Plant Materials and Growth Conditions
The S12 (anther indehiscent) and F142 (normal plant) S. melongena L. lines were provided and grown at the Institute of Vegetables and Flowers, Chongqing Academy of Agricultural Sciences (Chongqing, China) from 2017 to 2019. The eggplant seeds were sterilized and sown in trays. Then, the seedlings were transferred and grown under normal conditions. Selected anthers were immediately frozen in liquid nitrogen and stored at -80°C until they were used for further analysis.
RNA extraction, library construction, and RNA-Seq
Total RNA of each sample was extracted from the anther of eggplant according to the instruction manual of the TRlzol Reagent (Life technologies, California, USA). RNA integrity and concentration were examined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). The mRNA was isolated by NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB, E7490). The cDNA library was constructed following the manufacturer’s instructions of NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, E7530) and NEBNext Multiplex Oligos for Illumina (NEB, E7500). In brief, the enriched mRNA was fragmented into approximately 200nt RNA inserts, which were used to synthesize the first-strand cDNA and the second cDNA. The double-stranded cDNA were performed end-repair/dA-tail and adaptor ligation. The suitable fragments were isolated by Agencourt AMPure XP beads (Beckman Coulter, Inc.), and enriched by PCR amplification. Finally, the constructed cDNA libraries of the eggplant were sequenced on a flow cell using an Illumina HiSeq™ sequencing platform.
Transcriptome analysis using reference genome-based reads mapping
Low quality reads, such as only adaptor, unknown nucleotides>5%, or Q20 <20% (percentage of sequences with sequencing error rates <1%), were removed by perl script. The clean reads that were filtered from the raw reads were mapped to eggplant genome (SME_r2.5.1) using Tophat2 [50] software. The aligned records from the aligners in BAM/SAM format were further examined to remove potential duplicate molecules. Gene expression levels were estimated using FPKM values (fragments per kilobase of exon per million fragments mapped) by the Cufflinks software [51].
Identification of differential gene expression
DESeq and Q-value were employed and used to evaluate differential gene expression between F142 and S12. After that, gene abundance differences between those samples were calculated based on the ratio of the 2 FPKM values. The false discovery rate (FDR) control method was used to identify the threshold of the P-value in multiple tests in order to compute the significance of the differences. Here, only gene with an absolute value of log2 ratio ≥2 and FDR significance score <0.01 were used for subsequent analysis.
Sequence Annotation
Genes were compared against various protein database by BLASTX, including the National Center for Biotechnology Information (NCBI) non-redundant protein (Nr) database, Swiss-Prot database with a cut-off E-value of 10-5. Furthermore, genes were searched against the NCBI non-redundant nucleotide sequence (Nt) database using BLASTn by a cut-off E-value of 10-5. Genes were retrieved based on the best BLAST hit (highest score) along with their protein functional annotation.
To annotate the gene with gene ontology (GO) terms, the Nr BLAST results were imported into the Blast2 GO program. GO annotations for the genes were obtained by Blast2 GO. This analysis mapped all of the annotated genes to GO terms in the database and counted the number of genes associated with each term. Perl script was then used to plot GO functional classification for the unigenes with a GO term hit to view the distribution of gene functions. The obtained annotation was enriched and refined using TopGo (R package). The gene sequences were also aligned to the Clusters of Orthologous Group (COG) database to predict and classify functions [52]. KEGG (kyoto encyclopedia of genes and genomes) pathways were assigned to the assembled sequences by perl script.
Hormone extraction and determination
Endogenous hormone extraction and purification are based on the method of Yu mei et al. The high performance liquid chromatograph is Shimadzu LC-60A. Chromatographic conditions: mobile phase was methanol 0.8% glacial acetic acid solution = 55/45, column temperature was 30°C, flow rate was 0.8 mL/min, detection wavelength was 254 nm, injection volume was 10 μL. Each sample was tested 3 times and averaged. The data was statistically analyzed using Graphpad Prism 8 and SPSS 22.0.
