Proteomic Analysis of Glutathione Transferases from Lucilia Cuprina

10.26686/wgtn.16984750.v1 ◽

2021 ◽

Author(s):

◽

Ramavati Pal

Keyword(s):

Insecticide Resistance ◽

Proteomic Analysis ◽

Developmental Stages ◽

Adult Stage ◽

Glutathione Transferases ◽

Lucilia Cuprina ◽

Main Body ◽

Post Translational Modification ◽

2D Page ◽

Gst Activity

<p>The glutathione transferases are a family of multifunctional enzymes involved in detoxification of xenobiotic and endogenous electrophilic compounds. Interest in insect GSTs has primarily focused on their role in insecticide resistance. The sheep blowfly, Lucilia cuprina is a major economic problem for the sheep meat and wool industries in Australasia and hence this thesis has attempted the study of the Lucilia cuprina GST family, using proteomics, with a view to eventually determining their role in insecticide resistance. Combinations of different affinity matrices (glutathione-Sepharose matrix (GSH) followed by dinitrophenyl-glutathione-Sepharose matrix (DNP-GSH)) and two-dimensional electrophoresis has successfully isolated members from major four insect GST classes: Sigma, Delta, Epsilon and Omega. Drosophila melanogaster has been used as a model insect throughout as a basis for comparison. To characterise Lucilia GSTs, the whole metazoan fragmentation database was used for sequence alignment with Lucilia peptides. This approach is broad and speculative but predicts a possible classification of the GSTs based on % similarity and % identity. This method of characterisation yielded match scores that provided a basis for classification, which must at present be regarded as tentative and in need of confirmation. In D. melanogaster and L. cuprina, GSH affinity-purified extracts showed the presence of only Sigma and Delta GSTs. In D. melanogaster, the DNP-GSH affinity-purified GSTs showed mostly the presence of Epsilon and Omega GSTs whereas in L. cuprina no Omega GSTs were detected. In both species, the migration pattern of Delta GST on 2D PAGE gel indicated possible post-translational modification. The results from analysis of LC-MS/MS data by the software PEAKS suggested deamidation at asparagine and glutamine residues in a limited number of the matched peptides of Delta GST. GST activity was present in all developmental stages of L. cuprina. The number of isoenzymes and their extent of expression vary as the insect develops. Delta GSTs were present in all developmental stages. The Sigma GST started expressing from the larval stage and was abundantly present in adult stage. The DNP-GSH affinity matrix purified GSTs which have been tentatively classified as Mu-like GSTs were present in egg, larvae and pupae but totally absent in adult stage. The GST families were characterised by proteomics in the main body sections of L. cuprina. Higher GST activity towards 1-chloro-2, 4-dinitrobenzene (CDNB) was found in the thorax (65.2 %) followed by the abdomen (19.6%) and the head (15.2%). The cytosolic GSTs of a resistant strain (PY81) of L. cuprina had significantly higher (2.26- and 2.6- fold) activity than the susceptible strains (NSW and CSIRO) towards CDNB and 2, 3-dichloro, 4-nitrobenzene (DCNB) respectively. The proteomic analysis of DNP-GSH purified extract from susceptible and resistant strains showed quantitatively higher expression of GSTs on 2D PAGE gel of the PY81 strain. The in vitro interaction of purified GSTs and model insecticides studied by high performance liquid chromatography revealed that Delta and DNP-GSH affinity-purified GSTs catalyse the conjugation of the insecticides to reduced glutathione but Sigma GST had almost no activity.</p>

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Integrated transcriptomic and proteomic analysis reveals the complex molecular mechanisms underlying stone cell formation in Korla pear

Scientific Reports ◽

10.1038/s41598-021-87262-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Aisajan Mamat ◽

Kuerban Tusong ◽

Juan Xu ◽

Peng Yan ◽

Chuang Mei ◽

...

Keyword(s):

Cell Differentiation ◽

Proteomic Analysis ◽

Molecular Mechanisms ◽

Developmental Stages ◽

Cell Formation ◽

Parenchyma Cell ◽

Lignin Biosynthesis ◽

Cell Content ◽

Stone Cell ◽

Fruit Samples

AbstractKorla pear (Pyrus sinkiangensis Yü) is a landrace selected from a hybrid pear species in the Xinjiang Autonomous Region in China. In recent years, pericarp roughening has been one of the major factors that adversely affects fruit quality. Compared with regular fruits, rough-skin fruits have a greater stone cell content. Stone cells compose sclerenchyma tissue that is formed by secondary thickening of parenchyma cell walls. In this work, we determined the main components of stone cells by isolating them from the pulp of rough-skin fruits at the ripening stage. Stone cell staining and apoptosis detection were then performed on fruit samples that were collected at three different developmental stages (20, 50 and 80 days after flowering (DAF)) representing the prime, late and stationary stages of stone cell differentiation, respectively. The same batches of samples were used for parallel transcriptomic and proteomic analysis to identify candidate genes and proteins that are related to SCW biogenesis in Korla pear fruits. The results showed that stone cells are mainly composed of cellulose (52%), hemicellulose (23%), lignin (20%) and a small amount of polysaccharides (3%). The periods of stone cell differentiation and cell apoptosis were synchronous and primarily occurred from 0 to 50 DAF. The stone cell components increased abundantly at 20 DAF but then decreased gradually. A total of 24,268 differentially expressed genes (DEGs) and 1011 differentially accumulated proteins (DAPs) were identified from the transcriptomic and proteomic data, respectively. We screened the DEGs and DAPs that were enriched in SCW-related pathways, including those associated with lignin biosynthesis (94 DEGs and 31 DAPs), cellulose and xylan biosynthesis (46 DEGs and 18 DAPs), S-adenosylmethionine (SAM) metabolic processes (10 DEGs and 3 DAPs), apoplastic ROS production (16 DEGs and 2 DAPs), and cell death (14 DEGs and 6 DAPs). Among the identified DEGs and DAPs, 63 significantly changed at both the transcript and protein levels during the experimental periods. In addition, the majority of these identified genes and proteins were expressed the most at the prime stage of stone cell differentiation, but their levels gradually decreased at the later stages.

