Sequence analysis of sundew chitinase gene (original) (raw)
Isolation and Characterization of Chitinase Gene from the Untraditional Plant Species
2013
Round-leaf sundew (Drosera rotundifolia L.) from Droseraceae family belongs among a few plant species with strong antifungal potential. It was previously shown that chitinases of carnivorous plant species may play role during the insect prey digestion, when hard chitin skeleton is being decomposed. As many phytopathogenic fungi contain chitin in their cell wall our attention in this work was focused on isolation and in silico characterization of genomic DNA sequence of sundew chitinase gene. Subsequently this gene was fused to strong constitutive CaMV35S promoter and cloned into the plant binary vector pBinPlus and tested in A. tumefaciens LBA 4404 for its stability. Next, when transgenic tobacco plants are obtained, increasing of their antifungal potential will be tested.
Proceedings of the National Academy of Sciences, 1991
Tobacco contains different isoforms of chitinase (EC 3.2.1.14), a hydrolase thought to be involved in the defense against pathogens. Deduced amino acid sequences for putatively vacuolar, basic chitinases differ from the homologous extracellular, acidic isoforms by the presence of a C-terminal extension. To examine the role of this C-terminal extension in protein sorting, Nicotiana silvestris plants were stably transformed with chimeric genes coding for tobacco basic chitinase A with and without the seven C-terminal amino acids. In plants expressing unmodified chitinase A, the enzyme activity was low in the intercellular wash fluid but high in protoplasts and isolated vacuoles. In contrast, in plants expressing mutant chitinase lacking the C terminus, the activity
Structural and functional evolution of chitinase-like proteins from plants
Protein and Peptide Letters, 2015
The plant genome contains a large number of sequences that encode catalytically inactive chitinases referred to as chitinase-like proteins (CLPs). Although CLPs share high sequence and structural homology with chitinases of glycosyl hydrolase 18 (TIM barrel domain) and 19 families, they may lack the binding/catalytic activity. Molecular genetic analysis revealed that gene duplication events followed by mutation in the existing chitinase gene have resulted in the loss of activity. The evidences show that adaptive functional diversification of the CLPs has been achieved through alterations in the flexible regions than in the rigid structural elements. The CLPs plays an important role in the defense response against pathogenic attack, biotic and abiotic stress. They are also involved in the growth and developmental processes of plants. Since the physiological roles of CLPs are similar to chitinase, such mutations have led to plurifunctional enzymes. The biochemical and structural characterization of the CLPs is essential for understanding their roles and to develop potential utility in biotechnological industries. This review sheds light on the structure-function evolution of CLPs from chitinases.
The Plant Journal, 1994
A novel chitinese gene of tobacco was isolated and characterized by DNA sequence analysis of a genomic clone and a cDNA clone. Comparative sequence analysis of both clones showed an identity of 94%. The proteins encoded by these sequences do not correspond to any of the previously characterized plant chitinases of classes I-IV and are designated as class V chitinaeas. Comparison of the chitinese class V paptide sequence with sequences in the Swiss Protein databank revealed significant sequence similarity with bacterial exo-chitinases from Bacillus circulans, Serratla marcescens and Streptomyces plicatus. It was demonstrated that class V chitinase gene expression is Induced after treatment of tobacco with different forms of stress, like TMV-infection, ethylene treatment, wounding or ultraviolet irradiation.
Structural and Evolutionary Relationships Among Chitinases of Flowering Plants
Journal of Molecular Evolution, 1997
The analysis of nuclear-encoded chitinase sequences from various angiosperms has allowed the categorization of the chitinases into discrete classes. Nucleotide sequences of their catalytic domains were compared in this study to investigate the evolutionary relationships between chitinase classes. The functionally distinct class III chitinases appear to be more closely related to fungal enzymes involved in morphogenesis than to other plant chitinases. The ordering of other plant chitinases into additional classes mainly relied on the presence of auxiliary domains—namely, a chitin-binding domain and a carboxy-terminal extension—flanking the main catalytic domain. The results of our phylogenetic analyses showed that classes I and IV form discrete and well-supported monophyletic groups derived from a common ancestral sequence that predates the divergence of dicots and monocots. In contrast, other sequences included in classes I* and II, lacking one or both types of auxiliary domains, were nested within class I sequences, indicating that they have a polyphyletic origin. According to phylogenetic analyses and the calculation of evolutionary rates, these chitinases probably arose from different class I lineages by relatively recent deletion events. The occurrence of such evolutionary trends in cultivated plants and their potential involvement in host–pathogen interactions are discussed.
