Transcriptome sequencing
On average, 28.38 million paired-end reads were produced by sequencing the cDNA libraries constructed from the papillae and body samples. After adapter cleaning, quality trimming, and length filtering, a total of 28.00 million clean reads (98.67% of the total reads) were obtained. For the papillae cDNA library, an average of 62.75% of the quality-filtered reads were mapped to the reference genome of C. robusta and 2.85% of the reads were mapped to multiple regions of this genome. For the body cDNA library, the correspondence mapping ratios were 75.47% and 3.61%, respectively (Table S2). These multiple mapping reads were excluded in the subsequent analysis by HiSAT2. A total of 18,191 genes were annotated into the genome of the ascidian C. robusta (Satou et al. 2019), and an average of 17,633 (±173) genes were found to be expressed in six samples.
Identification of DEGs
After filtering the low-expressed genes, a total of 16,001 genes were obtained for subsequent DEGs analysis. A total of 5,051 genes were found to be differentially expressed in both papillae and body samples, with 1,875 genes were highly expressed in papillae and 3,176 genes were highly expressed in body (Fig. S1; Table S3). Analysis showed that the fold changes for the DEGs in RT-qPCR and transcriptome sequencing had good correlations, confirming the accuracy and validity of our transcriptome sequencing results (Fig. S2).
Functional enrichment of DEGs
A total of 1,875 genes highly expressed in papillae compared with body were arranged for GO and KEGG enrichments. A total of 174 terms were obtained from GO enrichment and 20 terms were significantly enriched (Fig. 2a; Table S4). In the biological process classification, the DEGs were annotated to the terms ‘excretion’, ‘calcium ion transport’, ‘detection of external stimulus’, ‘face morphogenesis’, ‘protein dephosphorylation’, ‘aminoglycan catabolic process’, and ‘multivesicular body organization’. In the cellular component classification, the DEGs were enriched into the terms ‘basement membrane’, ‘photoreceptor outer segment’, ‘external encapsulating structure’, ‘pseudopodium’, ‘non-motile cilium’, and ‘neuron projection membrane’. In addition, the terms ‘serine hydrolase activity’, ‘transmission system’, ‘iron ion’, and ‘proteoglycan and glycosaminoglycan binding’ were enriched into the classification of molecular function. Taken together, the enriched GO terms could be divided into four categories including neural recognition, environmental perception, metabolism, and ion transport.
The KEGG enrichment results could also be divided into four categories: neural recognition, environmental perception, metabolism, and ion transport (Fig. 2b, Table S5). The pathways ‘serotonergic synapse’ and ‘neuroactive ligand-receptor interaction’ were enriched in neural recognition category. The pathways ‘sensory system’ and ‘interaction of environmental information processing’ were associated with environmental perception. The metabolism category included ‘xenobiotics biodegradation’, ‘linoleic acid’, ‘purine’, and ‘nucleotide and arachidonic acid’ pathways. The ‘calcium signaling pathway’ was classified into the ion transport category. The KEGG pathways with higher values of rich factor were ‘non-homologous end-joining’, ‘linoleic acid metabolism’, and ‘ovarian steroidogenesis’ (Table S5).
Genes involved in papillary adhesion
A total of six genes, encoding candidate papilla adhesive proteins with molecular size ranging from 18.54 kDa to 116.29 kDa were identified (Table 1). The proteins encoded by these genes were typically characterized by a high proportion of serine, cysteine, glycine, and threonine, while most of these proteins had a proportion of hydrophobic amino acids such as leucine. Functionally conserved domains such as TSP-1, vWC, and EGF associated with the underwater adhesion of marine fouling organisms were distributed in the sequences of candidate papilla adhesive proteins. Some proteins were predicted to be of repetitive sequences, including TSP-1 and LDLa (cysteine-rich) repetitive sequences in ‘SCO-spondin’ (Gene id: KY.Chr1.200.v3.ND3-3), Fibronectin type 1 (FN1) repeat sequence in ‘Chordin-like protein 1’ (Gene id: KY.Chr10.1268.v1.ND1-1), and several internal repeats in ‘Protein delta homolog 2’ (Gene id: KY.Chr11.146.v1.SL1-1). In addition, some specific domains were also found in these candidate papilla adhesive proteins. The ‘Sushi, von Willebrand factor type A, EGF and pentraxin domain-containing protein 1’ (Gene id: KY.Chr10.1028.v1.nonSL5-1) possessed an antimicrobial peptide-like domain, a protease activity inhibitory structural domain Somatomedin-B (SO) and a Bowman-Birk domain (BowB). Similarly, there was a leucine-rich SCAN domain in ‘Fibronectin’ (Gene id: KY.Chr2.475.v2.SL2-1), a cysteine-rich domain Defensin (DEFSN) and an ADAM Cysteine-Rich domain (ACR) in ‘Chordin-like protein 1’.
In addition to the genes directly associated with underwater adhesion, two genes, ‘opsin 1, long wave sensitive’ (OPN1LW, KY.Chr1.1186.v2.ND1-2) and ‘G protein-coupled receptor 84’ (GPR84, KY.Chr7.1150.v1.ND1-1), we screened on those that might be involved in neural perception in papillae (Table 1). Both proteins encoded by these genes had high percentage of leucine and carried 7 transmembrane receptor (rhodopsin family, 7tm_1) domains.
Adhesive protein expression and purification
A total of two proteins (Gene ID: KY.Chr10.1028.v1.nonSL5-1 and KY.Chr10.1268.v1.ND1-1) were successfully expressed and purified in vitro. We named ascidian papilla adhesive protein 1 (APAP-1) and ascidian papilla adhesive protein 2 (APAP-2). After removing signal peptides and decahistidine tag labels, the purified product of recombinant APAP-1 had an SDS-PAGE band size of 46.47 kDa (Fig. 3a). The most abundant amino acid of this protein was Cystine, followed by Asparticacid, Glycine, and Asparagine in turn. Sequence analysis showed that APAP-1 had a large number of negatively charged amino acids (Fig. 3c). Furthermore, a total of six EGF-related domains, including one EGF, one EGF-like, and four EGF_CA domains, were found in APAP-1 (Fig. 3e). All the EGF-related domains were arranged in series with equal spacing, and there was only one amino acid spacing between two adjacent EGF-related domains. The purified product of recombinant APAP-2 had a band size of 65.01 kDa (Fig. 3b). The most abundant amino acid in this protein was Serine, followed by Cystine, Arginine, and Lysine. Many amino acids in APAP-2 are phosphorylable, especially for serine (Fig. 3d). APAP-2 contained three vWC domains in tandem (Fig. 3f), which were distributed at the positions of 13-70 aa, 123-184 aa, and 210-270 aa of this protein sequences.
Adhesive ability
Surface coating experiment showed that all the positive controls (Cell-Tak) were stained obviously and the negative controls (BSA) were not stained (Fig. 4a). The APAP-1 on all the material surfaces were not stained, while the APAP-2 on the corresponding surfaces were stained obviously. The adhesive abilities of APAP-2 on PVC, ABS, mica, stainless steel, aluminum, hydrophilic glass, and hydrophobic glass surfaces were strong, especially on aluminum surface where the color of APAP-2 spot was uniquely deeper than the corresponding positive control. There were no observed effects of metal ions on the adhesive ability of APAP-1 to PVC surface except iron. The additions of amino acids also had no interference with the adhesion of APAP-1 to PVC surface. (Fig. 4b). The iron also influenced the adhesive ability of APAP-2 on PVC surface (Fig. 4c). The changes in peak plots in all the gray analyses also showed similar trends with staining results (Fig. S3).