Determination of pollen vigor
The pollen viability was determined by TTC method [53]. A small amount of pollen was placed on a clean glass slide, a drop of 0.5% TTC solution was added, stirred, covered with a cover glass, and stained in a 35°C incubator for 20 min. Observe with a microscope and calculate pollen vigor.
Quantitative real-time PCR
The qRT-PCR was performed as previously described [54]. The primers used to test the transcript levels of all genes are shown in Table S4, using GAPDH as the internal reference. The qRT-PCR mixtures contained 2 μL primers, 2 μL cDNA, 10 μL SsoFast™EvaGreen®Supermix (Bio-Rad) and distilled water to a final of 20 μL. The reaction conditions were as follows: 95 ℃ for 3 min (one cycle); 95 ℃ for 10 s, 59 ℃ for 30 s and 72 ℃ for 30 s (39 cycles).
Yeast one-hybrid assay
The SmLOX, SmAOC, SmOPR3, SmCOI1, SmJAZ1 genes were ligated into the pGADT7 vector to construct the recombinant plasmid of pGADT7-SmLOX, pGADT7-SmAOC, pGADT7-SmOPR3, pGADT7-SmCOI1, pGADT7-SmJAZ1. The promoter of SmDAD1 in eggplant was inserted individually into the pAbAi vector and formed plasmids of pAbAi-SmDAD1 which was transformed into Y1HGold for screening the optimal resistance concentrations of AbA. Then the pGADT7-SmLOX, pGADT7-SmAOC, pGADT7-SmOPR3, pGADT7-SmCOI1, pGADT7-SmJAZ1 were respectively transformed into Y1H (pAbAi-DAD1) for examing the interactions between pGADT7-SmLOX, pGADT7-SmAOC, pGADT7-SmOPR3, pGADT7-SmCOI1, pGADT7-SmJAZ1 protein and promoter of SmDAD1 on media lacking Leu (SD/-Leu) supplemented with the optimal AbA of 400 ng/mL to identify the motif or sequence region of DNA-protein interactions
Dual-Glo® Luciferase assay
The interactions of SmLOX, SmAOC, SmOPR3, SmCOI1, SmJAZ1 protein with promoter of SmDAD1 were verified using the Dual-Glo® Luciferase Assay system (Promega, Madison, USA). The SmLOX, SmAOC, SmOPR3, SmCOI1, SmJAZ1 were subcloned and then were recombined into pGreenII 62-SK. Meanwhile, the promoters of SmDAD1 was ligated into pGreenII 0800-LUC. All the above recombinants were transformed into agrobacterium (GV3101) then co-infiltrated the leaves of Nicotianabenthamian for measuring the activities of fluorescein enzyme via GLOMAX® multifunctional instrument (Promega, USA).
Yeast two-hybrid assay
SmDAD1, SmLOX, SmAOC, SmOPR3, SmCOI1, SmJAZ1 were separately sub-cloned into the activation domain of pGADT7 and pGBKT7 using the BamHI and XhoI sites and then ligated into pGADT7 or pGBKT7 to construct the recombinant plasmids. Using MatchmakerTM Gold Yeast Two-Hybrid System, the recombinants were respectively transformed into Y187 and Y2H (Clontech) for detecting the protein-protein interactions. In addition, toxicity and self-activation detection of the recombinant plasmids had been performed previously.
Pull-down assay
SmDAD1 was sub-cloned into the pET32a(+) vector, while SmLOX, SmAOC, SmOPR3, SmCOI1, SmJAZ1 were cloned into the pGEX-4T-1 vector. Then, the plasmids were transformed into E. coli Rosetta (DE3) competent cells and 1.0 mM isopropyl β-D-thiogalactoside was added before incubation at 37°C for 3.5 h. The SmDAD1-HIS protein was purified using BeaverBeads IDA-Nickel Kit-10 (Beaver). The SmLOX-GST, SmAOC-GST, SmOPR3-GST, SmCOI1-GST and SmJAZ1-GST proteins were purified by BeaverBeads GSH (Beaver). The protein-protein interactions were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).