scholarly journals Tobacco Cutworm (Spodoptera Litura) Larvae Silenced in the NADPH-Cytochrome P450 Reductase Gene Show Increased Susceptibility to Phoxim

2019 ◽  
Vol 20 (15) ◽  
pp. 3839 ◽  
Author(s):  
Hong-Yi Ji ◽  
Christian Staehelin ◽  
Yan-Ping Jiang ◽  
Shi-Wei Liu ◽  
Zhi-Hui Ma ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Amino Acid ◽  
Spodoptera Litura ◽  
Fat Body ◽  
Larval Mortality ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Amino Acid Sequences ◽  
Cytochrome P450 Monooxygenases ◽  
Expression Levels ◽  
Nadph Cytochrome

Nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductases (CPRs) function as redox partners of cytochrome P450 monooxygenases (P450s). CPRs and P450s in insects have been found to participate in insecticide resistance. However, the CPR of the moth Spodoptera litura has not been well characterized yet. Based on previously obtained transcriptome information, a full-length CPR cDNA of S. litura (SlCPR) was PCR-cloned. The deduced amino acid sequence contains domains and residues predicted to be essential for CPR function. Phylogenetic analysis with insect CPR amino acid sequences showed that SlCPR is closely related to CPRs of Lepidoptera. Quantitative reverse transcriptase PCR (RT-qPCR) was used to determine expression levels of SlCPR in different developmental stages and tissues of S. litura. SlCPR expression was strongest at the sixth-instar larvae stage and fifth-instar larvae showed highest expression in the midgut. Expression of SlCPR in the midgut and fat body was strongly upregulated when fifth-instar larvae were exposed to phoxim at LC15 (4 μg/mL) and LC50 (20 μg/mL) doses. RNA interference (RNAi) mediated silencing of SlCPR increased larval mortality by 34.6% (LC15 dose) and 53.5% (LC50 dose). Our results provide key information on the SlCPR gene and indicate that SlCPR expression levels in S. litura larvae influence their susceptibility to phoxim and possibly other insecticides.

scholarly journals CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2

2016 ◽  
Vol 82 (22) ◽  
pp. 6507-6517 ◽  
Author(s):  
Birgit Unterweger ◽  
Dieter M. Bulach ◽  
Judith Scoble ◽  
David J. Midgley ◽  
Paul Greenfield ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Amino Acid ◽  
Amino Acid Sequences ◽  
Cytochrome P450 Monooxygenases ◽  
Content Type ◽  
E Coli ◽  
P450 Monooxygenases ◽  
Isolation And Characterization ◽  
Starting Point

ABSTRACTWe report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived fromSphingobium yanoikuyaeB2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6tagged) inEscherichia coliBL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation inE. colidemonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners fromE. colito yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found inNovosphingobium aromaticivoransandPseudomonas putida. Compared to P450cin(CYP176A1), a 1,8-cineole-hydroxylating P450 fromCitrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications.IMPORTANCECYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases fromS. yanoikuyaeB2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101 family. This could eventually provide enough bacterial parental enzymes with similar amino acid sequences to enablein vitroevolution via DNA shuffling.

scholarly journals Natural selections on both amino acid sequences and expression levels are determinants of ohnolog retention

2021 ◽  
Author(s):  
Lin Miao ◽  
Miaoxin Li
Keyword(s):  
Natural Selection ◽  
Amino Acid ◽  
Complex Traits ◽  
Evolutionary Biology ◽  
Amino Acid Sequences ◽  
Regulatory Sequences ◽  
Unified Framework ◽  
Retention Model ◽  
Coding Sequences ◽  
Expression Levels

