Construction of antimicrobial peptide VPS vector
Penaeidins 3a (Pen3a) cDNA was obtained from RNA of Litopenaeus vannensis by reverse transcription as a template. The product was connected to CaMV-35S promoter and NOS terminator of Tobacco Mosaic virus by PCR and enzyme digestion. After double enzyme (BglⅡand Pst Ⅰ) digestion, the plant expression vector p1301 -Pen 3a was constructed (Fig. 11).
Duckweed culture condition
The experimental material Lemna turionifera 5511 was collected from Xiqing District, Tianjin. The culture method refers to the liquid medium described by Wang and Kandeler. Briefly, the liquid culture medium consisted of 0.4 mM MgSO4·7H2O, 1.4 mM Ca(NO3)2·4H2O, 1.0 mM KNO3, 0.4 mM KH2PO4, 0.4 mM Mg(NO3)2·6H2O, 50 µM CaCl2·2H2O, 50 µM KCl, 6.1 µM Na2MoO4·2H2O, 69 µM H2BO3, 30 µM K2H2EDTA·2H2O, 56.7 µM FeNH4-EDTA, 13.8 µM MnCl2·4H2O, 2.8 µM ZnNa2EDTA·4H2O, 4.8 µM CoSO4·7H2O, 18.6µM Na2-EDTA·2H2O and the pH was adjusted to 5.8. Duckweed was cultured at 23 ± 2℃ with a light intensity of 95 µmol m−2 s−1 and the photoperiod was 16 h of light and 8 h of darkness.
Plant transformation and GUS activity detection
The methods of callus induction and regeneration of duckweed were referred to Yang et al. (2012). Briefly, the p1301-pen 3a vector has been transformed to Agrobacterium tumefaciens EHA105. The duckweed callus was co-cultured with Agrobacterium (OD600 = 0.6) and vacuumed for 30 s three times, then cultured with slow shaking at 28°C for 30 minutes under dark conditions. The callus was transferred to B5 medium at 28°C and cultivated under dark conditions overnight. The callus was cultivated on a selection medium containing 1 mg L−1 Hygromycin and 160 mg L−1 Cefalexin for 3 days. Then transfer to regeneration medium containing 1 mg L−1 Hygromycin, 160 mg L−1 Cefalexin, and 0.1 M Serine Cultivate for 30 days until the callus regenerates. Histochemicalstable b-glucuronidase (GUS) stain has been performed to determine the transformation of duckweed by Biosharp GUS Staining Kit (Biosharp; according to instruction).
RNA isolation and quantification
RNA samples from duckweed callus were extracted by the Tiangen kit (Tiangen RNAsimple total RNA kit). mRNA was purified from total RNA using poly-T oligo-attached magnetic beads and detected on 1% agarose gel to ensure the sample quality. The purity of RNA was measured by the Nanophotometer (IMPLEN, CA, USA). RNA concentration was analyzed using Qubit® RNA Assay Kit in Qubit® 2.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 assay kit of the Agilent BioAnalyzer 2100 system (Agilent Technologies, CA, USA). A total amount of 1.5 µg RNA was used for library preparation for transcriptome sequencing. Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina®® (NEB, USA).
Sequencing data filtering and transcript assembly
Data images of sequencing fragments measured by high-throughput sequencers are transformed into sequence data (readings) by CASAVA base recognition. The raw data obtained from sequencing included a small number of reads with sequencing adaptors or low sequencing quality. Filtered content: removed adapters; removed reads whose proportion of N is greater than 10%; remove low-quality reads. The clean reads were assembled by the Trinity de novo assembly program with min_kmer_cov set to 2 by default, otherwise, it was set to default. Overall, a reference sequence with an average length of 1928 bp and a total length of 282527137 bp was obtained for subsequent analysis. The combination comparison of differential clustering gene analysis can obtain the differential gene sets, and the FPKM values of the union of all comparative combinations of differential gene sets in the six samples are used for hierarchical clustering analysis.