Current View on Chitinase for Plant Defence
Plant pathogen is serious problem worldwide amongst the crop cultivars. Large number of plants suffers from range of infectious diseases caused by different pathogens, amongst them fungi are responsible for majority of infectious plant diseases that limiting the crop yield and impact the post-harvest quality of food. So it is important to protect the plants from fungal infection. Substantial approaches have been done with fungicides or heavy metals and by conventional breeding for combating the fungal diseases but so far no conclusive solution has been developed. Genetic engineering paves a new way to protect the plants from harmful fungal afflictions by introducing genes encoding chitinase enzyme that degrade the chitin, which are the key component of fungal cell wall.
Journal of Molecular Modeling, 2012
Glycoside hydrolase family 19 chitinases (EC 3.2.1.14) widely distributed in plants, bacteria and viruses catalyse the hydrolysis of chitin and play a major role in plant defense mechanisms and development. Rice possesses several classes of chitinase, out of which a single structure of class I has been reported in PDB to date. In the present study an attempt was made to gain more insight into the structure, function and evolution of class I, II and IV chitinases of GH family 19 from rice. The three-dimensional structures of chitinases were modelled and validated based on available X-ray crystal structures. The structural study revealed that they are highly α-helical and bilobed in nature. These enzymes are single or multi domain and multi-functional in which chitinbinding domain (CBD) and catalytic domain (CatD) are present in class I and IV whereas class II lacks CBD. The CatD possesses a catalytic triad which is thought to be involved in catalytic process. Loop III, which is common in all three classes of chitinases, reflects that it may play a significant role in their function. Our study also confirms that the absence and presence of different loops in GH family 19 of rice may be responsible for various sized products. Molecular phylogeny revealed chitinases in monocotyledons and dicotyledons differed from each other forming two different clusters and may have evolved differentially. More structural study of this enzyme from different plants is required to enhance the knowledge of catalytic mechanism and substrate binding.
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1995
Complementary and genomic DNAs coding for a Brassica napus chitinase have been cloned and sequenced. The genomic DNA contains one intron and encodes a 322-amino acid basic chitinase with a 20-amino acid N-terminal signal peptide followed by a 40-amino acid cysteine-rich domain, linked by a hinge region to the main domain of the enzyme. The sequence of the cDNAs is identical to the exon sequence deduced from the genomic DNA. A probe derived from this gene identified a 1.2-kb transcript present in high amount in roots, moderate in floral tissues and low in stems and leaves. The synthesis of these transcripts is regulated during development and is induced in roots by wounding and ethephon. This type of chitinase is encoded by two sequences in Brassica napus, as shown either by Southern hybridizations or by genomic amplification and sequencing using the polymerase chain reaction. These genes are homologous to one sequence found in the Brassica oleracea genome.
An increase in urbanization and industrialization has increased the demand of rapid and sustainable agricultural practices. Alternate practices offering better agricultural productivity are necessary to meet the world food demand. Biological control offers a suitable substitute towards chemical pesticides which have their drastic effects on both man and environment. Several lytic enzymes play a key role in these alternative strategies on control of pathogens and pests. Chitinase is one such enzyme complex that has been effectively used in biological control. These enzymes breakdown chitin efficiently into N-acetylglucosamine and its oligomers called chitooligosac-charides. Chitin forms the exoskeleton of arthropods, nematodes and cell wall component in fungi. Due to this, chitin has been targeted for the development of biological control agents against plant pathogens. Plants have known to implicate chitinases in defence against plant pathogens thus chitinases provide an alternate solution to harmful chemicals to combat plant pathogens. This review throws light on the current status of agriculture and further deals with chitinases native to many plants and genetically modified ones, which have been designed in defence against many plant pathogens.
Substrate specificity and antifungal activity of recombinant tobacco class I chitinases
Plant molecular biology, 2001
Endochitinases contribute to the defence response of plants against chitin-containing pathogens. The vacuolar class I chitinases consist of an N-terminal cysteine-rich domain (CRD) linked by a glycine-threonine-rich spacer with 4-hydroxylated prolyl residues to the catalytic domain. We examined the functional role of the CRD and spacer region in class I chitinases by comparing wild-type chitinase A (CHN A) of Nicotiana tabacum with informative recombinant forms. The chitinases were expressed in transgenic N. sylvestris plants, purified to near homogeneity, and their structures confirmed by mass spectrometry and partial sequencing. The enzymes were tested for their substrate preference towards chitin, lipo-chitooligosaccharide Nod factors of Rhizobium, and bacterial peptidoglycans (lysozyme activity) as well as for their capacity to inhibit hyphal growth of Trichoderma viride. Deletion of the CRD and spacer alone or in combination resulted in a modest <50% reduction of hydrolytic ...
