scholarly journals Efficient Transformation of Catalpa bungei Shows Crystal Genes Conferring Resistance to the Shoot Borer Omphisa plagialis

2021 ◽  
Vol 12 ◽  
Author(s):  
Fenni Lv ◽  
Peng Wang ◽  
Enliang Zhang ◽  
Lingling Ma ◽  
Lulu Gao ◽  
...  
Keyword(s):  
Function Analysis ◽  
Expression System ◽  
Great Promise ◽  
Resistant Variety ◽  
Stem Segment ◽  
Transformation System ◽  
Embryogenic Calli ◽  
Shoot Borer ◽  
Catalpa Bungei ◽  
Insect Resistant

Although Catalpa bungei is a forest plant with considerable economic and ornamental value in China, its wood and decorative qualities are constrained by insect pests such as the shoot borer Omphisa plagialis (Lepidoptera). Overexpressing insect resistance genes such as crystal genes to develop an insect-resistant variety of C. bungei is an environmental and ecological approach. However, genotype limitations and low regeneration rates of embryogenic calli (EC) inhibit the development of transformation and the insect-resistant gene expression system in C. bungei. Here, we first established embryogenic callus induction and regeneration systems of five genotypes using mature seed and stem segment explants; the highest induction and regeneration rates of EC were 39.89 and 100%, respectively. Next, an efficient and stable Agrobacterium-mediated genetic transformation system was developed from EC and its positive frequency was up to 92.31%. Finally, using the transformation system, 15 and 22 transgenic C. bungei lines that expressed Cry2A and Cry9Aa-like were generated, respectively. These transgenic lines that exhibited significantly higher resistance to O. plagialis in the laboratory and field have great promise for meeting the challenge of future pest management under changing climatic conditions. Additionally, this efficient, fast, and stable transformation system could be a potential tool for gene function analysis and forest tree genetic improvement.

scholarly journals A Toxoplasma gondii Class XIV Myosin, Expressed in Sf9 Cells with a Parasite Co-chaperone, Requires Two Light Chains for Fast Motility

2014 ◽  
Vol 289 (44) ◽  
pp. 30832-30841 ◽  
Author(s):  
Carol S. Bookwalter ◽  
Anne Kelsen ◽  
Jacqueline M. Leung ◽  
Gary E. Ward ◽  
Kathleen M. Trybus
Keyword(s):  
Toxoplasma Gondii ◽  
Light Chain ◽  
Function Analysis ◽  
Expression System ◽  
Host Cells ◽  
Light Chains ◽  
Essential Light Chain ◽  
Heterologous System ◽  
Tetratricopeptide Repeat Domain ◽  
Cell Expression

Many diverse myosin classes can be expressed using the baculovirus/Sf9 insect cell expression system, whereas others have been recalcitrant. We hypothesized that most myosins utilize Sf9 cell chaperones, but others require an organism-specific co-chaperone. TgMyoA, a class XIVa myosin from the parasite Toxoplasma gondii, is required for the parasite to efficiently move and invade host cells. The T. gondii genome contains one UCS family myosin co-chaperone (TgUNC). TgMyoA expressed in Sf9 cells was soluble and functional only if the heavy and light chain(s) were co-expressed with TgUNC. The tetratricopeptide repeat domain of TgUNC was not essential to obtain functional myosin, implying that there are other mechanisms to recruit Hsp90. Purified TgMyoA heavy chain complexed with its regulatory light chain (TgMLC1) moved actin in a motility assay at a speed of ∼1.5 μm/s. When a putative essential light chain (TgELC1) was also bound, TgMyoA moved actin at more than twice that speed (∼3.4 μm/s). This result implies that two light chains bind to and stabilize the lever arm, the domain that amplifies small motions at the active site into the larger motions that propel actin at fast speeds. Our results show that the TgMyoA domain structure is more similar to other myosins than previously appreciated and provide a molecular explanation for how it moves actin at fast speeds. The ability to express milligram quantities of a class XIV myosin in a heterologous system paves the way for detailed structure-function analysis of TgMyoA and identification of small molecule inhibitors.

Isoflavones from an Insect-Resistant Variety of Soybean and the Molecular Structure of Afrormosin

1986 ◽  
Vol 49 (6) ◽  
pp. 1126-1129 ◽  
Author(s):  
Porfirio Caballero ◽  
C. Michael Smith ◽  
Frank R. Fronczek ◽  
Nikolaus H. Fischer
Keyword(s):  
Molecular Structure ◽  
Resistant Variety ◽  
Insect Resistant

Bionomics of the Manchurian catalpa shoot borer, Sinomphisa plagialis (Lepidoptera: Pyralidae) in Shandong, China

1994 ◽  
Vol 84 (4) ◽  
pp. 533-540
Author(s):  
Qi Cheng-Jin ◽  
Li De-Wei ◽  
Zhang Bing-Xin
Keyword(s):  
Mountainous Area ◽  
Typical Symptom ◽  
Shoot Borer ◽  
Summer Generation ◽  
Jiaodong Peninsula ◽  
Catalpa Ovata ◽  
Two Generations ◽  
Lepidoptera Pyralidae ◽  
Catalpa Bungei

