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The supernatant was loaded onto the nickel nitrilotriacetic acid resin Cwbiotech, cat. The enzyme was eluted by mM imidazole in elution buffer 20 mM sodium phosphate buffer, pH 7.
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Subsequently, the biochemical properties of the purified AmCDase were tested. The substrate was further dissolved in various buffers at different pH values to determine the pH-dependency of AmCDase: mM acetate buffer, pH 3. The specific activity was obtained by standard curves with known concentrations of D-erythro-CNBD-ceramide and all the measurements for each sample were repeated three times independently. At first, a total of 31,, raw sequencing reads were produced, then 30,, The size of the sequencing data was approximately 4.
Trinity efficiently reconstructed the transcriptome for the de novo assembly [ 16 ]. Based on the high-quality reads, a total of 85, transcripts were assembled with an average length of The longest transcript was selected as a unigene in every subgraph.
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The reads of A. Transcript lengths ranged from bp to bp. Transcripts with lengths between to nt accounted for As is known to all, longer sequences were more conducive to bioinformatic analysis. Consequently, further analysis was mainly based on the longest sequences, which were also called all-unigenes. The results indicated that 21, all-unigenes exhibited gene annotation in at least one database S2 Table. A total of 10, all-unigenes can be annotated in all the databases, whereas 36, all-unigenes The non-annotated all-unigenes may be considered new genes.
KOG is eukaryote specific version of the Clusters of Orthologous Groups database, which was used to align the all-unigenes for predicting their potential functions. Per the Nr hits, a total of sequences were specified to 25 categories in the KOG database. The smallest categories included the cell motility, lipid transport and metabolism 3; 0. GO functional annotation can be derived from the Nr annotation information. The GO classification system includes three broad categories: molecular function, biological process, and cell components.
These categories were further divided into 52 small categories, and a total of 14, As seen from the result, the biological process category had the largest number of genes, including signalling processes , reproduction , and cellular component biogenesis Only a few all-unigenes were assigned to molecular transducer activity 3 , antioxidant activity 1 , proteasome regulator activity 1 and structural molecule activity 1 Fig 1. To give a better understanding of the biological pathways in A. These all-unigenes were divided into five categories: cellular processes, metabolism, organismal systems, genetic information and environmental information processing.
As shown in Fig 2 , the KEGG metabolic pathways involved 32 subcategories, such as carbohydrate metabolism, signal transduction, translation, lipid metabolism and amino acid metabolism. These results provided valuable information for investigations on the cell processes and enzyme functions of A. In recent years, because of the outbreak of zoonoses such as bovine spongiform encephalopathy BSE , foot-and-mouth disease FMD , and avian influenza, the security of animal origin ceramide was of universal concern [ 22 ].
According to the annotation and analysis results of the transcriptome, the possible metabolic pathway of sphingolipids in A. Then, we analysed the nucleotide sequence. The information of the nucleotide was submitted to GenBank accession no. The results showed that the sequence was bp in length, with an open reading frame bp encoding a protein of amino acids.
All available protein sequences of neutral ceramidases from different species were downloaded from GenBank database Till Phylogenetic analysis of neutral ceramidases from various species show the distance between the plant neutral ceramidase and animal neutral ceramidase. The result also showed that AmCDase was highly related to the neutral ceramidase of many other plant species, such as Anthurium amnicola , Arabidopsis thaliana , and Sesamum indicum.
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Nevertheless, low correlations could be found between AmCDase and the neutral ceramidase of animals such as humans, Fukomys damarensis and Microcebus murinus. Meanwhile, the cDNA sequence obtained by RACE was consistent with the related all-unigenes sequences, which showed the ability of de novo assembly in discovering transcripts for species without a reference genome. The MrBayes analyses were performed with a mixed amino acid model. Species names are Amorphophallus muelleri; Jatropha curcas; Prunus persica; Sesamum indicum; Arabidopsis thaliana; Anthurium amnicola; Oryza sativa Japonica; Hordeum vulgare; Triticum aestivum; Dicchanthelium oligosanthes; Human; Fukomys damarensis; Microcebus murinus; Castor Canadensis.
