Classification and Identification of Bacteria in the Soil Treated by AcMNPV Using High-throughput Sequencing Technique

Yuejun Fu,Xing Li,Shuhua Zheng,Jun Du,Aihua Liang 한국생물공학회 2015 Biotechnology and Bioprocess Engineering Vol.20 No.5

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) have been applied in the pesticide prevention as new bioinsecticides. Many studies have been carried out to assess the effects of insecticide on microorganism communities in different environments. However, little is known about whether the pesticides affect the microbial community in soil. Therefore, we performed high-throughput sequencing of V3–V4 hypervariable regions of the 16S rRNA genes from the bacteria in soil treated with AcMNPV and compared the difference of microbiota in these soil samples. In the study, a total of 80,301 validated reads were obtained, and the bacteria found belonged to 31 phyla and 748 genera. Statistical analysis showed that AcMNPV contributed the growth of Fusobacteria, Tenericutes, Cyanobacteria/Chloroplast, Actinobacteria, Gemmatimonadetes. AcMNPV inhibited the growth of Fibrobacteres, Crenarchaeota, Firmicutes, DeinococcusThermus, TM7, Chlorobi, Synergistetes, BRC1, Chlamydiae, Euryarchaeota, Planctomycetes, Acidobacteria, Verrucomicrobia, Bacteroidetes, Elusimicrobia, Nitrospira, Armatimonadetes, Proteobacteria, WS3, OD1, Chloroflexi, Spirochaetes. AcMNPV had no effect on SR1, OP11, Thermodesulfobacteria, Aquificae. Alpha Diversity analysis showed that the diversity of bacterial community for the soil treated with AcMNPV was lower than that of the soil before treatment or the control group. Meanwhile, the similarity of soil samples from AcMNPV treated group compared with samples from either untreated or prior treatment group was low as shown by Beta Diversity analysis. These findings provide previously unknown information about the impact of AcMNPV on the soil bacterial community structure and also lay a foundation for further investigations of AcMNPV how influences the development and progression of bioinsecticides.

Functional Study of Active Residues Scorpion Insect Toxin BmK IT from Buthus martensii Karsch

Yuejun Fu,Renjia Yang,Wujian Chen,Zhiyi Wu,Ai-Hua Liang,Fengyun Hu 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.2

Chinese scorpion Buthus martensii Karsch(BmK) venom is a rich source of neurotoxins which bindto various ion channels with high affinity and specificityand thus widely used as compounds to modulate channelgating. An excitatory insect toxin, BmK IT, is not conservedwith a glutamate residue at the preceding position of thethird Cys residue, and is a toxin with a non-glutamateresidue at the relevant position in the excitatory scorpion β-toxin subfamily. In this study, the mutants of recombinantBmK IT (BmK IT (I25E), BmK IT (E15G), BmK IT Cterminal(TKSYCDVQIN) truncated) were achieved bysite-directed mutagenesis. Biological activity of BmK ITand its mutants confirmed these residues or peptides playedkey roles in BmK IT. BmK IT (I25E) could increase thesensitivity of BmK IT, but BmK IT(E15G) could decreasethe sensitivity of BmK IT on Sf9 cells. BmK IT truncatedC-terminal hydrophobic amino acids could cross thespecies boundaries and was effective on mammalian C6cells. To date, several excitatory insect toxins have beenisolated and identified from the venom of Buthus martensiiKarsch. However, no functional data are available andtherefore its classification in the family of excitatory insecttoxins remains putative and is just based on its highsimilarity with the other toxins of this family. These resultsverified I25, E15 and C-terminal (TKSYCDVQIN) inBmK IT played key roles in the interaction of the BmK ITand its receptor- sodium channels on the surface of insectcells and laid a foundation for further structural andfunctional analysis of BmK IT.

