Cloning, characterization, and expression analysis of the CHITINASE gene family in Helice tientsinensis

Experimental materials

H. tientsinensis samples and nearby soil as well as water samples were excavated from coastal beach areas (33°13′38.62″N, 120°49′58.59″) of Dafeng Port in Yancheng, Jiangsu Province, China. H. tientsinensis was temporarily cultured in the laboratory at 25 °C. During the period of temporary cultivation, an appropriate amount of mineral water was added to the plastic box every day to keep the soil moist, and the dead samples were removed in time.

RNA extraction and cDNA library construction

Hepatopancreas, gill, muscle, heart, stomach and eyestalk tissues/organs of H. tientsinensis were ground to powder under liquid nitrogen. 0.02 g powder was used to extract RNA from all tissues using RNA prep Pure Animal Tissue Total RNA Extraction Kit (TIANGEN, China). According to the instructions of PrimeScript™ Master Mix (Takara Bio Inc., Shiga, Japan), cDNA libraries of different tissues were constructed.

Cloning of conserved sequence of cDNA

cDNA library of the hepatopancreas in H. tientsinensis was used as a template, and primers were designed to clone the conserved region of CHITINASE genes according to the RNA sequencing results and some known CHITINASE gene homology sequences from the shrimp and crab of Decapoda in NCBI GenBank (Table S1). The PCR system is as follows: 12.5 µL PCR MIX (Takara Bio Inc., Shiga, Japan), 2 µL cDNA template (50 times diluted from cDNA library), 1 µL forward primer (10 µmol/L), 1 µL reverse primer (10 µmol/L) and adding ddH₂O to 25 µL. The PCR mixture was put into the PCR instrument (Eppendorf, Hamburg, Germany), and the reaction parameters were: pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 1 kb/1 min, 35 cycles; final extending at 72 °C for 10 min. PCR bands were separated by 1% agarose gel electrophoresis, and the gel was cut under TGreen Gel Cutter (Tiangen, China). The gel was purified according to the instructions of AxyPrep™ DNA Gel Extraction Kit (Axygen, Union City, CA, USA).

The target fragment was introduced into the pMD19T vector and connected at 16 °C for 2 h. The ligating system was as follows: 2.5 µL ligase buffer, 0.5 µL pMD19T Simple vector (Takara Bio Inc., Shiga, Japan) and 2 µL gel for DNA recovery. The ligation products were transformed into DH5 α competent cells by heat shock method, and appropriate amount of bacterial liquid was pipetted and evenly coated on LB carbenicillin resistance solid medium (10 g peptone, 5 g yeast extract, 10 g NaCl and 15 g Agar dissolved in 1 L ddH₂O). After sterilization at 121 °C for 21 min, carbenicillin antibiotic with final concentration of 100 µg/L was added. Monoclone was selected and cultured with 700 µL LB liquid medium (carbenicillin resistance, 100 mg/L) at 250 rpm for 3 h at 37 °C for further PCR detection. The positive clones were sent to Shanghai Boshang Biotechnology Company (Shanghai, China) for sequencing.

Cloning of CHITINASE cDNA from 5′ to 3′ ends RACE

High quality of hepatopancreas RNA in H. tientsinensis was used as a template, following the instructions for SMARTer^® RACE 5′/3′Kit (Clotech, Palo Alto, CA, USA) and cDNA libraries were constructed for RACE 5′and 3′ ends of H. tientsinensis. Gene specific primer (GSP) which is based on known sequences of conserved regions (Table 1) was designed for RACE and cDNA ends were cloned follow the user manual. PCR bands were separated by 1% agarose gel electrophoresis, and the gel was cut under TGreen Gel Cutter. The gel was recovered according to the instructions of AxyPrep™ DNA Gel Extraction Kit (Axygen). The gel recovery products obtained by RACE were connected to the pJET vector according to the instructions of CloneJET™ PCR Cloning Kit, and ligation products were incubated at 22 °C for 30 min. The connection system is as follows: 2.5 µL 2 × Reaction buffer, 0.25 µL pJET vector, 0.25 µL T4 DNA ligase (Thermo Fisher, Waltham, MA, USA) and 2 µL gel recovery product of DNA. The ligation products were transformed into competent DH5 α cells and identified by bacterial liquid PCR. The positive clones were selected and sent to Shanghai Boshang Biotechnology Company (Shanghai, China) for sequencing.