AbstractThe mechanism of ohnolog retention is a subject of concern in evolutionary biology. Natural selections on coding sequences and gene dosages have been proposed to be determinants of ohnolog retention. However, the relationship between the two models is not widely accepted, and the role of regulatory sequences on ohnolog retention has long been neglected. In this study, based on a model of complex traits’ genetic architecture, we compared the natural selection’s strength on corresponding sequences between ohnologs and non-ohnologs by comparing complex traits’ heritability enrichments. We showed that complex traits’ regulatory sequences’ heritability enrichments (p = 1.1 × 10−5 in 5 kb flanking regions) and expression-mediated heritability enrichments (p = 2.1 × 10−5) of ohnologs were significantly higher than non-ohnologs. Then, we deduced that regulatory sequences of ohnologs were under substantial natural selection, which was also a determent of ohnolog retention. Meanwhile, we showed that in coding sequences, the complex traits’ heritability enrichments of ohnologs were significantly higher than of non-ohnologs (p = 9.9 × 10−5), supporting the ohnolog retention model of natural selection on coding sequences. We also showed that complex traits’ causal gene expression effect sizes of ohnologs were significantly larger than of non-ohnologs (p = 8.8 × 10−6), supporting the ohnolog retention model of natural selection on gene dosages. In conclusion, we provide the first unified framework to show that both amino acid sequences and expression levels of ohnologs are under substantial selection, which may end the long-standing debate on ohnolog retention models.

Molecular Characterization of Primary Juvenile Hormone Responders Methoprene-Tolerant (Met) and Krüppel Homolog 1 (Kr-h1) in Grapholita molesta (Lepidoptera: Tortricidae) with Clarification of Their Roles in Metamorphosis and Reproduction

2019 ◽  
Vol 112 (5) ◽  
pp. 2369-2380 ◽  
Author(s):  
Jing Zhang ◽  
Xiaoxia Liu ◽  
Yichen Liu ◽  
Yueqing An ◽  
Haibo Fang ◽  
...  
Keyword(s):  
Fat Body ◽  
Larval Mortality ◽  
Adult Emergence ◽  
Amino Acid Sequences ◽  
Grapholita Molesta ◽  
Preoviposition Period ◽  
Krüppel Homolog 1 ◽  
Lepidoptera Noctuidae ◽  
Dsrna Injection

AbstractMethoprene-tolerant (Met) is a putative JH intracellular receptor that transduces JH signal by activation of the inducible Krüppel homolog 1 (Kr-h1). We analyzed the gene sequences of Met and Kr-h1 and their patterns of expression in Grapholita molesta (Busck) immature and adult stages in order to better understand the roles of these primary JH responders in regulating the metamorphosis and reproduction of this global pest of fruit crops. The deduced amino acid sequences of both GmMet and GmKr-h1 were highly homologous to those of other Lepidoptera, especially the cotton bollworm, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae). Peak expression of GmMet occurred during the last 3 to 5 d of the final instar, followed by that of GmKr-h1, in the last 3 d of final instar. Similar patterns of GmMet and GmKr-h1 expression were detected across various tissue types in the fifth-instar larvae, with the highest expression observed in the head, followed by the epidermis, and the fat body. When expression of GmMet and GmKr-h1 was knocked down via dsRNA injection in the fifth instar, the results were increased larval mortality, abnormal pupation, delayed pupal duration, reduced adult emergence, extended preoviposition period, and reduced fecundity. We infer that both GmMet and GmKr-h1 participated in regulation of metamorphosis and reproduction in G. molesta, the former acting upstream of the latter, and could present biorational targets for novel pest control compounds.

scholarly journals Asatone and Isoasatone A Against Spodoptera litura Fab. by Acting on Cytochrome P450 Monoxygenases and Glutathione Transferases

Molecules ◽  
2019 ◽  
Vol 24 (21) ◽  
pp. 3940
Author(s):  
Ruimei Ling ◽  
Renyue Yang ◽  
Ping Li ◽  
Xiongfei Zhang ◽  
Tunkai Shen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cytochrome P450 ◽  
Spodoptera Litura ◽  
Glutathione Transferases ◽  
Structural Deformation ◽  
Relative Gene Expression ◽  
Expression Levels ◽  
Relative Expression ◽  
Insect Activity ◽  
Relative Gene

