Molecular Cloning and Expression Analysis of the Typical Class III Chitinase Genes from Three Mangrove Species under Heavy Metal Stress

Chitinases are considered to act as defense proteins when plants are exposed to heavy metal stresses. Typical class III chitinase genes were cloned from Kandelia obovate, Bruguiera gymnorrhiza, and Rhizophora stylosa by using RT-PCR and RACE and named KoCHI III, BgCHI III, and RsCHI III. Bioinformatics analysis revealed that the three genes encoding proteins were all typical class III chitinases with the characteristic catalytic structure belonging to the family GH18 and located outside the cell. In addition, there are heavy metal binding sites in the three-dimensional spatial structure of the type III chitinase gene. Phylogenetic tree analysis indicated that CHI had the closest relationship with chitinase in Rhizophora apiculata. In mangrove plants, the balance of the oxidative system in the body is disrupted under heavy metal stress, resulting in increased H2O2 content. Real-time PCR illustrated that the expression level under heavy metal stress was significantly higher than that in the control group. Expression levels of CHI III were higher in K. obovate than in B. gymnorrhiza and R. stylosa. With the increase in heavy metal stress time, the expression level increased continuously. These results suggest that chitinase plays an important role in improving the heavy metal tolerance of mangrove plants.


Introduction
Mangroves are a wetland woody plant community that grows in the intertidal zone of tropical and subtropical coasts and also play an important role in purifying seawater, preventing wind and waves, storing blue carbon, and maintaining biodiversity [1][2][3]. In recent years, with the rapid development of industry and agriculture and the expansion of coastal cities, large amounts of sewage and waste are discharged into estuaries and coastal areas every year, and the continuous development of the marine transportation industry causes water pollution in the bay area. Environmental pollution in the mangrove growing area has become an environmental problem that cannot be ignored, especially heavy metal pollution as a typical persistent pollutant; heavy metals can not only endanger the security of the mangrove ecosystem, but also pass through the food chain to other consumers and continuously accumulate in organisms, eventually posing a threat to the health of human beings and other organisms. This harm should not be ignored. Heavy metal pollution in estuaries and bays has become increasingly acute, which has attracted the attention of ecologists and environmental protection departments [4,5]. Excessive metal can inhibit seed germination and seedling growth, destroy antioxidant enzymes and membrane systems, cause chromosomal aberrations, and lead to plant death. Moreover,

The Full-Length cDNA of the CHI III Gene Cloning
In Figure 1A, the integrity of the extracted total RNA is good. The OD 260 /OD 280 values of the total RNA samples were between 1.8 and 2.1, indicating that the extracted total RNA Plants 2023, 12, 1681 3 of 14 had a high purity. After obtaining the intermediate fragment, the nucleotide sequences were obtained via sequencing. The nucleotide sequences were carried out in the NCBI database via BLAST comparison, and the similarity with the existing chitinase sequence reached 99%, which indicated that it was a chitinase sequence. The 3 and 5 end-specific fragments ( Figure 1B) were obtained by using the RACE method. After sequencing, the full length was finally determined after splicing.