Chitinase sequence alignment and functional domain analysis

The open reading frame (ORF) sequences and translation protein sequences were predicted by ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/). The protein sequences were submitted to online analysis software SignalP−6.0 Server (https://services.healthtech.dtu.dk/service.php?SignalP) and SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1) for prediction and analysis of protein signal peptides and domains, respectively. MEGA7 software was used to compare and analyze the coding sequence (CDS) of H. tientsinensis and CDS regions of other homologous genes in NCBI GenBank (https://www.ncbi.nlm.nih.gov/). Neighbor-joining (NJ) method was conducted to construct phylogenetic trees and analyze the evolutionary relationships among different species of chitinase in crustacean by MEGA6.0 and FigTree v1.4.4.

Tissue-specific expression analysis

Quantitative primers were designed based on the full-length cDNA sequences (Table 1), and the expression profiles of CHITINASE genes in different tissues (hepatopancreas, gill, muscle, heart, stomach and eye stalk) of H. tientsinensis were detected and analyzed by qRT-PCR. The reaction system is as follows: 10 µL 2 × SuperReal PreMix Plus (TIANGEN, China), 2 µL cDNA template (50 times diluted), 0.6 µL forward primer (10 µmol/L), 0.6 µL reverse primer (10 µmol/L), and ddH₂O to 20 µL. Mixture was put into ABI QuantStudio 3 quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA), and executed the procedure: pre-denaturation at 95 °C for 15 min; denaturation at 95 °C for 10 s, annealing at 55 °C for 20 s, elongation at 72 °C for 20 s, 45 cycles; Dissolution curves were 95 °C for 10 s, 65 °C for 60 s, 97 °C for 1 s. The 2^−ΔCT method was used to calculate the relative expression level of target gene relative to internal reference GAPDH, and significance analysis was conducted using one way ANOVA by SPSS Statistics 23. The calculation method is as follows: ΔCT = CT^T-CT^G; relative transcript level = 2^−ΔCT (CT, cycle threshold; CT^T is CT value of target gene; CT^G is CT value of internal reference gene GAPDH).

V. parahaemolyticus challenges in H. tientsinensis

V. parahaemolyticus strain (ATCC 17802) was bought in China general microbiological culture collection center (CGMCC) and stored at the −80 °C freezer. V. parahaemolyticus strain was cultured in the 3%NaCl nutrient agar liquid medium (10 g peptone, 3 g beef extract, 30 g NaCl dissolved in 1 L ddH₂O, pH = 7.0), and then the bacteria cells were centrifuged at 4,000 rpm for 10 min and suspended by phosphate buffer (PBS) for the further infection experiment.

H. tientsinensis were collected in the mudflat and randomly divided into the experiment group (V. parahaemolyticus group) and control group (PBS group) after temporary rearing for two days. Each mudflat crab in the experiment group was injected with 50 µL V. parahaemolyticus suspension containing 1 ×10⁶ bacteria copies in the second walking leg, while 50 µL PBS was injected into each mudflat crab in the control group. The hepatopancreas of six mudflat crabs were samples at each time point (0 h before injection, and 6 h, 12 h, 24 h, 48 h, 72 h in the post-infection period) in the experiment and control group, respectively. Hepatopancreas was immediately frozen in the liquid nitrogen and stored in the −80 °C freezer for RNA isolation until use, followed by RNA extraction and cDNA libraries construction. CHITINASE genes at different time points after injection in hepatopancreas of H. tientsinensis were detected and analyzed by qRT-PCR. The relative expression level of target genes at different time points after injection were calculated by the 2^−ΔΔCT method as described previously (Livak & Schmittgen, 2001). GAPDH was used as the internal reference gene to standardize the results. All primers used were listed in Table 1.

CHITINASE cDNA cloning

The primers were designed based on the RNA sequencing results of H. tientsinensis and homologous genes of NCBI GenBank, and the conserved regions of HtCHT1, HtCHT3 and HtCHT4 genes were cloned. The conserved regions of cDNA were 1,132 bp, 1,027 bp and 980 bp, respectively (Figs. 1A, 1C, Fig. S1), and the 5′/3′ ends of each cDNA were cloned by RACE (Figs. 1D, 1E, Fig. S1).