Asatone and isoasatone A from Asarum ichangense Cheng were determined to be defensive compounds to some insects in a previous investigation. However, the anti-insect activity mechanisms to caterpillar are still unclear. The compounds asatone and isoasatone A from A. ichangense were induced by Spodoptera litura. The anti-insect activity of asatone and isoasatone A to S. litura was further tested by weight growth rate of the insect through a diet experiment. Isoasatone A showed a more significant inhibitory effect on S. litura than asatone on the second day. The concentration of asatone was higher than isoasatone A in the second instar larvae of S. litura after 12 h on the feeding test diet. Both compounds caused mid-gut structural deformation and tissue decay as determined by mid-gut histopathology of S. litura. Furthermore, some detoxification enzyme activity were measured by relative expression levels of genes using a qPCR detecting system. Asatone inhibited the gene expression of the cytochrome P450 monooxygenases (P450s) CYP6AB14. Isoasatone A inhibited the relative expression levels of CYP321B1, CYP321A7, CYP6B47, CYP6AB14, and CYP9A39. Asatone increased the relative gene expression of the glutathione transferases (GSTs) SIGSTe1 and SIGSTo1, in contrast, isoasatone A decreased the relative gene expression of SIGSTe1 by about 33 fold. Neither compound showed an effect on acetylcholinesterase SIAce1 and SIAce2. The mechanism of anti-insect activity by both compounds could be explained by the inhibition of enzymes P450s and GSTs. The results provide new insights into the function of unique secondary metabolites asatone and isoasatone A in genus Asarum, and a new understanding of why A. ichangense is largely free of insect pests.

scholarly journals Comparative Analysis of NADPH-Cytochrome P450 Reductases From Legumes for Heterologous Production of Triterpenoids in Transgenic Saccharomyces cerevisiae

2021 ◽  
Vol 12 ◽  
Author(s):  
Pramesti Istiandari ◽  
Shuhei Yasumoto ◽  
Pisanee Srisawat ◽  
Keita Tamura ◽  
Ayaka Chikugo ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Higher Plants ◽  
Lotus Japonicus ◽  
Class Ii ◽  
Expression Level ◽  
Class I ◽  
Cytochrome P450 Monooxygenases ◽  
Heterologous Production ◽  
Nadph Cytochrome ◽  
Transgenic Yeast

Triterpenoids are plant specialized metabolites with various pharmacological activities. They are widely distributed in higher plants, such as legumes. Because of their low accumulation in plants, there is a need for improving triterpenoid production. Cytochrome P450 monooxygenases (CYPs) play critical roles in the structural diversification of triterpenoids. To perform site-specific oxidations, CYPs require the electrons that are transferred by NADPH-cytochrome P450 reductase (CPR). Plants possess two main CPR classes, class I and class II. CPR classes I and II have been reported to be responsible for primary and specialized (secondary) metabolism, respectively. In this study, we first analyzed the CPR expression level of three legumes species, Medicago truncatula, Lotus japonicus, and Glycyrrhiza uralensis, showing that the expression level of CPR class I was lower and more stable, while that of CPR class II was higher in almost all the samples. We then co-expressed different combinations of CYP716As and CYP72As with different CPR classes from these three legumes in transgenic yeast. We found that CYP716As worked better with CPR-I from the same species, while CYP72As worked better with any CPR-IIs. Using engineered yeast strains, CYP88D6 paired with class II GuCPR produced the highest level of 11-oxo-β-amyrin, the important precursor of high-value metabolites glycyrrhizin. This study provides insight into co-expressing genes from legumes for heterologous production of triterpenoids in yeast.

scholarly journals Sequence comparisons of cytochrome P450 aromatases from Australian animals predict differences in enzymatic activity and/or efficiency†