The Full-Length cDNA of the CHI III Gene Cloning
In Figure 1A, the integrity of the extracted total RNA is good. The OD260/OD280 values of the total RNA samples were between 1.8 and 2.1, indicating that the extracted total RNA had a high purity. After obtaining the intermediate fragment, the nucleotide sequences were obtained via sequencing. The nucleotide sequences were carried out in the NCBI database via BLAST comparison, and the similarity with the existing chitinase sequence reached 99%, which indicated that it was a chitinase sequence. The 3 and 5′ end-specific fragments ( Figure 1B) were obtained by using the RACE method. After sequencing, the full length was finally determined after splicing. Through SMART TM RACE cDNA amplification, the full-length cDNA sequence of chitinase was isolated from mangrove and named BgCHI III (Figure 2A), KoCHI III ( Figure  2B), and RsCHI III ( Figure 2C). The nucleotide sequence of BgCHI III was 1067 bp, containing a 711 bp open reading frame (ORF) encoding the deduced protein length of 236 amino acids. The ORF starts at the 109 ATG start codon and ends at the 819 TGA stop codon ( Figure 2A). The calculated molecular weight of the protein was 25.3 kDa and the isoelectric point was 6.06. Amino acid composition analysis showed that BgCHI III contained higher levels of Ser (11.4%), Gly (11.0%), and Ala (8.9%). The stability coefficient was 43.50, and the hydrophilic coefficient was −0.252. Through SMART TM RACE cDNA amplification, the full-length cDNA sequence of chitinase was isolated from mangrove and named BgCHI III (Figure 2A), KoCHI III ( Figure 2B), and RsCHI III ( Figure 2C). The nucleotide sequence of BgCHI III was 1067 bp, containing a 711 bp open reading frame (ORF) encoding the deduced protein length of 236 amino acids. The ORF starts at the 109 ATG start codon and ends at the 819 TGA stop codon ( Figure 2A). The calculated molecular weight of the protein was 25.3 kDa and the isoelectric point was 6.06. Amino acid composition analysis showed that BgCHI III contained higher levels of Ser (11.4%), Gly (11.0%), and Ala (8.9%). The stability coefficient was 43.50, and the hydrophilic coefficient was −0.252.
The nucleotide sequence of KoCHI III was 1071 bp, containing a 600 bp open reading frame (ORF) encoding the deduced protein length of 199 amino acids. The ORF starts at the 225 ATG start codon and ends at the 824 TGA stop codon ( Figure 2B). The calculated molecular weight of the protein was 21.7 kDa and the isoelectric point was 5.69. Amino acid composition analysis showed that KoCHI III contained higher levels of Ser (11.6%), Gly (10.6%), and Ala (8.5%). The stability coefficient was 42.31, and the hydrophilic coefficient was −0.284.
The nucleotide sequence of RsCHI III was 1063 bp, containing a 600 bp open reading frame (ORF) encoding the deduced protein length of 199 amino acids. The ORF starts at the 216 ATG start codon and ends at the 815 TGA stop codon ( Figure 2C). The calculated molecular weight of the protein was 21.6 kDa and the isoelectric point was 5.69. Amino acid composition analysis showed that RsCHI III contained higher levels of Ser (11.6%), Gly (10.6%), and Ala (8.5%). The stability coefficient was 41.98, and the hydrophilic coefficient was −0.285.
Analysis indicated that the protein was an unstable hydrophilic protein with no transmembrane helix signals. Subcellular localization predictions using the online software Plant-mPLoc [32] pairs showed that three genes encoding type III chitinase proteins may be non-secretory proteins localized extracellularly, which may be related to the defense mechanism of chitinase in mangrove plants. This is consistent with the previous conclusion that type I and V chitinases are located in the vacuole, and type III chitinase is located extracellularly. In addition, chitinase belongs to the 18th family of glycohydrolase (GH18) in terms of structure. Without a chitinase binding domain (CBD), it is a typical type III chitinase. Secondary structure analysis showed that the chitinase consisted of seven α-helices and seven β-turns ( Figure 3). Based on SWISS-MODEL analysis [33], ribbon cartoon and space-filling models of CHI III are presented in Figure 4. Taking the threedimensional spatial structure of pomegranate as a template, the three-dimensional spatial structure of the mangrove was established. GH 18 chitinase from pomegranate has the crystal structure of pomegranate chitinase III. In this study, there are heavy metal binding sites in the three-dimensional spatial structure of the type III chitinase gene. The type III chitinase gene may combine with heavy metal ions to reduce the entry of heavy metal into cells, reducing the toxicity of heavy metals to plant cells. This also provides insights on its metal storage capacity. The nucleotide sequence of KoCHI III was 1071 bp, containing a 600 bp open reading frame (ORF) encoding the deduced protein length of 199 amino acids. The ORF starts at the 225 ATG start codon and ends at the 824 TGA stop codon ( Figure 2B). The calculated molecular weight of the protein was 21.7 kDa and the isoelectric point was 5.69. Amino acid composition analysis showed that KoCHI III contained higher levels of Ser (11.6%), Gly (10.6%), and Ala (8.5%). The stability coefficient was 42.31, and the hydrophilic coeffi- stylosa belonging to Rhizoporaceae were clustered together, while the chitinase type III genes of A. marina belonging to Myrsinaceae and A. corniculatum belonging to Verbenaceae evolved another branch that was quite different. Phylogenetic tree analysis indicated that CHI had the closest relationship with chitinase in Rhizophora apiculate (94.40% similarity) ( Figure 5). Although B. gymnorrhiza, K. obovate, and R. stylosa belong to oriental mangrove species and R. apiculate belongs to western mangrove species, their chitinase genes are closely related.     The phylogenetic tree was constructed via the NJ method using MEGA6 software. In this study, we chose Vitis vinifera (AB105374), Arabidopsis thaliana (NM-122314.3), Glycine max (NM-001249780.1), Rhizophora apiculate (KY795335.1), Hevea brasiliensis (DQ873889.2), Citrullus colocynthis (JQ756124.1), Citrullus lanatus (DQ180495.1), Nepenthes rafflesiana (GQ338259.1), Avicennia marina (JQ655770), Aegiceras corniculatum (JQ655771), Bruguiera gymnorrhiza, Kandelia obovate, and Rhizophora stylosa. The amino acid sequences of chitinase type III in these plants were selected to construct the phylogenetic tree. The phylogenetic analysis results show that the chitinase type III genes of B. gymnorrhiza, K. obovate, and R. stylosa belonging to Rhizoporaceae were clustered together, while the chitinase type III genes of A. marina belonging to Myrsinaceae and A. corniculatum belonging to Verbenaceae evolved another branch that was quite different. Phylogenetic tree analysis indicated that CHI had the closest relationship with chitinase in Rhizophora apiculate (94.40% similarity) ( Figure 5). Although B. gymnorrhiza, K. obovate, and R. stylosa belong to oriental mangrove species and R. apiculate belongs to western mangrove species, their chitinase genes are closely related.