2003
Chitinase catalyses the hydrolysis of β-1,4-N-acetyl-D-glucosamine linkages of the fungal cell wall polymer chitin and is involved in the inducible defenses of plants. The aim of this research was to isolate and clone chitinase cDNAs from the seed of winged bean. Chitinase gene fragments were isolated from a winged bean seed cDNA library using two sets of degenerate primers corresponding to the conserved regions of chitinase class I and IV. The poly A + mRNA was reversed transcribed and further amplified using RT-PCR. A 1.1 Kb fragment was selected, cloned and sequenced. A nucleotide se- quence comparison identified the fragment as a Class I basic chitinase cDNA; this fragment was subsequently used as a probe to screen for a full length transcript from the cDNA library. Library screening resulted in the isolation of a 1324 bp clone designated CHRZP; encoding a polypeptide of 289 amino acids containing the diagnostic N-terminal cysteine-rich domain of class 1 chitinases. CHRZP showed...
Elicitor-specific induction of one member of the chitinase gene family in Arachis hypogaea
MGG Molecular & General Genetics, 1990
Chitinases are believed to play an important role in plant defence against bacterial and fungal attack. In peanut (Arachis hypogaea)chitinase genes form a small multigene family. Four chitinase cDNAs (chit 1-4) were isolated from cultured peanut cells. Expression of individual chit genes was assayed by the polymerase chain reaction (PCR) followed by analysis of restriction fragment length polymorphisms (RFLP). UV irradiation, dilution of cell cultures and treatment with Phytophthora megasperma (Ping) elicitor or yeast extract were used to induce expression of chit genes. The chit 3 gene is constitutively expressed at a low level in untreated as well as in treated cultures; the expression of chit 4 gene is induced by each of the stimuli tested, whereas the chit i gene is activated by cell culture dilution and by yeast extract treatment. The chit 2 gene is strongly activated by treatment with cell wall components from the fungus Phytophthora megasperrna but not by the other stimuli. These results indicate that chit 2 gene expression may be controlled by pathogen-specific regulatory elements.
Plant chitinases use two different hydrolytic mechanisms
FEBS Letters, 1996
Bacterial, fungalG animal, and some plant ehNnases form fmily 18 of glycosyl hydrelases. Most plant ehitinases form the family 19, While sense ¢kitinases also have lysozyme activity, animal ~mzymes belong to different families. For 81ycesyl hydrobmes, two reaction mechanisms are possible, lendin8 to either retention or inversion of the anomerlc conflauratlon. We analyzed by HPLC the stereoehemleal ontcome of the hydrolysis catalyzed by cucumber and bean chitlnaes~ belonging to families 18 and 19, respectively. Cucumber ehltlnue used the reteJnlng mechanism as known for bacterial cldtlmmeJ and hen eag white lysozyme for which the mechanlmn hun been determined. In contrast, bean ¢ltltinase utalyzed the hydrolysis of cldtoollgosaeehafldes with overall inversion of anomerle configuration.
Plant physiology
The vacuolar chitinases of class I possess an N-terminal cysteinerich domain homologous to hevein and chitin-binding lectins such as wheat germ agglutinin and Urfica dioica lectin. To investigate the significance of this domain for the biochemical and functional characteristics of chitinase, chimeric genes encoding the basic chitinase A of tobacco (Nicotiana fabacum) with and without this domain were constructed and constitutively expressed in transgenic Nicotiana sylvestris. The chitinases were subsequently isolated and purified t o homogeneity from the transgenic plants. Chromatography on colloidal chitin revealed that only the form with the Nterminal domain, and not the one without it, had chitin-binding properties, demonstrating directly that the domain is a chitinbinding domain (CBD). Under standard assay conditions with radioactive colloidal chitin, both forms of chitinase had approximately the same catalytic activity. However, kinetic analysis demonstrated that the enzyme without CBD had a considerably lower apparent affinity for its substrate. The p H and temperature optima of the two chitinases were similar, but the form with the CBD had an approximately 3-fold higher activation energy and retained a higher activity at low p H values. Both chitinases were capable of inhibiting growth of Trichoderma viride, although the form with the CBD was about three times more effective than the one without it. Thus, the CBD i s not necessary for catalytic or antifungal activity of chitinase.