This is essential for the eukaryotic expression of recombinant AmCDase and could minimize the influence of endogenous yeast ceramidase activity, which is likely to interfere with the following experiments.
The results showed that the expression quantity of recombinant AmCDase increased gradually, and the amount of recombinant AmCDase peaked at approximately 21 h, and decreased thereafter. The different induction time yeast with the AmCDase-vector was used in the reactions marked group A, and the yeast with empty vector at the same timing points marked group B.
Samples of these two groups reacted with the same amount of substrate D-erythro-CNBD-ceramide and cell lysates. The enzymatic reaction was continued for the same time under identical conditions, so the difference in the remnant amount of D-erythro-CNBD-ceramide could illustrate the degree of reactivity. It showed that D-erythro-CNBD-ceramide concentration remained essentially unchanged from the initial concentration in group B after the reaction.
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The apparent K m and the V max were estimated to be Moreover, AmCDase showed the highest activity between pH 6. The purified recombinant AmCDase was used for enzyme activity detection, and the reaction continued for 1 h. The buffers were used as described in the experimental procedures. At present, a great variety of enzymes, which are related to the metabolism of ceramide in plants, have been cloned and characterized.
The increasing information makes people more familiar with plant sphingolipids. Numerous studies revealed that the neutral ceramidase widely existed in plants, and it is crucial to ceramide and the whole metabolic pathway of sphingolipids. However, only a few studies about the neutral ceramidase of plants were reported, such as oryza sativa , triticum aestivum , arabidopsis thaliana [ 9 , 26 , 27 ]. In contrast, most of the studies were focused on animals and yeast.
In our study, an A. Moreover, the observation indicated that a pH optimum of AmCDase was 6. There are several studies that have reported the neutral ceramidase of plants, but none of them determined its properties at the protein level by using purified protein. In wheat, the full-length gene of the neutral ceramidase Ta-CDase was cloned, and the analyses of the biochemical characterization at the DNA level using Southern blot showed that Ta-CDase was a multi-copy gene and located on wheat chromosome 4D and 5A [ 25 ].
Pata et al. The detailed analysis based on the purified enzyme was more accurate and persuasive. In our study, we improved the method of Pata et al. As early as nine years ago, the experts of this field proposed that deeper understanding of the roles that sphingolipids play in plant physiological function would come from a better understanding at the transcriptomic level [ 11 ].
This study was exactly based on the data of transcriptome analysis. Furthermore, with mounting transcriptome data from various species, it was expected that valuable roles of plant sphingolipids would emerge as increasing plant sphingolipidomes are characterized accurately and quickly. In this study, we found the possible sphingolipid metabolic pathway and 13 enzymes involved in it. Then, the key enzyme-neutral ceramidase AmCDase was cloned and its biochemical characterization was analysed. The results showed that the reaction catalysed by AmCDase presented classical Michaelis—Menten kinetics, and the influences of cations on the enzyme activity of the neutral ceramidase varied with the difference in species.
In addition, the transcriptome analyses can provide accurate and specific data for the gene clone, which revealed the powerful ability of high-throughput sequencing in identifying genes from non-model organisms. Our research demonstrated that de novo transcriptome assembly can be considerable for certain purposes, such as transcriptome data mining, gene cloning and so on.
In addition, sequencing data can provide basic information for further research about neutral ceramidases and a comprehensive sequence resource for A. The authors would like to thank Yang Han for technical help with the figures and valuable suggestions. We also thank the editors and anonymous reviewers for their comments. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.
Abstract Amorphophallus is a perennial herbaceous plant species mainly distributed in the tropics or subtropics of Asia and Africa. Introduction Amorphophallus is a member of the family Araceae , order of Alismatales , and mainly distributed in the tropics or subtropics of West Africa and South Asia [ 1 — 3 ].
Materials and methods Material preparation No specific permissions were required for the locations where we collected wild materials because A.
Download: PPT. Results and discussion Preliminary analysis of A.
knowledgebrief.vvinners.com/gady-conocer-personas-en.php Transcript functional annotation. Metabolic pathways analysis by KEGG. Fig 2. Pathway annotation and classification of contigs KEGG. Construction of the possible sphingolipid metabolic pathway.