构建NJ进化树对这23个候选基因种内同源性进行分析,为该虫的生物防治提供一定理论基础.

Ectropis oblique Prout (Lepidoptera: Geometridae) is one of the main pests that damages the tea crop in Southeast Asia. To understand the molecular mechanisms of its feeding biology, transcriptomes of the alimentary tract (AT) and of the body minus the AT of E. oblique were successfully sequenced and analyzed in this study. A total of 36,950 unigenes from de novo sequences were assembled. After analysis using six annotation databases (e.g., Gene Ontology, Kyoto Encyclopedia of Genes and Genome, and NCBI nr), a series of putative genes were found for this insect species that were related to digestion, detoxification, the immune system, and Bacillus thuringiensis (Bt) receptors. From this series of genes, 21 were randomly selected to verify the relative expression levels of transcripts using quantitative real-time polymerase chain reaction. These results will provide an invaluable genomic resource for future studies on the molecular mechanisms of E. oblique, which will be useful in developing biological control strategies for this pest.

Globally, Aedes aegypti is one of the most dangerous mosquitoes that plays a crucial role as a vector for human diseases, such as yellow fever, dengue, and chikungunya. To identify (1) transcriptomic basis of midgut (2) key genes that are involved in the toxicity process by a comparative transcriptomic analysis between the control and Bacillus thuringiensis (Bt) toxin (LLP29 proteins)-treated groups. Next-generation sequencing technology was used to sequence the midgut transcriptome of A. aegypti. A total of 17130 unigenes, including 574 new unigenes, were identified containing 16358 (95.49%) unigenes that were functionally annotated. According to differentially expressed gene (DEG) analysis, 557 DEGs were annotated, including 226 upregulated and 231 downregulated unigenes in the Bt toxin-treated group. A total of 442 DEGs were functionally annotated; among these, 33 were specific to multidrug resistance, 6 were immune-system-related (Lectin, Defensin, Lysozyme), 28 were related to putative proteases, 7 were lipase-related, 8 were related to phosphatases, and 30 were related to other transporters. In addition, the relative expression of 28 DEGs was further confirmed through quantitative real time polymerase chain reaction. The results provide a transcriptomic basis for the identification and functional authentication of DEGs in A. aegypti.

Bacillus thuringiensis (Bt) is one of the most widely used and studied biopesticides. However, it is vulnerable to the influence of ultraviolet (UV) radiation, causing shorter persistence under field conditions. To obtain a high-active and effective Bt new product, the main objective of this study is to obtain a highly UV-resistant Bt mutant from the mosquitocidal Bt LLP29 through UV exposure. After 19 rounds of UV exposure, a Bt mutant named LLP29-M19 was obtained, showing resistance to UV radiation for up to 67 min. The mosquitocidal fatality rate of LLP29-M19 was 95%, which was slightly higher than that of LLP29 (90%). Comparative characterization showed that there were no substantial differences in morphology between LLP29-M19 and the original strain, LLP29. However, some changes were detected in physiological and biochemical characteristic reactions, including fructose, glucose, and xylose metabolism. Furthermore, although both LLP29-M19 and LLP29 showed negative zeta potentials, the surface charge of LLP29 was -28.1 mV and that of LLP29-M19 was -42.8 mV. The size distribution of LLP29-M19 was also slightly larger than that of LLP29. Fourier transform infrared analysis indicated that amide functional groups might be involved in the resistance mechanism of LLP29-M19. Quantitative analysis using inductive coupled plasma emission spectrometry showed that some elements increased greatly in LLP29-M19, such as K. All of these results will be highly valuable for better understanding the mechanism of Bt resistance. Explanations regarding the resistance mechanism of this novel Bt mutant may lead to the development of new biopesticides with high mosquitocidal activity and persistence.

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.

Tea production has been significantly impacted by the false-eye leafhopper, Empoasca vitis (Göthe), around Asia. To identify the key genes which are responsible for nutrition absorption, xenobiotic metabolism and immune response, the transcriptome of either alimentary tracts or bodies minus alimentary tract of E. vitis was sequenced and analyzed. Over 31 million reads were obtained from Illumina sequencing. De novo sequence assembly resulted in 52,182 unigenes with a mean size of 848nt. The assembled unigenes were then annotated using various databases. Transcripts of at least 566 digestion-, 224 detoxification-, and 288 immune-related putative genes in E. vitis were identified. In addition, relative expression of highly abundant transcripts was verified through quantitative real-time PCR. Results from this investigation provide genomic information about E. vitis, which will be helpful in further study of E. vitis biology and in the development of novel strategies to control this devastating pest.