Isolation and characterisation of chitinase gene from Oryza rufipogon
Malaysian applied biology, 2010
The chitinase gene has been shown to play a role in plant defence mechanisms. Here, the Oryza rufipogon chitinase gene was isolated from genomic DNA of the plant through polymerase chain reaction using specific primers designed with Oryza sativa japonica cv. Nipponbare chitinase gene sequence as template. A 1096 nucleotide long product was obtained and this was translated into a 347 amino acid protein with a molecular weight of 37.7kDa and a pI value of 5.06. The O. rufipogon chitinase gene shared 97% sequence identity at the nucleotide level and 98% at the amino acid level with the chitinase gene sequence of O. sativa japonica cv. Nipponbare (D55711). A motif analysis on the protein sequence showed the presence of glycoside hydrolases and chitinase 18 motifs that are common in chitinase gene architecture. The protein contained two catalytic domains which suggest that it is a class III chitinase. Further, the presence of six cystein residues classed this chitinase into IIIb. In the ...
1989
Plants respond to an attack by potentially pathogenic organisms and to the plant stress hormone ethylene with an increased synthesis of hydrolases such as chitinase and 8-1,3-glucanase. We have studied the subcellular localization of these two enzymes in ethylene-treated bean leaves by immunogold cytochemistry and by biochemical fractionation techniques. Our micrographs indicate that chitinase and 8-1,3-glucanase accumulate in the vacuole of ethylene-treated leaf cells. Within the vacuole label was found predominantly over ethylene-induced electron dense protein aggregates. A second, minor site of accumulation of 8-1,3-glucanase was the cell wall, where label was present nearly exclusively over the middle lamella surrounding intercellular air spaces. 60th kinds of antibodies labeled Golgi cisternae of ethylene-treated tissue, suggesting that the newly synthesized chitinase and p1,3glucanase are processed in the Golgi apparatus. Biochemical fractionation studies confirmed the accumulation in high concentrations of both chitinase and ,B-1,3-glucanase in isolated vacuoles, and demonstrated that only 8-1,sglucanase, but not chitinase, was present in intercellular washing fluids collected from ethylene-treated leaves. Based on these results and earlier studies, we propose a model in which the vacuole-localized chitinase and 8-1,3glucanase are used as a last line of defense to be released when the attacked host cells lyse. The cell wall-localized p-1,8glucanase, on the other hand, would be involved in recognition processes, releasing defense activating signaling molecules from the walls of invading pathogens.
Introduction Chitin, a homopolymer of N-acetyl glucosamine (GlcNAc) residues linked by β-1,4 bonds, is widely distributed in nature as a component of crustacean exoskeleton, insect outer shell, diatoms, fungal cell walls, and squid pens. It is the second most abundant biopolymer next to cellulose, as well as a constant source of renewable raw materials on earth (Tharanathan and Kittur, 2003). Two enzymes catalyzing hydrolysis of the chitin chain to its monomer by synergistic and consecutive action are endochitinases (EC 3.2.1.14), which randomly hydrolyze the β-1,4 glycosidic bonds of chitin, and N-acetylglucosaminidases (chitobiase, EC 3.2.1.30), which preferentially break lower chitooligomers to produce N-acetyl glucosamine (GlcNAc) monomers (Patil et al., 2002). Chitinases play important roles in biological activities such as nutrient intake, morphological change, defense, and attack. Fish and squid can digest chitinous substances as food by using chitinases in the stomach and liver (Matsumiya and Mochizuki, 1996; Matsumiya et al., 1998), whereas insects and shellfish use chitinases to degrade chitinous substances in the exoskeleton during ecdysis (Kramer and Koga, 1986). In plants, chitinases serve to attack fungal pathogens that contain chitinous substances for self-defense (Singh et al., 2007). Chitinases have received considerable attention due to potential applications in the biocontrol of plant pathogenic fungi and insects (Patil et al., 2002) as a target for biopesticides. Chitinases play an important role in the virulence of many bacteria and fungi for insects and fungi by lysing their cell walls. In addition, chitinases inhibit spore germination and germ tube elongation of the phytopathogenic fungi (Mathivanan et al., 1998). Chitinases can be used widely in the production of (GlcNAc)n and GlcNAc, the formation of yeast and fungal spheroplast and protoplast, and the bioconversion of chitin waste to single cell protein for animal feed (Mathivanan et al., 1998). Chitinases are produced by various microorganisms such as bacterial (Singh et al., 2008) and fungal antagonists of other fungal mycoparasistes, nematophagous and entomopathogenic fungi, and others (Rocha-Pino et al., 2011). Because of their wide range of biotechnological applications, chitinases have been purified and characterized.