Cloning and expression of class I chitinase gene from four mangrove species under heavy metal stress

Aims Cu, Pb and Cd are common heavy metals in mangroves. Objective to clone chitinase I gene in mangrove plants and explore the role of chitinase I gene in plants under heavy metal stress. Methods Homologous cloning and RACE cloning were used to clone chitinase type I gene in mangrove plants, and bioinformatics analysis software was used to analyze and predict gene structure and functional domain. The mRNA expression pattern of chitinase gene in mangrove plants under heavy metal stress was analyzed by real-time quantitative PCR analysis.

It has been con rmed that chitinase is one of the related proteins (PR proteins) in plants. Plant chitinases have been divided into at least ve classes (I, II, III, IV, and V) based on their sequence similarities (Collinge et al., 1993;Melchers et al., 1994). Most chitinases are also induced by some biological or no-biological factors, such as mechanical damage, chitin, ethylene, salicylic acid, heavy metals, UV, osmotic pressure, low temperature, and drought stress (Kasprzewska, 2003). In normal conditions, chitinase gene expression is very low or not expressed in most higher plants. However, when plants are infected by pathogenic fungi, bacteria or viruses, mechanical trauma or ethylene treatment, their expression activity is greatly increased, and they are often induced to express at the same time with glucanase (EC3.2.1.39), which plays an important role in plant disease resistance and defense response. Peas of three ecological types were treated with 3mg/kg Cd sand for one week, then RNA was extracted to clone stress-related genes. The results showed that chitinase gene expression was higher than that of the control (Rivera et al., 2005a). Mycorrhizal and non-mycorrhizal peas were cultured in100mg/ kg Cd sand for 3 weeks. Gene expression analysis showed that chitinase, heat shock protein, metallothionein and glutathione synthetase were signi cantly higher than the control group without Cd treatment (Rivera et al., 2005b). It has been reported that heavy metal ions can induce oxidative stress in plants, indicating that the accumulation of reactive oxygen species in plants under heavy metal stress leads to the accumulation of H 2 O 2 . The accumulation of H 2 O 2 diffused and induced the transcription of chitinase gene, the accumulation of corresponding mRNA and the increase of corresponding enzyme activity (Lamb and Dixon, 1997;Fang and Kao, 2000;Tewari et al., 2002). At present, chitinase gene has been cloned from terrestrial plants, such as tobacco (Shinshi et al., 1990), potato (Ancillo et al., 1999), pear (Xiao et al., 2007), rice (Hang et al.,1991;Xu et al. 1996), etc. And studies of regulation are also deeper. However, as a defense protein, it may not directly participate in metal binding, but also plays an important role in metal tolerance of plants, which is little known. In mangrove plants, a class chitinase gene was cloned form in A. corniculatum (Wang et al., 2015b). It was also cloned with the full-length cDNA of a class III chitinase gene (AmCHI III) from A. marina (Wang et al., 2015a).
In the paper, the class I chitinase was rstly cloned by using RT-PCR and RACE methods in Bruguiera gymnorrhiza, Rhizophora stylosa, Kandelia obovata, Avicennia marina. Also elucidated for the mRNA expression pattern of CHI I in response to heavy metal stress.

Plant material and Treatments
The six-month-old seedlings of Rhizophora stylosa, Bruguiera gymnorrhiza, Kandelia obovata, and Avicennia marina were purchased from Guangdong Mangrove Ecological Development Co. LTD (China). Each of the species planted 3 seedlings in each pot and divided them into 5 pots (control group CK, C1, C2, C3, C4). Each pot was irrigated with 500 mL of 1/2 Hoagland solution (containing 10% NaCl) every 3 days. The plants were watered with heavy metal sewage of different pollution levels prepared (Table1). Fresh leaves of plants were harvested after 0 day, 3 day, 7 day, 14 day, 28 day under heavy metal treatment. All the harvested samples were immediately frozen in liquid nitrogen, and stored at − 80°C before use.

Cloning the full-length cDNA of chitinase gene
The sequences of primers used were shown in Table 2

Bioinformatic analysis
The full-length sequence was subjected to bioinformatics analysis using the following software or online tools ( Table  3), validation of the full-length sequence and analysis of the functional domains. 3 Results