2020 ◽  
Vol 102 (6) ◽  
pp. 1261-1269
Author(s):  
Anam Fatima ◽  
Jessica K Holien ◽  
Chandni Tiwari ◽  
Michael W Parker ◽  
Raymond J Rodgers ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Amino Acid ◽  
Active Sites ◽  
Amino Acid Sequences ◽  
Slight Variation ◽  
Or Efficiency ◽  
Sequence Comparisons ◽  
High Amino Acid ◽  
Bearded Dragon ◽  
Human Aromatase

Abstract Aromatase (P450arom, CYP19A1) is the terminal enzyme in the synthesis of the steroid hormone family of estrogens. Not surprisingly, this enzyme has structural similarities between the limited number of species studied thus far. This study examined the structure of aromatases from four diverse Australian species including a marsupial (tammar wallaby; Macropus eugenii), monotreme (platypus; Ornithorhynchus anatinus), ratite (emu; Dromaius novaehollandiae) and lizard (bearded dragon; Pogona vitticeps). We successfully built homology models for each species, using the only crystallographically determined structure available, human aromatase. The amino acid sequences showed high amino acid sequence identity to the human aromatase: wallaby 81%, platypus 73%, emu 75% and bearded dragon at 74%. The overall structure was highly conserved among the five species, although there were non-secondary structures (loops and bends) that were variable and flexible that may result in some differences in catalytic activity. At the N-terminal regions, there were deletions and variations that suggest that functional distinctions may be found. We found that the active sites of all these proteins were identical, except for a slight variation in the emu. The electrostatic potential across the surfaces of these aromatases highlighted likely variations to the protein-protein interactions of these enzymes with both redox partner cytochrome P450 reductase and possibly homodimerization in the case of the platypus, which has been postulated for the human aromatase enzyme. Given the high natural selection pressures on reproductive strategies, the relatively high degree of conservation of aromatase sequence and structure across species suggests that there is biochemically very little scope for changes to have evolved without the loss of enzyme activity.

scholarly journals Haplotype Diversity of NADPH-Cytochrome P450 Reductase Gene of Ophiocordyceps sinensis and the Effect on Fungal Infection in Host Insects

2020 ◽  
Vol 8 (7) ◽  
pp. 968
Author(s):  
Zixian Xu ◽  
Yunguo Zhu ◽  
Lingyan Xuan ◽  
Shan Li ◽  
Zhou Cheng
Keyword(s):  
Cytochrome P450 ◽  
Haplotype Diversity ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Western China ◽  
Cytochrome P450 Reductase ◽  
Ophiocordyceps Sinensis ◽  
Nadph Cytochrome ◽  
P450 Reductase ◽  
Nadph Cytochrome P450 Reductase ◽  
Almost All

Ophiocordyceps sinensis Berk. is a fungal parasite that parasitizes the larvae of Hepialidae and is used as a traditional Chinese medicine. However, it is not clear how O. sinensis infects its host. The encoding gene haplotype diversity and predicted function of the nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) related to the fungal pathogenicity was analyzed for 219 individuals from 47 O. sinensis populations. Two NADPH CPR genes of O. sinensis were detected and their dominant haplotypes were widely distributed throughout the entire distribution range in Western China. Only 5.43% of all O. sinensis individuals possessed the specific private haplotypes of NADPH CPR-1 and CPR-2 genes. Bioinformatic analyses predicted that the phosphorylation sites, motifs, and domains of NADPH CPR of O. sinensis were different between those encoding by the dominant and private gene haplotypes. The one-to-one match fungus–host correspondence of the same individual suggested that the widely distributed O. sinensis with the dominant NADPH CPR gene haplotypes may strongly infect almost all host insects through a random infection by oral or respiratory pores. Conversely, O. sinensis with the specific private NADPH CPR gene haplotypes is likely to infect only a few corresponding host insects by breaching the cuticle, due to the changed NADPH CPR structure and function.

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