Experimental species and reagents
Kale-type oilseed rape Hua You No. 9, produced by GuchengShengguang Seed Industry Co. Hetero-chitooligosaccharides (HTCOS) were provided by the Institute of Process Engineering, Chinese Academy of Sciences.
Select full oilseed rape seeds and place them in a 9 cm Petri dish lined with filter paper, add 8 ml of sterile water and leave overnight at 4 ℃ to allow the seeds to absorb the water. The seeds are then placed in an artificial incubator at 26±2 ℃ for 24 h, selected for consistent germination potential, and planted in a 161-hole floating tray with seedling substrate and allowed to grow to 2 leaves and 1 heart. The seedlings are then transferred to a 10 cm diameter, 8.5 cm high seedling bowl with the seedling substrate, and 1 seedling of rape is planted in each pot. When growth reached 4 leaves and 1 heart, potted oilseed rape seedlings of uniform growth were selected and sprayed with 80 mg/L of HTCOS (recorded as DA treatment group), with an additional clear water treatment as a blank control. Each pot was sprayed with 10 mL, and 42 plants were sprayed at each concentration, divided into two groups, 12 of which were used for the two determinations, and the remaining 30 plants were used for phenotypic data statistics. Treated rape seedlings were placed in an artificial climate chamber at a temperature of 26±2 ℃, humidity RH 70±10%, and light L:D=14:10.
After 72 h of HTCOS spray once treatment. The third and fourth leaves of each plant, counted from bottom to top, were packed in 50 ml centrifuge tubes and frozen rapidly in liquid nitrogen, with one part sent to Shanghai Ouyi Biomedical Co Ltd for determination of transcriptome (3 biological replicates) and metabolome (6 biological replicates), and one part kept in an ultra-low temperature refrigerator at -80 ℃.
After 21 d of HTCOS spray once treatment, the growth of rape in pots seedling height, number of leaves, wet weight of above-ground parts and dry weight of above-ground parts were investigated uniformly in each treatment group and control group. Both treatment and control groups had 10 plants as one replication and were replicated three times, counting 30 plants each.
360 μL of cold methanol and 40 μL of 2-chloro-l-phenylalanine (0.3 mg/mL) dissolved in methanol as internal standard was added to each sample, samples were placed at -80 ℃ for 2 min. Then ground at 60 Hz for 2 min. The mixtures were ultrasonicated at ambient temperature for 30 min. 200 μL of chloroform was added to the samples, and the mixtures were vortexed, 400 μL water was added. Samples were vortexed again, then ultrasonicated at ambient temperature for 30 min. The samples were centrifuged at 12000 rpm for 10 min at 4 ℃ QC sample was prepared by mixing aliquots of all samples to be a pooled sample. And 80 μL of 15 mg/mL methoxylamine hydrochloride in pyridine was subsequently added. The resultant mixture was vortexed vigorously for 2 min and incubated at 37 ℃ for 90 min. 80 μL of BSTFA (with 1% TMCS) and 20 μL n-hexane were added into the mixture, which was vortexed vigorously for 2 min and then derivatized at 70 ℃ for 60 min. The samples were placed at ambient temperature for 30 min before GC-MS analysis.
The derivative samples were analyzed on an Agilent 7890B gas chromatography system coupled to an Agilent 5977A MSD system (Agilent Technologies Inc., CA, USA). A DB-5MS fused-silica capillary column (30 m ×0.25 mm ×0.25 μm, Agilent J & W Scientific, Folsom, CA, USA) was utilized to separate the derivatives. Helium (>99.999%) was used as the carrier gas at a constant flow rate of 1 mL/min through the column. The injector temperature was maintained at 260 ℃. The initial oven temperature was 60 ℃, ramped to 125 ℃ at a rate of 8 ℃/min, to 210 ℃ at a rate of 4 ℃/min, to 270 ℃ at a rate of 5 ℃/min, to 305 ℃ at a rate of 10 ℃/min, and finally held at 305 ℃ for 3 min. The temperature of MS quadrupole and ion source (electron impact) was set to 150, and 230 ℃, respectively. The collision energy was 70 eV. Mass data were acquired in a full-scan mode (50-500 m/z).
