Computational protocol: Functional characterization of a class III acid endochitinase from the traps of the carnivorous pitcher plant genus, Nepenthes

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[…] Total RNA from N. rafflesiana pitchers was isolated using the Concert™ Plant RNA Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. RNA was purified to eliminate genomic DNA using the Qiagen RNeasy Plant RNA kit (Qiagen, Hilden, Germany) and DNA was digested by TURBO™ DNase (Applied Biosystems/Ambion, Darmstadt, Germany). First-strand cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen, Darmstadt, Germany), oligo(dT)20 primer, and 1 μg of total RNA at 50 °C for 55 min.Degenerate primers, designed according to conserved protein sequences of known plant endochitinases (NCBI GenBank), were used to amplify a fragmental cDNA sequence. Cloning the 5′ and 3′ end of NrChit1 cDNA was accomplished by rapid amplification of cDNA ends (RACE) PCR using total RNA and the FirstChoice® RLM-RACE Kit (Applied Biosystems/Ambion) following the manufacturer's protocol. Primers were designed by using DNASTAR Lasergene® Software (GATC BIOTECH, Konstanz, Germany). The resulting amplified products were cloned into a pCR®-TOPO®-vector, and the resulting plasmid was subjected to nucleotide sequencing (Eurofins MWG Operon, Ebersberg, Germany). The complete NrChit1 cDNA sequence was amplified by PCR using Pfu DNA polymerase (Fermentas, St. Leon-Rot, Germany), and the primers: forward 5′-ATG AAG ACC CAT TAT TCA TCA GCA ATT C-3′ and reverse 5′-TTA AAC ACT ATC CTT GAT AGC TGA G-3′ (PCR: 3 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 60 °C, 60 s at 72 °C; and 10 min at 72 °C).For functional identification, cDNA was amplified with primers for an open reading frame (ORF) lacking the signal peptide. The cDNA was subcloned into the pHIS8-3 expression vector (). The recombinant vector was transformed into E. coli BL21(DE3) already transformed with the chaperone-coding plasmid pG-Tf2 (Takara Bio Europe S.A.S., Saint-Germain-en-Laye, France). The bacterial strain was grown to A600=0.6 at 37 °C in LB medium with kanamycin at 50 μg ml−1 and chloramphenicol at 20 μg ml−1. Cultures were induced with 1 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) for NrChit1 and with 10 ng ml−1 tetracycline for chaperone co-expression. Cultures were kept overnight at 16 °C while being shaken at 200 rpm. After expression, the protein was purified following the instructions of QIAexpressionist™ with a modification of the elution buffer (10 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8).The respective endochitinase sequences from genomic DNA of seven additional Nepenthes species were also cloned. Therefore, genomic DNA was isolated from pitchers using the CTAB (cetyltrimethylammonium bromide) method (). Amplification and cloning were done as described above. [...] For real-time PCR analyses, total RNA was isolated from either whole pitchers, glands, or the epidermal tissue of the digestive zone of closed and open pitchers from N. mirabilis or N. alata. When pitchers were fed with Drosophila melanogaster (30 individuals each; three replicates), they were packed in a full-fashioned stocking to avoid contamination and harvested after 7 d. Fifty glands or surrounding epidermal tissue were isolated using the aureka platform (aura optik, Jena, Germany; ). NrChit1 expression levels were analysed and compared as follows: for total RNA isolation, the RNAqueous®-Micro kit (Applied Biosystems/Ambion) was used following the manufacturer's protocol. DNA digestion and the reverse transcription reaction were done as described above. Actin was chosen as the internal control gene; the sequence was isolated using the primers published by , actinF 5′-ACC GAA GCC CCT CTT AAC CC-3′ and actinR 5′-GTA TGG CTG ACA CCA TCA CC-3′, coding for a 180 bp fragment with 93% identity to an actin gene from Quercus robur (GQ339769) (PCR: 3 min at 94 °C; 35 cycles of 30 s at 94 °C, 30 s at 57 °C, 60 s at 72 °C; and 10 min at 72 °C).Primers for real-time PCR were designed in order to obtain resulting PCR products of ∼100 bp using the Primer3Plus software (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi): for actin, 5′-CTC TTA ACC CCA AAG CAA ACA GG-3′ and 5′-GTG AGA GAA CAG CCT GGA TG-3′; and for endochitinase, 5′-AAG GGA TCA AGG TCC TCC TAT C-3′ and 5′- GAG GTA GTT ATT CCA AAG GTA AGC-3′.Real-time PCR was done on a Mx3000P Real-Time PCR System (Stratagene, La Jolla, CA, USA). The process was performed with 25 μl of reaction mixture containing 12.5 μl of 2× Brilliant II SYBR® Green QPCR Master Mix (Stratagene), cDNA (20–75 ng), 400 nM of each primer, and 30 nM ROX as a passive reference dye. The following protocol was used: initial polymerase activation for 10 min at 95 °C, 40 cycles of 30 s at 95 °C, 60 s at 61 °C, and 60 s at 72 °C. Actin levels were equal in every reaction independently of tissue type. PCR conditions were determined by a non-reverse transcriptase template control and a non-template control for each primer pair. Relative RNA levels were calibrated and normalized with the level of actin mRNA by determining the efficiency of every single reaction using the method of . Calculation of expression ratios and statistical analyses were performed with the Relative Expression Software Tool (REST© 2009, http://www.gene-quantification.de/rest.html; ). Data are from triplicates. [...] To infer phylogenetic relationships, a maximum parsimony analysis of the endochitinase sequence data set that had been obtained from genomic DNA as described above was carried out in PAUP* 4.0b10 () using the default exhaustive search settings. The 50% bootstrap majority rule consensus trees (Felsenstein, 1981) were calculated from 10 000 replicates under the default heuristic search settings with no maxtrees limit, and character states specified as unordered and equally weighted.Additionally, a Bayesian analysis was conducted using the Markov-Chain-Monte-Carlo algorithm of MrBayes 3.1.2 () under the assumption of the HKY model () and invariable sites (HKY+I), which was determined to be the best-fit model of sequence evolution by the likelihood ratio tests implemented in MrModeltest 2.2 (). Four Markov chains were calculated simultaneously, according to MrBayes’ default setting. Analysis was terminated after 1 000 000 generations; trees were summarized in a 50% majority rule consensus tree after discarding burn-in trees yielded before reaching likelihood stationarity. […]

Pipeline specifications

Software tools DNASTAR Lasergene, Primer3, REST
Applications Sanger sequencing, qPCR