• Title/Summary/Keyword: Transformed insect cells

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Enhanced Resistance to Botrytis cinerea Mediated by Transgenic Expression of the Spider Chitinase Gene AvChit in Arabidopsis

  • Hur, Yeon-Jae;Kim, Doh-Hoon
    • International Journal of Industrial Entomology and Biomaterials
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    • v.19 no.2
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    • pp.259-264
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    • 2009
  • The AvChit gene encodes for a chitinase from the spider, Araneus ventricosus. This spider, A. ventricosus, is an abundant species in Korea. Arabidopsis thaliana plants were transformed with the AvChit gene using Agrobacterium tumefaciens. Thirteen transgenic lines expressing the AvChit gene were obtained. Functional expression of the AvChit gene in transgenic Arabidopsis was confirmed by Southern, northern and western blot analysis. The AvChit cDNA was expressed as a 61 kDa polypeptide in baculovirus-infected insect Sf9 cells. AvChit protein extracted from transgenic Arabidopsis exhibited high levels of chitinase activity. Phytopathological tests showed that two transgenic Arabidopsis lines expressing the AvChit gene displayed high levels of resistance to gray mold disease (Botrytis cinerea).

Insect Juvenile Hormone Antagonists as Eco-friendly Insecticides (친환경 살충제로서의 곤충 유충호르몬 길항제)

  • Choi, Jae Young;Je, Yeon Ho
    • Korean journal of applied entomology
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    • v.61 no.1
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    • pp.101-108
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    • 2022
  • Because of their specificity to target insects and relatively low toxicity to non-target organisms, insect growth regulators (IGRs) have been regarded as attractive alternatives to chemical insecticides. Commercially available IGRs are classified into juvenile hormone agonists (JHAs), ecdysone agonists (EAs), and chitin synthesis inhibitors (CSIs) according to their mode of action. Recently, JH-mediated interaction of methoprene-tolerant (Met), which is JH receptor, and its binding partners have been replicated in vitro using yeast cells transformed with the Met and FISC/CYC genes of A. aegypti. Using this in vitro yeast two-hybrid β-galactosidase assay, juvenile hormone antagonists (JHANs) have been identified from various sources including chemical libraries, plants, and microorganisms. As juvenile hormone (JH) is an insect specific hormone and regulates development, reproduction, diapause and other physiological processes, JHANs fatally disrupt the endocrine signals, which result in abnormal development and larval death. These results suggested that JHANs could be efficiently applied as IGR insecticides with a broad insecticidal spectrum. This review discuses JH signaling pathway mediated by Met and future prospects of JHANs as environmentally benign IGR insecticides.

Basic Studies on the Apoptosis Mechanism of Trichoplusia ni Cell Line (Trichoplusia ni 세포의 apoptosis 메커니즘 규명을 위한 기초연구)

  • Lee, Jong-Min;Yang, Jai-Myung;Lee, Youn-Hyung;Chung, In-Sik
    • Applied Biological Chemistry
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    • v.44 no.1
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    • pp.1-6
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    • 2001
  • To elucidate the apoptosis mechanism of Trichoplusia ni cell, fundamental studies for apoptosis induction and suppression were performed. Hygromycin B, a known inducer of apoptosis, started the inhibition of T. ni cell growth at $200\;{\mu}/ml$ concentration. Furthermore, at $400\;{\mu}/ml$ concentration, DNA fragmentation was detected on day 2 of incubation. Although both dexamethasone and sodium butyrate inhibited T. ni cell growth, DNA fragmentation was not detected by both treatments. Also, when apoptosis induced T. ni cells with $200\;{\mu}/ml$ hygromycin B were treated with caspase inhibitor (Ac-DEVD-CHO), the apoptotsis was suppressed by 36%. In addition, N-acetylcysteine, another apoptosis repressor, also inhibited the apoptosis of T. ni cells. In order to express the anti-apoptosis gene (bcl-2), T. ni cells were transiently transformed with bcl-2 and its expression was confirmed by western blot analysis. These results showed the potential of developing new insect cell lines with suppressed apoptosis.