Sequence analysis showed that the full-length cDNA of the HtCHT1 was 2,229 bp (NCBI ID: OQ025399), including 96 bp 5′UTR, 1,884 bp CDS and 249 bp 3′UTR, encoding 627 amino acids (AA) (Fig. 2). The full-length cDNA of the HtCHT3 was 2,191 bp (NCBI ID: OQ025400), including 37 bp 5′UTR, 1,470 bp CDS and 684 bp 3′UTR, encoding 684 amino acids (Fig. 2). The full-length cDNA of the HtC HT4 was 3,312 bp (NCBI ID: OQ025401), including 30 bp 5′UTR, 1,995 bp CDS and 1,287 bp 3′UTR. The code produced 664 amino acids (Fig. 2).

Bioinformatics and phylogenetic analysis of chitinase

The analysis of signal peptides and functional domains of the HtCHT1, HtCHT3 and HtCHT4 protein showed that HtCHT1 protein had 23 AA signal peptides at the N-terminal. 47–398 AA was the catalytic domain of glycoside hydrolases 18 family (GH18) of the HtCHT1, and 470–525 AA was the chitin-binding domain (ChtBD2). The HtCHT3 had a GH18 catalytic domain between 19 and 367 AA, and 423–480 AA was chitin-binding domain. The N-terminal of the HtCHT4 had 26 AA signal peptides, while 29–385 AA was also GH18 catalytic domain, and HtCHT4 had two chitin-binding domain (548–606 AA and 607–663 AA) (Fig. 3). There were three highly conserved motif (motif II, motif III, motif IV) in the HtCHT1, HtCHT3 and HtCHT4. However, only HtCHT1 had the complete motif I, and the lysine (K) was replaced by valine (V) between the HtCHT3 and HtCHT4 (Fig. 4).

Phylogenetic analysis showed that different chitinases clustered in similar clusters, which were obviously divided into six categories and each kind of chitinases were concentrated in the same category. Crabs and shrimps further aggregated, and the internal genetic distance was relatively closer (Fig. 5).

NJ phylogenetic tree was constructed by alignment the chitinase protein sequences among H. tientsinensis, Eriocheir sinensis, Scylla serrata, S. paramamosain, P. trituberculatus, Macrobrachium nipponense, L. vannamei, Fenneropenaeus chinensis, Penaeus monodon, P. japonica, Marsupenaeus japonicus and Exopalaemon carinicauda. Phylogenetic analysis showed that HtCHT1, HtCHT3, and HtCHT4 of H. tientsinensis were all in the corresponding cluster, Group I, Group II, Group IV, respectively. The EsCHT1, EsCHT3 and EsCHT4 of E. sinensis (Varunidae) had the closest genetic distance with HtCHT1, HtCHT3, and HtCHT4, while M. nipponense (Palaemonidae) had the farthest genetic relationship (Fig. 5). Comparing the HtCHT1, HtCHT3, HtCHT4 with the EsCHT1, EsCHT3, EsCHT4, the conservations of protein sequences were 79.1%, 92.9% and 65.5%, respectively.

Tissue-specific expression analysis

The expression of the HtCHT1, HtCHT3 and HtCHT4 of different tissues/organs (hepatopancreas, gill, muscle, heart, stomach and eye stalk) in H. tientsinensis was analyzed by qRT-PCR. The expression of the HtCHT1, HtCHT3 and HtCHT4 showed the tissue-specific expression and similar expression profiles. They specifically expressed in the hepatopancreas, and the expression levels of three genes were significantly higher than other tissues and organs (P < 0.01) (Fig. 6).

Expression analysis of CHITINASES in H. tientsinensis after bacterial infection

To study the biological functions of three CHITINASES, the expression of the HtCHT1, HtCHT3 and HtCHT4 in hepatopancreas was detected at 6 h, 12 h, 24 h, 48 h and 72 h after V. parahaemolyticus infection (Fig. 7). HtCHT1 in hepatopancreas infected with the V. parahaemolyticus showed significant up-regulation expression by more than 14-fold at 48 h, compared to that in the PBS group, with a tendency to decrease at 72 h, showing up-regulation (11.3-fold), compared to the PBS group (P < 0.05) (Fig. 7A). Significant up-regulation of HtCHT3 was observed from the 48 h (23.2-fold) to 72 h (12.0-fold) after infected with the V. parahaemolyticus as compared with PBS solution (P < 0.05) (Fig. 7B). The expression of HtCHT4 was significantly up-regulated at 48 h (5.6-fold) and 72 h (7.3-fold) and kept a significantly up-regulated pattern until the tested 72 h compared to the PBS group in hepatopancreas (P < 0.05) (Fig. 7C).