Computational protocol: Amylases StAmy23, StBAM1 and StBAM9 regulate cold-induced sweetening of potato tubers in distinct ways

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Protocol publication

[…] The amylase sequences from different species were obtained from public databases (Supplementary Table S1 at JXB online). Phylogenetic analysis of glucosyl hydrolase domains of β-amylase was performed using Phylogeny.fr (), and the phylogram was displayed with iTOL online software (). Exon–intron structures were analysed using the Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/). Phylogenetic analysis of α-amylase was performed by MUSCLE. Multiple alignments of the glucosyl hydrolase domains of β-amylase were performed using the ClustalX program with default settings and displayed by Jalview (http://www.jalview.org/). The crystal structure of the soybean β-amylase GmBMY1 (PDB ID 1BYB) was used for alignment analysis. [...] Based on the Potato Genome Sequencing Consortium (PGSC) database, StAmy23, StBAM1, and StBAM9 were cloned from CIS-sensitive cultivar E-potato 3 (E3) cDNA with specific primers shown in Supplementary Table S2. To analyse the locations of StAmy23, StBAM1, and StBAM9, the N-terminal regions of three proteins were analysed for the presence of possible chloroplast transit peptides or signal peptides using ChloroP (http://www.cbs.dtu.dk/services/ChloroP/), TargetP (http://www.cbs.dtu.dk/services/TargetP/) and iPSORT (http://ipsort.hgc.jp/index.html). The open reading frames (ORFs) of these genes without a termination codon were amplified with specific primers modified to contain the Gateway (Invitrogen) attB recombination sites. PCR products were purified and recombined into pDONR221 (Invitrogen) to generate entry clones via BP reactions. C-terminal green fluorescent protein (GFP) fusions of StAmy23–GFP, StBAM1–GFP and StBAM9–GFP were performed by recombining the entry clones with pK7FWG2 driven by the 35S silencing promoter using LR clonase (Invitrogen). For a starch granule marker, full-length granule-bound starch synthase I (StGBSS) without a stop codon was amplified from E3 cDNA with specific primers and cloned into pJCV55 obtained by restriction with BglII using Exnase II (Vazyme). Primer sequences are shown in Supplementary Table S2. Agrobacterium tumefaciens (GV3101) containing StAmy23–GFP, StBAM1–GFP, StBAM9–GFP and StGBSS–red fluorescent protein (RFP) were pressure infiltrated into the leaves of 4-week-old Nicotiana benthamiana plants. A. tumefaciens was resuspended in agroinfiltration medium at a final concentration of OD600=0.1. For coexpression, Agrobacterium cultures carrying the appropriate vectors were mixed before infiltration. Two days after infiltration, cells expressing fluorescent protein fusions were observed using a Carl Zeiss AXIO Observer A1 inverted fluorescence microscope. [...] For amylase extraction, 1 ml of 0.1 M citric acid buffer, pH 5.6, was added to 100 mg of powdered tuber tissue and extracted for 20 min on ice. The extract was centrifuged for 10 min at 4 °C and 2000 g, and the amylase activity in the supernatant was determined using assay kits from Megazyme (Bray, Ireland) as previously described (). Protein quantification in the extraction was performed with a bicinchoninic acid kit (PPLYGEN; P1511). Starch, glucose, fructose and sucrose contents were determined as described previously (). The soluble starch content was determined by measuring the amount of glucose released by treatment with amyloglucosidase (). The fry test was carried out, and crisp colour was determined according to with modifications. Briefly, each sampled tuber was peeled and cut in half longitudinally. One part was used for the fry test; the tubers were cut into about 1 mm slices and fried at 170 °C for 3 min or until the cessation of bubbles in a Frymaster (USA) H14 electric fryer. The crisp colour was visually determined by using the Color Standards Reference Chart for Potato Chips from scale 1 (light) to 10 (dark) (Snack Food Association, USA). The crisp colour index (CCI) was calculated for each line with 15 crisps from three tubers by applying the colour scale value (C) to the formula CCI = [∑(Ci×Ni)]/N, where Ci is colour scale i, Ni is number of crisps of Ci and N is the total number of the crisps tested. For the statistical analyses, biological triplicates were used for each measurement, and all the data are presented as means±SD. Significance was determined by Student’s t test and LSD test with the software SPSS Statistics v. 20.0 for Windows. […]

Pipeline specifications

Software tools Phylogeny.fr, iTOL, Clustal W, Jalview, ChloroP, TargetP, iPSORT, SPSS
Applications Miscellaneous, Phylogenetics, Protein sequence analysis
Organisms Solanum tuberosum
Chemicals Carbohydrates