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F-Box Genes in the Wheat Genome and Expression Profiling in Wheat at Different Developmental Stages

Genes ◽

10.3390/genes11101154 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1154

Author(s):

Min Jeong Hong ◽

Jin-Baek Kim ◽

Yong Weon Seo ◽

Dae Yeon Kim

Keyword(s):

Developmental Stages ◽

Brachypodium Distachyon ◽

Triticum Aestivum L ◽

Wheat Genome ◽

Post Translational Modification ◽

Genome Wide ◽

A Genome ◽

High Sequence Homology ◽

Wide Scale

Genes of the F-box family play specific roles in protein degradation by post-translational modification in several biological processes, including flowering, the regulation of circadian rhythms, photomorphogenesis, seed development, leaf senescence, and hormone signaling. F-box genes have not been previously investigated on a genome-wide scale; however, the establishment of the wheat (Triticum aestivum L.) reference genome sequence enabled a genome-based examination of the F-box genes to be conducted in the present study. In total, 1796 F-box genes were detected in the wheat genome and classified into various subgroups based on their functional C-terminal domain. The F-box genes were distributed among 21 chromosomes and most showed high sequence homology with F-box genes located on the homoeologous chromosomes because of allohexaploidy in the wheat genome. Additionally, a synteny analysis of wheat F-box genes was conducted in rice and Brachypodium distachyon. Transcriptome analysis during various wheat developmental stages and expression analysis by quantitative real-time PCR revealed that some F-box genes were specifically expressed in the vegetative and/or seed developmental stages. A genome-based examination and classification of F-box genes provide an opportunity to elucidate the biological functions of F-box genes in wheat.

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Insecticide resistance status of Lucilia cuprina in New Zealand using biochemical and toxicological techniques

Veterinary Parasitology ◽

10.1016/0304-4017(94)90194-5 ◽

1994 ◽

Vol 53 (3-4) ◽

pp. 301-308 ◽

Cited By ~ 10

Author(s):

D.M. Gleeson ◽

S.C. Barry ◽

A.C.G. Heath

Keyword(s):

New Zealand ◽

Insecticide Resistance ◽

Lucilia Cuprina

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Growth, development and allometry of Philophthalmus nocturnus in the eyes of domestic chicks

Journal of Helminthology ◽

10.1017/s0022149x00012669 ◽

1992 ◽

Vol 66 (2) ◽

pp. 100-107 ◽

Cited By ~ 5

Author(s):

V. G. M. Swarnakumari ◽

R. Madhavi

Keyword(s):

Growth Rate ◽

Developmental Stages ◽

Adult Stage ◽

The Body ◽

Lag Phase ◽

Allometric Growth ◽

Body Width ◽

Rapid Phase ◽

Growth Development ◽

Two Phases

ABSTRACTFifty day-old chicks were each infected with 10 excysted metaccreariae of Philophthalimus nocturnus Looss. 1907 around each orbit and growth, development and allometry were studied. The growth rate showed two phases over a period of 35 days, a limited lag phase lasting two days post-infection in which flukes did not exceed 440 μm in length, and a rapid phase during which growth was rapid and flukes reached a size of 3·008–3·504 mm on day 35. Five developmental stages were noticed during the course of development of the metacercaria to the egg-producing adult stage. Eggs appeared in the uterus on day 14 and oculate miracidia on day 25. The hindhody, testes and ovary showed positive allometric growth, the pharnyx less so, whereas negative allometric growth was shown by the forebody. Body width, oral sucker and ventral sucker were close to isometry, growing at the same rate as the body length.

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Translation initiation in Drosophila melanogaster is reduced by mutations upstream of the AUG initiator codon.

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.2149 ◽

1991 ◽

Vol 11 (4) ◽

pp. 2149-2153 ◽

Cited By ~ 12

Author(s):

Y Feng ◽

L E Gunter ◽

E L Organ ◽

D R Cavener

Keyword(s):

Translation Initiation ◽

Larval Stage ◽

Developmental Stages ◽

Germ Line ◽

Adult Stage ◽

Mutant Gene ◽

Start Codon ◽

Fold Reduction

The importance to in vivo translation of sequences immediately upstream of the Drosophila alcohol dehydrogenase (Adh) start codon was examined at two developmental stages. Mutations were introduced into the Adh gene in vitro, and the mutant gene was inserted into the genome via germ line transformation. An A-to-T substitution at the -3 position did not affect relative translation rates of the ADH protein at the second-instar larval stage but resulted in a 2.4-fold drop in translation of ADH at the adult stage. A second mutant gene, containing five mutations in the region -1 to -9, was designed to completely block translation initiation. However, transformant lines bearing these mutations still exhibit detectable ADH, albeit at substantially reduced levels. The average fold reduction at the second-instar larval stage was 5.9, while at the adult stage a 12.5-fold reduction was observed.

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