Five-year-old sandalwood trees that had been growing in South China Botanical Garden, Guangzhou, China, were used. The young and mature leaves, heartwood, sapwood, roots and shoots were collected and wrapped in tin foil paper, frozen immediately in liquid nitrogen, and stored at -80℃ for later use. Two-month-old young seedlings (6-8 leaves) of S. album were sprayed with 100 μM MeJA until the leaf surfaces were wet. 2% alcohol served as the control for each treatment. Samples were collected at 0 h, 2 h, 6 h, 12 h, 24 h, 48 h and 72 h after treatment and stored at -70°C for further analyses. Each treatment was repeated three times.
Cloning of the full-length putative cDNA of SaMK and SaPMK by RACE
The total RNA of sandalwood leaves was extracted using Column Plant RNAOUT (Tiandz, Beijing, China) according to the manufacturer’s instruction. The concentration and quality of RNA were measured using a NanoDrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, NC, USA) and agarose gel electrophoresis.
First-strand cDNA synthesis was obtained through the PrimeScript first-strand cDNA synthesis kit (Takara Bio Inc., Dalian, China). 5′ and 3′ RACE was performed according to the SMARTer RACE cDNA Amplification Kit (Clontech Laboratories Inc., CA, USA) manual. Primers were designed on the basis of the initial data of MK and PMK unigenes in the transcriptome (Zhang et al. 2015) (Table 1). The sequence information of 5′ and 3′ RACE PCR product clones were used to design primers from the start and stop codon to obtain the internal fragments. The amplified PCR products were purified by a gel DNA purification kit (Tiangen, Beijing, China) and ligated into pMD18-T vector (Takara Bio Inc.). The recombined plasmids were transformed into Escherichia coli DH5α competent cells (Takara Bio Inc.) and sequenced at the Beijing Genomics Institution (BGI, Shenzhen, China).
Bioinformatics analysis and molecular evolution analysis of SaMK and SaPMK
SaMK and SaPMK gene sequences were assembled and translated into amino acid sequences using DNAMAN software. The open-reading frame of the SaMK and SaPMK genes were predicted by ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/). Sequence comparison was performed with NCBI BLAST online tools (http://www.ncbi.nlm.nih.gov/BLAST/). The physicochemical properties of the deduced SaMK and SaPMK proteins were calculated by ExPASy (http://cn.expasy.org). Protein domains and active sites were predicted by the CDD database in NCBI (http://www.ncbi.mlm.nih.gov/Structure/cdd/wrpsb.cgi). Transmembrane domains and signal peptides were predicted by the TMHMM Server (http://www.cbs.-dtu.dk/services/TMHMM/) and SignalP (http://www.cbs.dtu.dk/services/SignalP/), respectively. Multiple sequence alignment was performed with CLUSTALX 2.0 (Conway Institute, UCD Dublin, Dublin, Ireland) and phylogenetic tree of SaMK and SaPMK proteins from S. album and other plants were constructed by MEGA 7 through the neighbor-joining (NJ) method with 1000 bootstrap replicates (Saitou and Nei 1987).
Subcellular localization of SaMK and SaPMK proteins.
A vector pSAT6-EYFP containing enhanced yellow fluorescent protein (EYFP) open reading frame was used in this study. The cDNA encoding SaMK and SaPMK were amplified with two pairs of primers YFP-MK-F and YFP-MK-R, YFP-PMK-F and YFP-PMK-R, respectively (Table 1). The PCR products and pSAT6-EYFP vector were digested with corresponding endonuclease restriction enzymes. The digested fragment was ligated into pSAT6-EYFP linearized vector to generate pSAT6-EYFP-SaMK and pSAT6-EYFP-SaPMK fusion constructs. The fusion expression vectors and the pSAT6-EYFP vector were transformed into arabidopsis mesophyll protoplasts through PEG-mediated transformation followed the method described previously (Yoo et al. 2007). Using a confoca laser-scanning microscope (Leica TCS SP8 STED 3X, Wetzlar, Germany) to observe YFP fluorescence in transformed protoplasts after overnight incubation at 22℃.
Functional complementation of SaMK and SaPMK in yeast
The two recombined plasmids, pYES2-SaMK and pYES2-SaPMK, were constructed by the In-Fusion HD Cloning Kit (Takara Bio Inc.) according to the manufacturer’s instructions. The pYES2 vectors (Invitrogen, Carlsbad, CA, USA), containing a yeast galactose-dependent promoter that can promote high levels of expression of target genes, were used as carriers for target genes in this study. The recombined plasmids (pYES2-SaMK and pYES2-SaPMK) were extracted and transformed into YMR208W (ΔERG12) and YMR220W (ΔERG8) (Dharmacon, Chicago, IL, USA), respectively with the Frozen-EZ Yeast Transformation II Kit (Zymo Research, Irvine, CA, USA). Transformants were spotted on SC (-Ura) medium (6.7% yeast nitrogen base without amino acids, 2% galactose) (Chen et al. 2017). Positive clones were further confirmed by PCR. Subsequently, transformed diploid cells were induced to sporulate and formed haploid cells containing pYES2-SaMK and pYES2-SaPMK. To further observe their growth conditions, the diploid Saccaromyces cerevisiae strain YSC1021 and transformed haploid strains YMR208W and YMR220W were grown separately on YPD (1% yeast extract, 2% bacto peptone, 2% glucose) and YPG (1% yeast extract, 2% bacto peptone, 2% galactose) media, respectively (Albers and Larsson 2009; Tao et al. 2016).
Tissue-specific analysis and expression profiles of SaMK and SaPMKinduced by MeJA
To investigate the expression levels of SaMK and SaPMK genes in different tissues (roots, sapwood, heartwood, young leaves, mature leaves and shoots) and their expression profiles after MeJA treatment, qRT-PCR was carried out according to the manufacturer’s instructions. About 1.0 μg of total RNA was reverse transcribed into first-strand cDNA using the PrimeScript RT Reagent Kit (Takara Bio Inc.) according to the manufacturer’s protocols. The reactions were performed on ABI7500 fluorescence quantitative PCR (Applied Biosystems, Thermo Fisher Scientific, MA, USA) using iTaq Universal SYBR Green supermix as the buffer (Applied Biosystems, USA). PCR amplification was performed under the following conditions: 95℃ for 30 s, followed by 35 cycles of 95 ℃ for 15 s and 60 ℃ for 60 s and melting curve analyses were performed. The housekeeping gene, β-actin, was selected as the internal control (Zhang et al. 2015) for normalization of all the reactions. All experiments were performed in triplicate and the mean value was analyzed. The 2-ΔΔCT method was used to analyze the relative expression level of genes (Schmittgen et al. 2008).