DNA extraction and polymerase chain reaction(PCR)
Total DNA was isolated from duckweed using TAKARA MiniBEST Plant Genomic DNA Extraction Kit (TAKARA; according to the manufacture’s instruction). DNA concentration was quantified by NanoDrop spectrophotometer. Experimental setup and execution were conducted using a Veriti® 96-Well Thermal cycler, according to the protocol provided by the manufacturer (ABI, USA). PCR products were analyzed by agar gel electrophoresis. The forward primer and reverse primers are TACGCGGAGCACCAGACGGA and TCAACCGGAATATCCCTTT.
Bacteriomic and bioinformatics analysis
Firstly, raw data FASTQ files were imported into the format which could be operated by the QIIME2 system using qiime tools import program. Then, the QIIME2 DADA2 plug-in was used for quality control, pruning, denoising, splicing, and removal of chimera to obtain the final feature sequence table. The QIIME2 feature-classifier plugin was then used to align ASV sequences to a pre-trained GREEN GENES 13_8 99% database (trimmed to the V3V4 region bound by the 338F/806R primer pair) to generate the taxonomy table. Any contaminating mitochondrial and chloroplast sequences were filtered using the QIIME2 feature-table plugin. Secondly, ANCOM, ANOVA, Kruskal Wallis, LEfSe, and DEseq2 were used to identify bacteria that differed in abundance between groups and samples[24–26]. Thirdly, diversity metrics were calculated using the core-diversity plugin within QIIME2. Feature level alpha diversity indices, such as observed OTUs, Chao1 richness estimator, Shannon diversity index, and Faith’s phylogenetic diversity index were calculated to estimate the microbial diversity within an individual sample. Beta diversity index was used to assess the differences in microbial community structure among samples and was subsequently demonstrated by PCoA and NMDS maps. Finally, redundancy analysis (RDA) was performed to reveal the association of microbial communities to environmental factors based on relative abundances of microbial species at different taxa levels using the R package “vegan”.
Antibacterial test of transgenic duckweed extract
Pen 3a duckweed cultured for 14 days was frozen in liquid nitrogen and thoroughly ground. And then, it has been diluted by 2 g to 5 ml phosphate buffer saline (PBS, PH 7.2-7.4, 10 mM). The inoculation rings were used to pick the strains on the inclined surface of the test tube in sterile water with glass beads, and the spores were dispersed by shaking with the hand for several minutes. The mixed spore suspension was prepared after filtration. Mix the bacteria liquid with 15-20 ml melted agar medium, then self-cooling it. The filter paper was soaked in antimicrobial peptides and placed in the center of the plate with bacteria. After culture for 2-3 days, the presence and size of the bacteriostatic circle around the filter paper were observed.
Label-free Quantification Proteomics
Protein was extracted from tissue samples using SDT lysis buffer (4% SDS, 100 mM DTT, 100 mM Tris-HCl pH 8.0), which were boiled for 5 min and further ultrasonicated and boiled again for another 5 min. Undissolved cellular debris was removed by centrifugation at 16000g for 15 min. The supernatant was collected and quantified with a BCA Protein Assay Kit (Bio-Rad, USA). Protein digestion (200 µg for each sample) was performed with the FASP method described. Then LC-MS/MS analysis was performed. LC-MS/MS was performed on a Q Exactive Plus mass spectrometer coupled with Easy 1200 nLC (Thermo Fisher Scientific). Sequence database searching and data analysis: The MS data were analyzed using MaxQuant software version 184.108.40.206. MS data were searched against the UniProtKB Rattus norvegicus database (36080 total entries, downloaded 08/14/2018). The quantitative protein ratios were weighted and normalized by the median ratio in Maxquant software. Only proteins with fold change ≥1.5-fold and a p-value <0.05 were considered for significantly differential expressions. Bioinformatics analysis of bioinformatics data was carried out with Perseus software, Microsoft Excel, and R statistical computing software.