ChemStation (version E.02.02.1431, Agilent, USA) software was used to convert the raw data to CDF format, and then the CDF data were imported into the Chroma TOF software (version 4.34, LECO, St Joseph, MI) for data processing. Metabolites were annotated through Fiehn or NIST database. After alignment with the Statistic Compare component, the ‘raw data array’ (.cvs) was obtained from raw data with three-dimension data sets including sample information, peak names (or retention time and m/z), and peak intensities. In the ‘data array’, all internal standards and any known pseudo positive peaks (caused by background noise, column bleed, or BSTFA derivatization procedure) were removed. The data were normalized to the total peak area of each sample, and multiplied by 10000, and the peaks from the same metabolite were combined.
Data were transformed by log2 (use 0.000001 to replace 0 before transforming), and the resulting data matrix was then imported into the SIMCA software package (v14.0). Principle component analysis (PCA) and (orthogonal) partial least-squares-discriminant analysis (OPLS-DA) was performed to visualize the metabolic difference among experimental groups, after mean centring and unit variance scaling. The Hotelling’s T2 region, shown as an ellipse in score plots of the models, defines the 95% confidence interval of the modeled variation. Variable importance in the projection (VIP) ranks the overall contribution of each variable to the OPLS-DA model, and those variables with VIP >1 are considered relevant for group discrimination.
The differential metabolites were selected based on the combination of a statistically significant threshold of variable influence on projection (VIP) values obtained from the OPLS-DA model and p-values from a two-tailed Student’s t-test on the normalized peak areas from different groups, where metabolites with VIP values larger than 1.0 and p-values less than 0.05 were considered as differential metabolites.
RNA extraction and establishment of cDNA library
The total RNA of B. napus leaf under the HTCOS conditions was extracted using an RNA extraction kit and checking the quality of extracted RNA using Nanodrop 2000 spectrophotometer. The integrity of total RNA was checked using formamide denaturing gel electrophoresis, and mRNA isolated from total RNA using Dynabeads Oligo (dT) 25 isolation beads. The RNA of the extracted sample was used for cDNA synthesis using a reverse transcription kit based on the manufacturer’s instruction (NEBNext UltraTM RNA Library PrepKit for Illumina), and the establishment library of cDNA. The insert size of the cDNA library was checked by Agilent 2100 bioanalyzer. The cDNA library was sequenced on the Illumina sequencing platform using the paired-end (PE) technology within a single run, in which 150 bp PE reads were obtained.
Sequencing and differentially expressed genes (DEGs) analysis
The reference genome sequence of Brassica napus was downloaded from NCBI (GenBank: GCA_000686985.2). The raw transcriptome data of all samples in this trial were uploaded to the NCBI database (BioProject: PRJNA781006). The cDNA library of high quality was sequenced on the Illumina sequencing platform based on the second technology of sequencing. To obtain localization information of reads in reference genomic, compare clean reads with reference genomic using HISAT2-2.0.5, and the expression level was calculated using the FPKM method (fragments per kilobase million). The difference expressed genes (DEGs) were analyzed using the DEseq2 package version 3.8.6. The genes with｜log2 Fold Change｜³1, and false discovery rate (FDR) <0.05 were considered as differentially expressed genes (DEGs). The KEGG enrichment analysis of functional significance terms based on KEGG (http://www.kegg.jp/kegg/pathway/html) database was conducted using a hypergeometric test to find significant KEGG terms in DEGs for comparison with the genome background.
Validation of gene expression by qRT-PCR
To verify genes that were differentially expressed in RM-challenged samples compared with unchallenged ones, qRT-PCR was performed, using an iQ SYBR Green SuperMix kit (Bio-Rad) on an iCycleriQ system (Bio-Rad, Hercules, CA, USA). Gene-specific primers of 3 candidate genes (Table 3) were designed using the Primer Premier 5.0 software. The ramie gene encoding actin, which displays a stable expression under different stress condition, was used as an internal control for data normalization. For each sample, first-strand cDNA was synthesized from 1 𝜇g from the pooled RNA sample of the CK or HTCOS plants, using a Revert Aid First-Strand cDNA Synthesis Kit (ThermoScientific, Fermentas, Vilnius, Lithuania), according to the manufacturer’s instructions. All reactions were performed in triplicate with six replicates. Expression levels of each gene are presented as the fold change relative to that of the control gene, calculated with the method.