The full -length cDNA of CHI I gene cloning
As shown in Figure 1A, obvious 28S and 18S bands were observed, indicating good RNA integrity. The UV detection results showed that the OD 260 / OD 280 ratios of the total RNA samples were between 1.8 and 2.2, indicating that the RNA purity was high. Therefore, the proposed RNA has high purity and good quality, which meet the quality requirements of subsequent experiments. With leaf cDNA as the template, a speci c fragment of more than 750 bp was obtained by ampli cation with degenerate primers (Fig. 1B). Blast was performed after sequencing, and the results showed that the fragment was highly homologous to the chitinase gene of other plants (84.73% -74.72%), indicating that the fragment was the intermediate fragment of CHII gene. According to the ampli ed intermediate fragment sequence, two pairs of primers for rapid ampli cation of 3' and 5' ends were designed. After the rst and second cycles of 3' and 5' RACE PCR, the 3' and 5' end-speci c fragments of the gene were ampli ed (Fig. 1C). Sequencing results of these fragments were spliced by MEGAE software, and submitted to NCBI for Blast homology analysis. Finally, we con rmed a correctly encoded nucleotide sequence.
3.2 Sequence and structure analysis of the full-length cDNA sequence of Chi I Cloning and characterization analysis of the full-length cDNA sequence of cDNA sequence analysis indicated that the full-length cDNA fragment encodes a novel basic chitinase gene, designated BgChi, KoChi, AmChi, RsChi (Fig. 2). Four cDNA all were 1092bp, including 831bp open-reading frame encoding a protein of 276 amino acids. There are different sequences among the four species with 6-30 bases different. BgChi with a predicted molecular mass of 29.50 kDa and a pI of 4.47 (Table 4). KoChi with a predicted molecular mass of 29.59 kDa and a pI of 4.74 (Table 4). AmChi with a predicted molecular mass of 25.57 kDa and a pI of 4.66 (Table 4). RsChi with a predicted molecular mass of 29.47 kDa and a pI of 4.65 (Table 4). And the Chi I polypeptide is hydrophilic based on the hydrophilicity values (Table 4).  , 2010), which was determined as a modeling template (Fig. 5). Four 3D models of Chi all contained seven α-helix and some random coils (Fig. 5).

CHI I mRNA expression in leaf in response to heavy metal
To realize the expression patterns of CHI I induced by heavy metal stress, Total RNA was isolated from four mangrove species seedlings leaves after heavy metal stimulation. The effects of heavy metal on the expression of CHI I mRNA in leaves were presented in Fig. 6 and Fig. 7.  (Santos et al., 2008). In this study, chitinase genes (CHI I) were cloned from B.gymnorrhiza, K.obovate, A.marina and R.stylosa for the rst time (Fig. 2). Using SMART software to predict chitinase protein structures including signal region, CBD and GH19 chitinase family catalytic domain. Most of them are small molecular proteins with molecular weight between 25 and 35 kDa (Arakane and Koga, 1999). In our study, we found that all four cDNA were 1092bp, including 831bp open-reading frame encoding a protein of 276 amino acids with molecular weight between 25.57 and 29.59 kDa ( Table 4). As a result, bioinformatics analysis revealed that BgChi, KoChi, AmChi, RsChi were a typical class I chitinase with the characteristic catalytic structure of chitinase.
The sequences among the four species are different (Fig. 2&3, Table 4). Compared with R.stylosa, there is one amino acid difference in B.gymnorrhiza, ve amino acid differences in K.obovate, and ten amino acid differences in A.marina (Fig. 3). BgChi showed very close homology to KoChi and RsChi in Fig. 4. We know that B.gymnorrhiza, K.obovate and R.stylosa belong to the same family of Rhizophora, while A.marina belongs to Verbenaceae. Phylogenetic tree analysis indicated that CHI had the closest relationship with chitinase in Camellia fraternal (75.05% similarity) (Fig. 8).
Phylogenetic clustering results are more consistent with the traditional morphological classi cation results. CHI I of A.corniculatum exhibited very close homology to the class I chitinase from Camellia sinensis (69% similarity) (Wang et al., 2015a). Camellia fraternal and Camellia sinensis are similar species. A.corniculatum is one species of mangroves.
Iseli et al. studied class I chitinase genes in tobacco suggesting that CBD is not catalytic and antifungal activity is necessary, but binding chitin is necessary and has enhanced antibacterial effect (Iseli et al., 1993). CBD of class I chitinases that acted as allergens in avocados and chestnuts may be associated with allergic reactions (Blanco et al.,1999). Therefore, we suppose that some of the cysteine residues in CHI I are essential for metal homeostasis in a harsh environment such as heavy metal stress.
Chitinases in plants are encoded by single genes. Both secreted outside and localized inside. In this study, the CHI protein was predicted to locate on vacuoles in cells according to Plant-mpLoc (Chou and Shen, 2008 (Fig. 9). Mangrove more tolerable to the heavy metal and it can be used as a potential phytoremediator in heavy metal polluted marine wetlands.

Conclusion
A new chitinase (CHI) gene was cloned from Bruguiera gymnorrhiza, Rhizophora stylosa, Kandelia obovata, Avicennia marina. And structure includes signal peptide region at its N-terminus, a chitin-binding domain (CBD), and a glycosyl hydrolase catalytic domain. CHI I belongs to glycosidase family 19. The chitinase gene sequences of four mangrove species were different. CHI I transcripts differentially express in four mangrove species under heavy metal. The gene expression of CHI I was highly induced in the B.gymnorrhiza leaves than other mangrove species. This study will provide more details on the molecular mechanisms or a scienti c basis for coastal wetland with heavy metal environmental remediation with mangrove plants.  Similarity and phylogenetic analysis of amino acid sequence of four species chitinase genes  Expression of CHI gene in leaves of four species in response to heavy metal stresses using real-time quantitative PCR analysis. Signi cant difference is indicated by an asterisk (P < 0.05).

Figure 7
The time effects of heavy metal exposure on the expression of CHI I mRNA in leaves of four mangrove species.
Signi cant difference is indicated by an asterisk (P < 0.05).

Figure 8
Phylogenetic tree of the CHI I. Multiple alignments of the sequences of CHI I and other selected plant chitinase-1 were performed using MEGA 6.

Figure 9
A schematic diagram of the mechanism of chitinase resistance to heavy metals.