A genome walking strategy for the identification of eukaryotic nucleotide sequences adjacent to known regions

Determination of nucleotide sequences adjacent to a known region is a recurring need in many genome scale studies. Various methods have been developed based on PCR techniques in order to fulfill th..

Purification, Characterization and Functional Analysis of a Serine Protease Inhibitor from the Pulps of Cicer arietinum L. (Chick Pea)

Serine proteinase inhibitors (SPIs) are present in high amount in legume seeds where they play an important role in plant defence mechanism against pests. In the present study, a serine protease inhibitor has been identified from the seeds of Cicer arietinum (L.) and characterized for its inhibitory potency against trypsin and chymotrypsin. Ammonium sulphate fractionation was executed as an initial step to purify the inhibitor. The fraction which was obtained from 30-60% relative ammonium sulphate saturation exhibited the maximum trypsin inhibition activity against 0.2% casein using radial diffusion method. The 30–60% fraction was further subjected to ion exchange chromatography using 1 mL HiTrap Q HP column. The peak fractions were analyzed for the enzymatic activity and also characterized on 12% SDS PAGE. The results indicated that the flow through fraction has retained a significant proteolytic inhibition towards trypsin with a relative molecular mass of approximately 12-13 kDa. The kinetic results have demonstrated that the purified inhibitor from Cicer arietinum L. not only inhibited trypsin but also chymotrypsin. The Circular Dichroism spectrum analysis of the purified inhibitor has revealed that the secondary structure content is highly composed of random coils which were supported by the reports of other low molecular weight trypsin inhibitors. To conclude, a low molecular weight serine protease inhibitor possessing both trypsin and chymotrypsin inhibition from the seeds of C. arietinum has been purified, characterized and the results are reported

Isolation of a serine Kunitz trypsin inhibitor from leaves of Terminalia arjuna

A serine Kunitz protease inhibitor was isolated from the semi-mature leaves of Terminalia arjuna, a host plant for Antheraea mylitta, using ammonium sulphate fractionation, gel permeation chromatography and trypsin–sepharose affinity chromatography. A 29-fold purification of T. arjuna Trypsin Inhibitor (TaTI) with a yield recovery of 3.2% was achieved. The purified protease inhibitor (TaTI) was resolved into a single protein band corresponding to molecular weight of 19.0 kDa on 12% SDS–PAGE under non-reducing conditions, whereas an additional band of 21.5 kDa was observed when the same fraction was resolved on SDS–PAGE under reducing conditions in the presence of 2-mercaptoethanol. TaTI inhibited both trypsin and chymotrypsin, but showed higher affinity for trypsin compared to chymotrypsin. However, it is more effective on bovine trypsin than midgut trypsin of tasar silkworm. TaTI retains its activity over a wide range of temperatures (0–100°C) and pH (2.0–8.0), with pH optimum of 8.0. These observations indicate that TaTI is not only specific to tasar silkworm but also to bovine serine proteases. Hence it can be considered as a generalist protease inhibitor

Expression of two barley proteinase inhibitors in tomato promotes endogenous defensive response and enhances resistance to Tuta absoluta

[EN] Background: For as long as 350 million years, plants and insects have coexisted and developed a set of relationships which affect both organisms at different levels. Plants have evolved various morphological and biochemical adaptations to cope with herbivores attacks. However, Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) has become the major pest threatening tomato crops worldwide and without the appropriated management it can cause production losses between 80 to 100%. Results: The aim of this study was to investigate the in vivo effect of a serine proteinase inhibitor (BTI-CMe) and a cysteine proteinase inhibitor (Hv-CPI2) from barley on this insect and to examine the effect their expression has on tomato defensive response. We found that larvae fed on the double transgenic plants showed a notable reduction in weight. Moreover, only 56% of the larvae reached the adult stage. The emerged adults showed wings deformities and reduced fertility. We also investigated the effect of proteinase inhibitors ingestion on the insect digestive enzymes. Our results showed a decrease in larval trypsin activity. Transgenes expression had no harmful effect on Nesidiocoris tenuis (Reuter) (Heteroptera: Miridae), a predator of Tuta absoluta, despite transgenic tomato plants attracted the mirid. We also found that barley cystatin expression promoted plant defense by inducing the expression of the tomato endogenous wound inducible Proteinase inhibitor 2 (Pin2) gene, increasing the production of glandular trichomes and altering the emission of volatile organic compounds. Conclusion: Our results demonstrate the usefulness of the co-expression of different proteinase inhibitors for the enhancement of plant resistance to Tuta absoluta.This work was partly supported by grants BIO2013-40747-R and AGL2014-55616-C3 from the Spanish Ministry of Economy and Competitiveness (MINECO)Hamza, R.; Pérez-Hedo, M.; Urbaneja, A.; Rambla Nebot, JL.; Granell Richart, A.; Gaddour, K.; Beltran Porter, JP.... (2018). Expression of two barley proteinase inhibitors in tomato promotes endogenous defensive response and enhances resistance to Tuta absoluta. BMC Plant Biology. 18. https://doi.org/10.1186/s12870-018-1240-6S18Oerke EC. Crop losses to pests. J Agric Sci. 2005;144(01):31.Jouanin L, Bonadé-Bottino M, Girard C, Morrot G, Giband M. Transgenic plants for insect resistance. Plant Sci. 1998;131(1):1–11.Markwick NP, Docherty LC, Phung MM, Lester MT, Murray C, Yao JL, Mitra DS, Cohen D, Beuning LL, Kutty-Amma S, et al. 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Proyección de extractos de diferentes plantas brasileñas como candidatos para el control de Spodoptera frugiperda (Lepidoptera: Noctuidae).