AbstractLarvae of the Manchurian catalpa shoot borer Sinomphisa plagialis (Wileman) mainly damaged young shoots of Manchurian catalpa (Catalpa bungei) and ovate catalpa (Catalpa ovata). The typical symptom was production of spindle galls on damaged parts of shoots or branches, usually confined to seedlings, vigorously growing shoots, twigs and branches, as well as young trees below 3 m in height. Feeding occurred from early April to late October. In the Jiaodong peninsula, where there were one to two generations in a year, whereas in the mountainous area of central and southern Shandong, there were always two generations. Larvae had five instars. The longevity of adults was 3–11 days; eggs of the summer generation 7–9 days, overwintering generation 4–6 days; larvae of the summer generation 42–48 days; pupae of the summer generation 13–16 days, of the overwintering generation 22–26 days. Most overwintered in the pith as fifth instar larvae.

scholarly journals Functional expression of P-glycoprotein in Saccharomyces cerevisiae confers cellular resistance to the immunosuppressive and antifungal agent FK520

1994 ◽  
Vol 14 (1) ◽  
pp. 277-286
Author(s):  
M Raymond ◽  
S Ruetz ◽  
D Y Thomas ◽  
P Gros
Keyword(s):  
Multidrug Resistance ◽  
Mammalian Cells ◽  
Drug Transport ◽  
Function Analysis ◽  
Expression System ◽  
Functional Expression ◽  
Yeast Cells ◽  
Mutant Form ◽  
Cellular Resistance ◽  
P Glycoprotein

We have recently reported that expression in yeast cells of P-glycoprotein (P-gp) encoded by the mouse multidrug resistance mdr3 gene (Mdr3) can complement a null ste6 mutation (M. Raymond, P. Gros, M. Whiteway, and D. Y. Thomas, Science 256:232-234, 1992). Here we show that Mdr3 behaves as a fully functional drug transporter in this heterologous expression system. Photolabelling experiments indicate that Mdr3 synthesized in yeast cells binds the drug analog [125I]iodoaryl azidoprazosin, this binding being competed for by vinblastine and tetraphenylphosphonium bromide, two known multidrug resistance drugs. Spheroplasts expressing wild-type Mdr3 (Ser-939) exhibit an ATP-dependent and verapamil-sensitive decreased accumulation of [3H]vinblastine as compared with spheroplasts expressing a mutant form of Mdr3 with impaired transport activity (Phe-939). Expression of Mdr3 in yeast cells can confer resistance to growth inhibition by the antifungal and immunosuppressive agent FK520, suggesting that this compound is a substrate for P-gp in yeast cells. Replacement of Ser-939 in Mdr3 by a series of amino acid substitutions is shown to modulate both the level of cellular resistance to FK520 and the mating efficiency of yeast mdr3 transformants. The effects of these mutations on the function of Mdr3 in yeast cells are similar to those observed in mammalian cells with respect to drug resistance and transport, indicating that transport of a-factor and FK520 in yeast cells is mechanistically similar to drug transport in mammalian cells. The ability of P-gp to confer cellular resistance to FK520 in yeast cells establishes a dominant phenotype that can be assayed for the positive selection of intragenic revertants of P-gp inactive mutants, an important tool for the structure-function analysis of mammalian P-gp in yeast cells.

scholarly journals Lotus japonicus Triterpenoid Profile and Characterization of the CYP716A51 and LjCYP93E1 Genes Involved in Their Biosynthesis In Planta

2019 ◽  
Author(s):  
Hayato Suzuki ◽  
Ery Odette Fukushima ◽  
Yuko Shimizu ◽  
Hikaru Seki ◽  
Yukiko Fujisawa ◽  
...  
Keyword(s):  
Function Analysis ◽  
Expression System ◽  
Lotus Japonicus ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Loss Of Function ◽  
In Planta ◽  
Metabolic Intermediate ◽  
Oxidation Activity ◽  
Model Legume

Abstract Lotus japonicus is an important model legume plant in several fields of research, such as secondary (specialized) metabolism and symbiotic nodulation. This plant accumulates triterpenoids; however, less information regarding its composition, content and biosynthesis is available compared with Medicago truncatula and Glycine max. In this study, we analyzed the triterpenoid content and composition of L. japonicus. Lotus japonicus accumulated C-28-oxidized triterpenoids (ursolic, betulinic and oleanolic acids) and soyasapogenols (soyasapogenol B, A and E) in a tissue-dependent manner. We identified an oxidosqualene cyclase (OSC) and two cytochrome P450 enzymes (P450s) involved in triterpenoid biosynthesis using a yeast heterologous expression system. OSC9 was the first enzyme derived from L. japonicus that showed α-amyrin (a precursor of ursolic acid)-producing activity. CYP716A51 showed triterpenoid C-28 oxidation activity. LjCYP93E1 converted β-amyrin into 24-hydroxy-β-amyrin, a metabolic intermediate of soyasapogenols. The involvement of the identified genes in triterpenoid biosynthesis in L. japonicus plants was evaluated by quantitative real-time PCR analysis. Furthermore, gene loss-of-function analysis of CYP716A51 and LjCYP93E1 was conducted. The cyp716a51-mutant L. japonicus hairy roots generated by the genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, the complete abolition of soyasapogenols and soyasaponin I was observed in mutant plants harboring Lotus retrotransposon 1 (LORE1) in LjCYP93E1. These results indicate that the activities of these P450 enzymes are essential for triterpenoid biosynthesis in L. japonicus. This study increases our understanding of triterpenoid biosynthesis in leguminous plants and provides information that will facilitate further studies of the physiological functions of triterpenoids using L. japonicus.

