Computational protocol: Expression pattern of four storage xyloglucan mobilization-related genes during seedling development of the rain forest tree Hymenaea courbaril L.

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Protocol publication

[…] RNA extraction was performed with different protocols according to the part of the seedling being analysed, i.e. cotyledon, hypocotyl, leaves, or root. For roots and leaves, the method used was the same as that described by , and for hypocotyls a Concert Total RNA extraction solution (Invitrogen) was used following the manufacturer's instructions. For cotyledons, a procedure described by was used with the following modifications. A 1 g aliquot of cotyledons was ground into a fine powder in liquid nitrogen and then 10 ml of buffer solution (0.5 M TRIS-HCl pH 8, 0.01 M EDTA, 0.25 M LiCl, 2.5% SDS, and 0.1% β-mercaptoethanol) was added. The mixture was vigorously stirred for 1 min and phenol:chloroform:isoamylic alcohol (25:24:1, v/v/v) was added and stirred again for 1 min. The material was centrifuged at 4 °C, 13 000 g, for 15 min. This step was repeated twice. The RNA was then precipitated twice. The first precipitation was performed by adding 0.5 ml of 12 M LiCl and kept for 2 h at –80 °C. After 30 min of centrifugation the pellet was washed with 6 M LiCl. After a further 15 min of centrifugation under the same conditions, the pellet was resuspended in 0.3 ml of diethylpyrocarbonate (DEPC)-treated water and a second precipitation was performed using 1/10 volume of 3 M sodium acetate, pH 5.2 and a 0.5 vol. of absolute ethanol. The mixture was stirred and kept overnight at –80 °C. After 30 min of centrifugation, the pellet was washed with 70% ethanol, resuspended in DEPC-treated water, and stored at –80 °C. RNA integrity was checked by 1% agarose gel electrophoresis with 6% formaldehyde in MOPS buffer (20 M MOPS, 0.6 M sodium acetate, 0.01 M EDTA, pH 8). The RNAs were stored at –80 °C until used for cDNA synthesis.The reverse transcription of total RNAs to cDNA was performed using Ready-To-Go RT-PCR beads (Amersham Biosciences). The reaction was carried out following the manufacturer's instructions, using 3.5 μg of total RNA samples and 1 mM primer Oligo dT12-18 (Invitrogen). The cDNA was stored at –20 °C until use.Coding sequences for actin, xyloglucan endo-transglycosylase, β-galactosidase, alkaline invertase, and sucrose synthase from eudicotyledons were obtained from GenBank (NCBI: http://www.ncbi.nlm.nih.gov/Genbank/index.html). Amino acid sequence alignments were performed with Clustal W (http://www.ebi.ac.uk/clustalw) using default parameters. Gaps were removed for phylogenetic analyses which were performed using the Prodist program to calculate distances among sequences, and the Neighbor–Joining tree constructing algorithm () was used to establish the evolutionary relationships among the homologous sequences.To amplify partial cDNAs, pairs of nested and degenerated primers were designed from conserved protein domains detected in the sequences from organisms phylogenetically closely related to Hymenaea, i.e. the Leguminosae. The primers synthesized had 17–24 nucleotides (Table S2).For PCR amplification of cDNAs, the following solution in a total volume of 50 μl was used: 10 μl of cDNA reaction, 5 μl of 10× PCR buffer, 1.5 μM MgCl2 (50 mM), 2.5 μl of dNTP mix (10 μM), 1 mM for the degenerated 3′ primer and 1 mM for the degenerated 5′ primer, and 0.3 μl of Taq DNA polymerase (5 U μl−1;– Invitrogen). The PCR conditions for β-galactosidase, xyloglucan transglycosylase hydrolase, alkaline invertase, and sucrose synthase were: initial denaturation for 2 min at 95 °C followed by 5 min at 72 °C and 36 cycles (94 °C for 5 s, 55 °C for 1 h 30 min, and 72 °C for 45 s), and a final extension of 10 min at 72 °C. Actin was used as an internal control in semi-quantitative RT-PCR experiments.The amplification products of the expected size were purified after separation by 2% agarose gel electrophoresis using the kit Wizard SV Gel and PCR Clean-Up System (Promega) according to the manufacturer's instructions.PCR products were cloned into the TA-Cloning vector (Invitrogen) following the manufacturer's instructions. Sequencing was performed using a DNA sequencing Big Dye Kit™ (Applied Biosystems) following the manufacturer's instructions, and analysed with an automatic sequencer ABI PRISM™ 377 (Perkin Elmer).The sequences that were obtained were then compared with GenBank sequences using the blastn tool (NCBI: http://www.ncbi.nlm.nih.gov/GenBank/index.html) to confirm their identities. [...] Arabidopsis and rice xyloglucan transglycosylase hydrolase, β-galactosidase, alkaline invertase, and sucrose synthase were identified by using the blast program and the query sequences at TAIR (The Arabidopsis Information Resource) in order to identify genes that encode in the Arabidopsis genome. Orthologues in rice (Oryza sativa) were also searched for using tbastn. Alignment was performed using Clustal W.Phylogenetic reconstruction was performed from distances calculated from the aligned amino acid sequences using the JTT matrix (), and the tree was inferred by the Neighbor–Joining method (). The numbers of amino acid positions used were 145, 111, 273, and 382 for β-galactosidase, xyloglucan transglycosylase hydrolase, alkaline invertase, and sucrose synthase, respectively. The analyses were carried out using the software MEGA4 ().In order to classify the cDNAs obtained from cotyledons of H. courbaril β-galactosidase (HcBGAL1; EU370969), xyloglucan transglycosylase hydrolases (HcXTH1; EU370971), alkaline invertase (HcAlkIN1; EU370968), and sucrose synthase (HcSUS1; EU370970), trees were generated using the representatives of the Leguminosae (see Supplementary Table S1 available at JXB online for accession numbers). […]

Pipeline specifications

Software tools Clustal W, BLASTN, MEGA
Application Phylogenetics
Diseases Gangliosidosis, GM1
Chemicals Sucrose