Flow Expansion and Deflection Downstream of a Symmetric Multi-Gate Sluice Structure

Yuejun Chen,James Yang,Jianzhong Yu,Zongfu Fu,Qingsheng Chen 대한토목학회 2020 KSCE JOURNAL OF CIVIL ENGINEERING Vol.24 No.2

A sluice structure with multiple gates is often followed by an enlarging channel downstream. Experiments are conducted for different enlargement ratios in cross-section and Froude numbers at the gates. A large-scale PIV system is adopted to capture the surface flow field for examination of the flow features. The study shows that, despite the centrally placed sluice structure with symmetric outflow, a hydraulic jump occurs and the main flow downstream exhibits, in terms of expansion and deflection, a high degree of asymmetry and formation of large circulation zones. The degree of deflection increases significantly along the longitudinal direction for all the enlargement ratios except the smallest. The toe of the hydraulic jump is controlled at the upper edge of the sloping surface downstream of the gates. In light of outflow width, Froude number and enlargement ratio, the flow is classified into three regions. Immediately downstream, the effect of outflow width gradually decreases with increasing Froude number. Further downstream, the degree of deflection augments with increasing enlargement ratio, with circulation zones. At a given cross-section, the degree of expansion is positively related to the enlargement ratio. The study is expected to provide guidance for examination of similar issues of flow pattern and erosion protection design.

Function and Application Analysis of Ac132 Protein in Autographa californica Multiple Nucleopolyhedrovirus

Yueyou Peng,Aihua Liang,Yuejun Fu 한국생물공학회 2018 Biotechnology and Bioprocess Engineering Vol.23 No.6

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is used as bac-to-bac eukaryotic protein expression system and biopesticide. However, AcMNPV isn’t widely used in protein expression system and biological control due to some drawbacks and it’s required to understand the function of AcMNPV genes comprehensively in the infection process. Ac132 is an improtant component of the budded virus (BV) nucleocapsid of AcMNPV and the function of Ac132 in regulating progeny virus generation is still unclear. In this study, the recombinant AcMNPV-Ac132-EGFP was constructed. Virus propagation assay indicated that overexpression of Ac132 could improve progeny virus production. Moreover, fluorescent microscope and western blot analysis showed AcMNPV-Ac132-EGFP could accelerate the aggregation of F-actin in the nucleus and delay the evacuation process. In addition, AcMNPV-Ac132-EGFP boosted the utilization of glucose in Sf9 cells at the aspect of energy metabolism. These results demonstrated that Ac132 played important roles in progeny virus production, F-actin formation, nucleocapsid transportion, and glucose metabolism in Sf9 cells, which provided an improved bac-to-bac eukaryotic protein expression system and biopesticide.

Insect-resistant Mechanism of Recombinant Baculovirus AcMNPVPK2- EGFP against Spodoptera exigua Larvae

Lili Wei,Chenggang Xu,Ai-Hua Liang,Yuejun Fu 한국생물공학회 2019 Biotechnology and Bioprocess Engineering Vol.24 No.4

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is a kind of potential biological insecticide. Ac-PK2 protein encoded by orf123 in AcMNPV genome can inhibit eIF2α family kinases to increase viral adaptation in vivo. In our previous study, it was found that recombinant AcMNPV-PK2-EGFP showed high virulence and anti-insect activity against Sf9 cells and Spodoptera exigua larvae. In this study, we investigated the function of Ac-PK2 protein in the insecticidal activity of AcMNPV and the anti-insect mechanism. AcMNPV-PK2- EGFP up-regulated the expression of Ac-pk2 gene in midgut and nerve cord in infected Spodoptera exigua larvae, compared with these in wild-type treatment group. Transcriptional level of BmK IT and detoxication related genes in epidermis, midgut and nerve cord was detected by quantitative PCR. Phenoloxidase activity assay showed that the insect’s humoral immunity was affected by AcMNPV-PK2-EGFP in a time-effect relationship. Western blot analysis of P53 protein indicated the apoptotic level in the midgut tissue was higher in AcMNPV-BmK IT and AcMNPV-PK2-EGFP co-infeced Spodoptera exigua larvae than that in other treatment groups at 4 h and 8 h postinfection. These results provided a theoretical basis to study the application of AcMNPV-PK2-EGFP as a biological insecticide.