Identification of species-specific genes from Leucaena leucocephala using interspecies suppression subtractive hybridisation

Negi, Vishal Singh; Pal, Archana; Singh, Ratnesh; Borthakur, Dulal

doi:10.1111/j.1744-7348.2011.00506.x

Cited by 18 publications

(7 citation statements)

References 41 publications

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“…Isolation of cDNA for a Mimosine-Degrading Enzyme from L. leucocephala Previously, we isolated a set of 406 clones from the cDNA library of L. leucocephala, made from young shoots from approximately 8-week-old seedlings, through interspecies suppression subtractive hybridization (iSSH) using cDNAs from L. leucocephala and a related tree legume, Acacia confusa, as the tester and driver, respectively (Negi et al, 2011). These cDNA clones represent either L. leucocephala-specific genes that are missing in A. confusa or genes that are highly expressed in L. leucocephala.…”

Section: Resultsmentioning

confidence: 99%

“…For the identification of a Leucaena leucocephala cDNA that encodes a mimosine-degrading enzyme, a set of 406 cDNA sequences obtained from the iSSH library of L. leucocephala (Negi et al, 2011) were screened for their homology with C-N lyase using the BLASTX tool and a reference protein database of the NCBI. The cDNA fragment of the iSSH clone that exhibited similarity with C-N lyase was amplified to full-length cDNA by using the FirstChoice RLM-RACE kit (Ambion) according to the manufacturer's instructions.…”

Section: Identification and Isolation Of A Cdna That Encodes A Mimosimentioning

confidence: 99%

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A Carbon-Nitrogen Lyase from Leucaena leucocephala Catalyzes the First Step of Mimosine Degradation

Negi

Bingham

et al. 2013

Plant Physiology

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The tree legume Leucaena leucocephala contains a large amount of a toxic nonprotein aromatic amino acid, mimosine, and also an enzyme, mimosinase, for mimosine degradation. In this study, we isolated a 1,520-bp complementary DNA (cDNA) for mimosinase from L. leucocephala and characterized the encoded enzyme for mimosine-degrading activity. The deduced amino acid sequence of the coding region of the cDNA was predicted to have a chloroplast transit peptide. The nucleotide sequence, excluding the sequence for the chloroplast transit peptide, was codon optimized and expressed in Escherichia coli. The purified recombinant enzyme was used in mimosine degradation assays, and the chromatogram of the major product was found to be identical to that of 3-hydroxy-4-pyridone (3H4P), which was further verified by electrospray ionization-tandem mass spectrometry. The enzyme activity requires pyridoxal 59-phosphate but not a-keto acid; therefore, the enzyme is not an aminotransferase. In addition to 3H4P, we also identified pyruvate and ammonia as other degradation products. The dependence of the enzyme on pyridoxal 59-phosphate and the production of 3H4P with the release of ammonia indicate that it is a carbon-nitrogen lyase. It was found to be highly efficient and specific in catalyzing mimosine degradation, with apparent K m and V max values of 1.16 3 10 24 M and 5.05 3 10 25 mol s 21 mg 21 , respectively. The presence of other aromatic amino acids, including L-tyrosine, L-phenylalanine, and L-tryptophan, in the reaction did not show any competitive inhibition. The isolation of the mimosinase cDNA and the biochemical characterization of the recombinant enzyme will be useful in developing transgenic L. leucocephala with reduced mimosine content in the future.

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Section: Resultsmentioning

confidence: 99%

Section: Identification and Isolation Of A Cdna That Encodes A Mimosimentioning

confidence: 99%

A Carbon-Nitrogen Lyase from Leucaena leucocephala Catalyzes the First Step of Mimosine Degradation

Negi

Bingham

et al. 2013

Plant Physiology

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“…were tested on first strand cDNA samples from the root and shoot with the primer sequences described in Negi et al [17]. The qRT-PCR protocol was performed as described above.…”

Section: Selection Of Internal Reference Gene For Qrt-pcr Analysismentioning

confidence: 99%

Transcriptome analysis of Leucaena leucocephala and identification of highly expressed genes in roots and shoots