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Histochemical Study on the Hemocytes during Metamorphosis (배추흰나비의 變態에 따른 血球細胞의 組織化學的 硏究)

  • Kim, Chang-Whan;Kim, Woo-Kap;Kim, Yong-Kuk
    • The Korean Journal of Zoology
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    • v.11 no.4
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    • pp.103-117
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    • 1968
  • Insect hemocytes during metamorphosis were studies by histchemical and autoradiographic methods is Pieris rapae (Lepidoptera). The hemocytes were classified into six types, prohemocytes, plasmatocytes, podocytes, granular hemocytes, spherule cells and oenocytoids on the basis of the transitions in shapes and inclnsions of cytoplasms. Proteins, nucleic acids, polysaccharides and lipids in the hemocytes were detected histochemically from larval to pupal stages to learn the rise and fall of them during growth and metamorphosis. Most of the granules consisted of glycogen, neutral mucopolysaccharides and mucoprotein in addition to some granules of neutral fats and phospholipids were found in the granular hemocytes and spherule cells. Mitotic figures and DNA synthetic activities were observed in every type of hemocytes from 2nd to 5th instars, suggesting the all types of hemocytes originated from the prohemocytes. The cytoplasmic filaments of plasmatocytes and pdocytes extended very long in prepupa and pupa and the vermiform cells were the transformed plasmatocytes due to their further differentiation.

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Metabolic Engineering of Indole Glucosinolates in Chinese Cabbage Plants by Expression of Arabidopsis CYP79B2, CYP79B3, and CYP83B1

  • Zang, Yun-Xiang;Lim, Myung-Ho;Park, Beom-Seok;Hong, Seung-Beom;Kim, Doo Hwan
    • Molecules and Cells
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    • v.25 no.2
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    • pp.231-241
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    • 2008
  • Indole glucosinolates (IG) play important roles in plant defense, plant-insect interactions, and stress responses in plants. In an attempt to metabolically engineer the IG pathway flux in Chinese cabbage, three important Arabidopsis cDNAs, CYP79B2, CYP79B3, and CYP83B1, were introduced into Chinese cabbage by Agrobacterium-mediated transformation. Overexpression of CYP79B3 or CYP83B1 did not affect IG accumulation levels, and overexpression of CYP79B2 or CYP79B3 prevented the transformed callus from being regenerated, displaying the phenotype of indole-3-acetic acid (IAA) overproduction. However, when CYP83B1 was overexpressed together with CYP79B2 and/or CYP79B3, the transformed calli were regenerated into whole plants that accumulated higher levels of glucobrassicin, 4-hydroxy glucobrassicin, and 4-methoxy glucobrassicin than wild-type controls. This result suggests that the flux in Chinese cabbage is predominantly channeled into IAA biosynthesis so that coordinate expression of the two consecutive enzymes is needed to divert the flux into IG biosynthesis. With regard to IG accumulation, overexpression of all three cDNAs was no better than overexpression of the two cDNAs. The content of neoglucobrassicin remained unchanged in all transgenic plants. Although glucobrassicin was most directly affected by overexpression of the transgenes, elevated levels of the parent IG, glucobrassicin, were not always accompanied by increases in 4-hydroxy and 4-methoxy glucobrassicin. However, one transgenic line producing about 8-fold increased glucobrassicin also accumulated at least 2.5 fold more 4-hydroxy and 4-methoxy glucobrassicin. This implies that a large glucobrassicin pool exceeding some threshold level drives the flux into the side chain modification pathway. Aliphatic glucosinolate content was not affected in any of the transgenic plants.

Gene functional analysis of Harmonia axyridis by in vitro transcription

  • Park, Sang-Eun;Youn, Young-Nam
    • Korean Journal of Agricultural Science
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    • v.46 no.3
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    • pp.471-488
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    • 2019
  • Random genes were screened in two transforming ways to investigate the new genes of a ladybug using the Harmonia axyridis cDNA library stock cell cloned in the LITMUS 28i vector in a previous study. Phenotypic variation was observed after injection of the synthesized double-stranded RNA through the in vitro transcription process. The cDNA library of H. axyridis was transformed into E. coli $DH5{\alpha}$ and 10B competent cells by heat shock. Analysis of the nucleotide sequences of the 42 clones with the insert DNAs revealed that 21 clones were homologous with the genes of insects, and only one clone had a gene from H. axyridis. Thirteen of the 21 insect genes were homologous with genes from coleopteran insects. Fourteen genes were selected, which were identified by the gene screening results, and were synthesized as double-stranded RNA through in vitro transcription. One microgram of the synthesized double-stranded RNA between segments T1 and T2 were injected using a syringe into each anesthetized fourth larvae which were under 2 days old. As a result, a phenotypic variation appeared in the larva injected with the two genes. While the eggs of H. axyridis injected with distilled water hatched out three days after oviposition, the eggs of H. axyridis injected with dsHma 06 did not hatch but become shrivel a week after oviposition. Most of the H. axyridis injected with dsHma 08 died and were unable to complete the pupation or eclosion during ecdysis.