Los efectos de extractos de las plantas Actinostemon concolor, Geonoma shottiana, Palicourea rigida y Rudgea viburnoides sobre Spodoptera frugiperda fueron evaluados en este estudio. Los extratos se incorporaron en la dieta artificial (1 mg mL-1 de la dieta) y se ofrecieron a larvas de S. frugiperda. El extracto de A. concolor causó 36,1 % de mortalidad. Se detectaron cambios ultraestructurales en los huevos de adultos alimentados en sus estados larvales con extractos de P. rigida y A. concolor. Se observó reducción de la actividad de la tripsina de S. frugiperda después del tratamiento con el extracto de A. concolor. Al cuantificar los fenoles totales en A. concolor, G. shottiana, P. rigida y R. viburnoides, se encontró que A. concolor fue la especie con mayor contenido. El residuo de metanol/agua extracción, que se obtuvo del extracto crudo de A. concolor, contenía compuestos fenólicos que causan cambios en la biología de S. frugiperda similar a los generados por el extracto crudo. En conclusión, A. concolor produce metabolitos secundarios con actividad insecticida sobre S. frugiperda

Purification of a trypsin inhibitor from Cocculus hirsutus and identification of its biological activity

Proteinase inhibitors play a significant role in plant defense against insect pests and phytopathogens by inhibiting their proteases. A thermotolerant monomeric trypsin inhibitor with molecular weight ∼18kD was purified from Cocculus hirsutus (ChTI) using trypsin sepharose affinity column. Western blot analysis using ChTI IgY revealed its presence in vegetative parts and seeds. The second and third instar larvae of H. armigera fed with ChTI (5000TIU/ml) resulted in 84.59 and 58.71% reduction in mean larval weight respectively. An increase in the larval growth period was observed in ChTI fed larvae at all instars and inhibitor fed larvae could not complete their life cycle. ChTI caused 74 and 59.53% inhibition of bovine trypsin and Helicoverpa gut proteases respectively. ChTI exhibited strain specificity and inhibited growth and development of plant fungal pathogens. Bioassay studies on yeast strains indicated that ΔYNK and MNN1 are more sensitive to ChTI. The results suggest that phosphodiester linkage in cell wall components is likely to be the key determinants for binding of ChTI. Taken together, these studies indicate that ChTI is a potential candidate for development of transgenic plants against foliar diseases and insect pests

Potential Use of a Serpin from Arabidopsis for Pest Control

Although genetically modified (GM) plants expressing toxins from Bacillus thuringiensis (Bt) protect agricultural crops against lepidopteran and coleopteran pests, field-evolved resistance to Bt toxins has been reported for populations of several lepidopteran species. Moreover, some important agricultural pests, like phloem-feeding insects, are not susceptible to Bt crops. Complementary pest control strategies are therefore necessary to assure that the benefits provided by those insect-resistant transgenic plants are not compromised and to target those pests that are not susceptible. Experimental GM plants producing plant protease inhibitors have been shown to confer resistance against a wide range of agricultural pests. In this study we assessed the potential of AtSerpin1, a serpin from Arabidopsis thaliana (L). Heynh., for pest control. In vitro assays were conducted with a wide range of pests that rely mainly on either serine or cysteine proteases for digestion and also with three non-target organisms occurring in agricultural crops. AtSerpin1 inhibited proteases from all pest and non-target species assayed. Subsequently, the cotton leafworm Spodoptera littoralis Boisduval and the pea aphid Acyrthosiphon pisum (Harris) were fed on artificial diets containing AtSerpin1, and S. littoralis was also fed on transgenic Arabidopsis plants overproducing AtSerpin1. AtSerpin1 supplied in the artificial diet or by transgenic plants reduced the growth of S. littoralis larvae by 65% and 38%, respectively, relative to controls. Nymphs of A. pisum exposed to diets containing AtSerpin1 suffered high mortality levels (LC50 = 637 µg ml−1). The results indicate that AtSerpin1 is a good candidate for exploitation in pest control

Uses of phage display in agriculture: A review of food-related protein-protein interactions discovered by biopanning over diverse baits

This review highlights discoveries made using phage display that impact the use of agricultural products. The contribution phage display made to our fundamental understanding of how various protective molecules serve to safeguard plants and seeds from herbivores and microbes is discussed. The utility of phage display for directed evolution of enzymes with enhanced capacities to degrade the complex polymers of the cell wall into molecules useful for biofuel production is surveyed. Food allergies are often directed against components of seeds; this review emphasizes how phage display has been employed to determine the seed component(s) contributing most to the allergenic reaction and how it has played a central role in novel approaches to mitigate patient response. Finally, an overview of the use of phage display in identifying the mature seed proteome protection and repair mechanisms is provided. The identification of specific classes of proteins preferentially bound by such protection and repair proteins leads to hypotheses concerning the importance of safeguarding the translational apparatus from damage during seed quiescence and environmental perturbations during germination. These examples, it is hoped, will spur the use of phage display in future plant science examining protein-ligand interactions