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RISS 인기검색어
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- 저자 : Tinghao Kang (검색결과 3 건)
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AvCystatin, a novel cysteine protease inhibitor from spider (Araneus ventricosus) venom
Hu Wan,Tinghao Kang,김보연,이광식,Jianhong Li,진병래 한국응용곤충학회 2015 Journal of Asia-Pacific Entomology Vol.18 No.1
Cystatins are involved in various physiological and cellular processes, including immune responses, protein homeostasis, signaling pathways, and apoptosis. Thus far, no Cystatins have been identified from spider venom. In this study, we report the cloning and characterization of a spider venom-derived Cystatin fromAraneus ventricosus (AvCystatin). The AvCystatin gene contains an open reading frame of 405 bp encoding a predicted protein of 134-amino acids with a 16-amino acid signal peptide. Sequence alignment and structural modeling indicated that AvCystatin is closely related to family 2 Cystatins. Endogenous AvCystatin was present as an 18-kDa peptide in spider venom. Recombinant AvCystatin, expressed in baculovirus-infected insect cells, showed inhibitory activity against papain (Ki 86.73 nM), but not trypsin and chymotrypsin, defining a role for AvCystatin as a spider venom-derived cysteine protease inhibitor. Furthermore, recombinant AvCystatin exhibited stability against high temperatures and pH extremes, but had no effects on the growth of the entomopathogenic fungi Beauveria bassiana. These data demonstrate that AvCystatin is a novel member of the family 2 Cystatins and provide new insight for the future investigations of spider venom-derived Cystatins.
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Muhammad Shakeel,Xun Zhu,Tinghao Kang,Hu Wan,Jian Hong Li 한국응용곤충학회 2015 Journal of Asia-Pacific Entomology Vol.18 No.2
An efficient technique for investigating gene expression is the real time quantitative reverse transcription PCR (qRT-PCR). Despite the fact that this technique has been extensively used to explore the gene function in Helicoverpa armigera, stability of the reference genes still requires validation. This research aims to validate the stability of expression of nine potential reference genes under different experimental conditions including temperature, mechanical injury, starvation, photoperiod, and developmental stage. An exhaustive system (RefFinder), available online, was employed to evaluate and grade the studied genes. Appropriateness of the reference genes as endogenous controls was determined through four computational algorithms (ΔCt, NormFinder, BestKeeper, and geNorm). According to the findings of this study, RPL28 and RPS15 were found to be the most stable reference genes in case of starved larvae, temperature stressed larvae, and different developmental stages. On the other hand, HSP90 and TUBB proved to be highly stable in case of photoperiod stressed larvae. Finally, TUBB and GAPDH were the most stable reference genes in case of larvae subjected to mechanical injury. These results can facilitate development of a standardized qRT-PCR technique and can also prove to be helpful for standard RT-PCR method which need reference gene for normalization.
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Yashu Zhang,Xiao Zhao,Jinyun Ji,Tinghao Kang,Jian Hong Li,Hu Wan 한국응용곤충학회 2016 Journal of Asia-Pacific Entomology Vol.19 No.2
In the present study, a phenylalanine hydroxylase (PAH) was cloned and characterized from the common cutworm Spodoptera litura (SlPAH). The SlPAH gene is an 1863 bp full-length cDNAwith a 1371 bp open reading frame that encodes a predicted 52.35 kDa protein of 457 amino acid residueswith an isoelectric point of 5.39. The SlPAH gene contains 6 exons and 5 introns. A multiple sequence analysis of SlPAH showed an 88–68% sequence identity to Papilio xuthus PAH and other insect PAHs. A phylogenetic tree revealed that SlPAH is a novel member of the PAH family. Tissue distribution analyses indicated that SlPAH is constitutively expressed in the epidermis, midgut, and fat body, with the highest expression occurring in the fat body on days 2 and 3 of the sixth-instar larval stage and day 1 of the prepupa stage. However, RNAi-mediated SlPAH gene silencing had no observable phenotype in S. litura larvae. Additionally, SlPAH expression is acutely up-regulated in the fat body after injection with Bacillus thuringiensis, Escherichia coli, or Beauveria bassiana. Therefore, these results provide a molecular basis for the functional roles of SlPAH during development and the innate immune response in S. litura.
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