Ishihara¹,

Honda²,

Pham

et al. 2016

Transcriptomics

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Leucaena leucocephala (leucaena) is a fast-growing tree legume highly tolerant to various abiotic and biotic stresses. Because of its abilities to withstand high temperature and prolonged drought and to grow as a disease-free plant, it is an interesting model plant to investigate genetics of stress resistance. The high-level stress resistance may be correlated with higher expression of certain genes in the root, which is the primary site for nutrient and water uptake and also infection by soil-borne pathogens. The objectives of this study were to characterize the transcriptome of leucaena and to identify root-specific genes that may be involved in drought tolerance and disease resistance. Transcriptomes of leucaena were analyzed through Illumina-based sequencing and de novo assembly, which generated 62,299 and 61,591 unigenes (≥500 bp) from the root and shoot, respectively. Through a 4 x 180k microarray analysis, the expression of 10,435 unigenes were compared between the root and shoot. Upregulated sequences in the root were mostly represented by unigenes that were related to secondary metabolism, while in the shoot, upregulated sequences were mostly represented by unigenes that were involved in carbohydrate and lipid metabolism. The unigenes sharing homology with terpenoid biosynthesis genes and a nicotianamine synthase gene were upregulated more than 100-fold in the root, which indicates that these genes may have important roles in high stress tolerance of leucaena. Cataloging of actively transcribed sequences in the root and shoot will lead to identification of genes for drought tolerance and disease resistance in leucaena.

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“…The outstanding ecological suitability of L. leucocephala indicated strong resistance against the environmental stresses, including drought, herbivores, and wounding. Six stress-related genes and 22 hypothetical proteins in L. leucocephala were identified and expressed at high levels under drought conditions [ 1 ].…”

Section: Introductionmentioning

confidence: 99%

“…Salicylic acid (simulating pathogen attack) applied in culture media of L. leucocephala induced mimosine accumulation in roots [ 14 ]. The L. leucocephala plant was well-known for its high level of tolerance to various stress conditions [ 1 ]. However, the study using jasmonate-elicited stress in L. leucocephala was very limited.…”

Section: Introductionmentioning

confidence: 99%

Jasmonate-Elicited Stress Induces Metabolic Change in the Leaves of Leucaena leucocephala

et al. 2018

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The plant Leucaena leucocephala was exposed to four jasmonate elicitors, i.e., jasmonic acid (JA), methyl jasmonic acid (MeJA), jasmonoyl-l-isoleucine (JA-Ile) and 6-ethyl indanoyl glycine conjugate (2-[(6-ethyl-1-oxo-indane-4-carbonyl)-amino]-acetic acid methyl ester) (CGM). The treatment was to mimic the herbivores and wounding stresses. By using NMR spectroscopy along with chemometric analysis, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), the changes of metabolites in the leaves of L. leucocephala were determined under the stress as induced by the four elicitors. The challenge of JA-Ile caused an accumulation of lactic acid (6), β-glucose (10), alanine (12), threonine (13), steroids (18), 3,4-dihydroxypyridine (19) and an unidentified compound 20. The chemometric analysis of the PCA and PLS-DA models indicated that the alternation of metabolites triggered by JA, MeJA, and CGM treatments were very minimum. In contrast, the treatment by JA-Ile could induce the most significant metabolic changes in the leaves. Moreover, there was very minimal new metabolite being detected in responding to the jasmonate-induced stresses. The results showed some metabolite concentrations changed after application of the elicitors, which may be related to a high level of tolerance to stress conditions as well as the strong ecological suitability of L. leucocephala.

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Identification of species-specific genes from Leucaena leucocephala using interspecies suppression subtractive hybridisation

Cited by 18 publications

References 41 publications

A Carbon-Nitrogen Lyase from Leucaena leucocephala Catalyzes the First Step of Mimosine Degradation

A Carbon-Nitrogen Lyase from Leucaena leucocephala Catalyzes the First Step of Mimosine Degradation

Transcriptome analysis of Leucaena leucocephala and identification of highly expressed genes in roots and shoots

Jasmonate-Elicited Stress Induces Metabolic Change in the Leaves of Leucaena leucocephala

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