MATERIALS AND METHODS
Sample collection, treatment and toxicity test. The samples
used in this study have been tested for the TTX toxicity in our previous
published research 22. Based on the toxicity test,N. succinctus and N. variciferus were selected for
transcriptional analysis since they included both toxic and non-toxic
specimens. The liver and pancreas were treated for RNA extraction and
sequencing since they contained more toxin. For RNA-seq protocol, the
toxic and non-toxic specimens were sequenced as treatment group and
control group respectively for each species. We selected three toxic
specimens and three non-toxic specimens as triple repeat for treatment
group and control group respectively, for each of N. succinctusand N. variciferus . The detailed sample collection was shown in
Fig. 1.
RNA library preparation, transcriptome and gene assembly. After
RNA extraction the mRNA were fragmented with fragmentation buffer and
were used to synthesize cDNA. Short cDNA fragments were purified and
then resolved with end reparation and adapter connection, from which
fragments with suitable length were selected for PCR enrichment. Agilent
2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System was used for
qualification. Finally, the library was sequenced using Illumina HiSeq
400023.
The Trinity (v2.0.6) 24 was used to perform de novo
assembly after filtering out raw reads. The assembly quality was
evaluated with N50 and ortholog hit ratios (OHR). Paired reads passing
the filter were then concatenated using Concatenate datasets (version
1.0.0) in both the right and left direction. Transcript abundancies were
calculated by RSEM version 1.1.175425-26 using the
pool of non-normalized reads with default settings.
Differentially expressed gene analysis. The functional
annotation was performed from databases of NT, NR, GO, COG, KEGG,
SwissProt and InterPro. With BLASTX the final assembly was submitted to
these databases with the threshold of E-value ≤
10−527. The NR and InterProScan5 were used to get GO
annotation and InterPro annotation respectively. Unigenes were obtained
from mapping clean reads by Bowtie228. The gene
expression level was calculated with RSEM. The PCA analysis was
performed with all samples by princomp, a function of R. We detected
DEGs (Differentially Expressed Genes) with Possion Dis as
requested29.
Q-PCR confirmation. Real-time PCR was performed with the SG
Fast qPCR Master Mix (High Rox) using the cDNA synthetized above. Cycle
threshold values were normalized to the gene 18S. A total of 18 DEGs
closely related with coding toxin-binding protein were selected as
target Q-PCR functional genes. Fold changes were determined with the
relative expression software tool.
Mutations in sodium channels genes. Based on the transcript
unigenes of all samples, we retrieved the s odium channels genes
to which the TTX binds. We downloaded available s odium channels
genes from mollusca, human, mouse and others in NCBI as reference
database. Then all unigenes of N. succinctus and N.
variciferus were mapped to the reference database. While the unigenes
were best matched to the reference sequences specific primers were
designed to sequence them for confirmation.