scholarly journals Evaluation of the Genotype-dependency of the Leafbased Regeneration and Transformation System in Maize

2018 ◽  
Vol 28 (2) ◽  
pp. 261-268 ◽  
Author(s):  
Saeideh Ebrahimzadeh ◽  
Mohammad Ahmadabadi
Keyword(s):  
Tissue Culture ◽  
Genetic Transformation ◽  
Callus Induction ◽  
Gus Expression ◽  
Transformation System ◽  
Embryogenic Calli ◽  
Maize Genotypes ◽  
Explant Source ◽  
Young Leaves ◽  
Embryogenic Callus Induction

Tissue culture and genetic transformation in maize are very laborious. The existing regeneration methods, which mainly use immature embryos as starting material, are highly genotype-dependent. Leaf segments can be used as an alternative explant source to produce embryogenic calli. Although a reliable leafbased regeneration and transformation system has been recently reported for maize, however, the genotype-dependency of this method has not been described yet. To this end, we evaluated the production of embryogenic calli from young leaves of several maize genotypes. The results showed that, overall callus induction potential as well as embryogenic callus induction rate is significantly different among the tested genotypes, demonstrating the genotypedependency of this system. However, induced embryogenic calli from different genotypes remained their embryogenic capability during several callus multiplication rounds. In addition, embryogenic calli showed high potential for biolistic-based genetic transformation, as revealed by transient GUS expression. Plant Tissue Cult. & Biotech. 28(2): 261-268, 2018 (December)

scholarly journals Cloning, expression, purification, characterization, crystallization and X-ray crystallographic analysis of recombinant Der f 21 (rDer f 21) fromDermatophagoides farinae

2015 ◽  
Vol 71 (11) ◽  
pp. 1396-1400 ◽  
Author(s):  
Sze Lei Pang ◽  
Kok Lian Ho ◽  
Jitka Waterman ◽  
Aik-Hong Teh ◽  
Fook Tim Chew ◽  
...  
Keyword(s):  
Function Analysis ◽  
Expression System ◽  
Crystallographic Analysis ◽  
Size Exclusion ◽  
Diffusion Method ◽  
Monoclinic Space Group ◽  
High Sequence Identity ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion

Dermatophagoides farinaeis one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in anEscherichia coliexpression system. The purified rDer f 21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19 MTris–HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å resolution using an in-house X-ray source. The crystal belonged to theC-centered monoclinic space groupC2, with unit-cell parametersa= 123.46,b= 27.71,c= 90.25 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å3 Da−1suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.

scholarly journals An Optimized Transformation System and Functional Test of CYC-Like TCP Gene CpCYC in Chirita pumila (Gesneriaceae)

2021 ◽  
Vol 22 (9) ◽  
pp. 4544
Author(s):  
Jing Liu ◽  
Juan-Juan Wang ◽  
Jie Wu ◽  
Yang Wang ◽  
Qi Liu ◽  
...  
Keyword(s):  
Expression System ◽  
Transient Gene Expression ◽  
Functional Test ◽  
Transformation System ◽  
Model Plant ◽  
Horizontal Orientation ◽  
Basal Group ◽  
The Family ◽  
Genetic Transformation System ◽  
Evo Devo

The development of an ideal model plant located at a key phylogenetic node is critically important to advance functional and regulatory studies of key regulatory genes in the evolutionary developmental (evo-devo) biology field. In this study, we selected Chirita pumila in the family Gesneriaceae, a basal group in Lamiales, as a model plant to optimize its genetic transformation system established previously by us through investigating a series of factors and further conduct functional test of the CYC-like floral symmetry gene CpCYC. By transforming a RNAi:CpCYC vector, we successfully achieved the desired phenotypes of upright actinomorphic flowers, which suggest that CpCYC actually determines the establishment of floral zygomorphy and the horizontal orientation of flowers in C. pumila. We also confirmed the activities of CpCYC promoter in dorsal petals, dorsal/lateral staminodes, as well as the pedicel by transferring a CpCYC promoter:GUS vector into C. pumila. Furthermore, we testified the availability of a transient gene expression system using C. pumila mesophyll protoplasts. The improved transformation system together with the inherent biological features would make C. pumila an attractive new model in functional and regulatory studies for a broad range of evo-devo issues.

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