The Effect on Mangrove under Heavy Metal Treatment
A plant's growth is slowed by heavy metal treatment, resulting in yellowing of the leaves. The heavy metal exposure affects the morphology of the mangrove plants. In this study, we observed that the leaf morphology of K. obovate and R. stylosa changed after exposure to high concentrations of heavy metals ( Figure 6). The content of H 2 O 2 can reflect the accumulation of ROS in plants [34]. The changing trend in H 2 O 2 content was obvious with the increase in the concentration of composite heavy metals. At the highest concentration of treatment, the H 2 O 2 content of the five mangrove plants increased significantly compared with the control group. H 2 O 2 content was 3.78 times higher than the control of B. gymnorrhiza at C3. H 2 O 2 content was 6.17 times higher than the control of K. obovate at C4. H 2 O 2 content was 7.57 times higher than the control of R.stylosa at C4. The order of H 2 O 2 content was K. obovate > R. stylosa > B. gymnorrhiza (Figure 7).

The Effect on Mangrove under Heavy Metal Treatment
A plant's growth is slowed by heavy metal treatment, resulting in yellowing of the leaves. The heavy metal exposure affects the morphology of the mangrove plants. In this study, we observed that the leaf morphology of K. obovate and R. stylosa changed after exposure to high concentrations of heavy metals ( Figure 6). The content of H2O2 can reflect the accumulation of ROS in plants [34]. The changing trend in H2O2 content was obvious with the increase in the concentration of composite heavy metals. At the highest concentration of treatment, the H2O2 content of the five mangrove plants increased significantly compared with the control group. H2O2 content was 3.78 times higher than the control of B. gymnorrhiza at C3. H2O2 content was 6.17 times higher than the control of K. obovate at C4. H2O2 content was 7.57 times higher than the control of R.stylosa at C4. The order of H2O2 content was K. obovate > R. stylosa > B. gymnorrhiza (Figure 7).

CHI III mRNA Expression in Leaf in Response to Heavy Metals
The effects of heavy metals on the expression of CHI III mRNA in leaves are shown in Figure 8. Moreover, there were different patterns of CHI III mRNA expression response to heavy metal stress among the three mangrove plants. After 3 days of exposure, BgCHI

CHI III mRNA Expression in Leaf in Response to Heavy Metals
The effects of heavy metals on the expression of CHI III mRNA in leaves are shown in Figure 8. Moreover, there were different patterns of CHI III mRNA expression response to heavy metal stress among the three mangrove plants. After 3 days of exposure, BgCHI III mRNA expression was 2.41 times higher than the control at C2. BgCHI III mRNA expression was 1.97 times higher than the control at C4. In addition, the maximum expression level was detected at C2 after 3 days in B. gymnorrhiza leaves ( Figure 8A). After 28 days of exposure, KoCHI III mRNA expression was 14.79 times higher than the control, and the maximum expression level was detected at C2 ( Figure 8B). Under the treatment of heavy metal C4 concentration, the expression levels of R. stylosa were relatively high, reaching a maximum value of 6.74 times that of the control group on the 28th day of treatment ( Figure 8C). Under treatment at C2 and C4 concentrations and with the increase in heavy metal stress time, the expression change had an upward trend in K. obovate. Under C1, C3, and C4 heavy metal treatment, the expression change had an upward trend in R. stylosa with the increase in heavy metal stress time. The expression levels of the CHI gene with C2 stress treatment in K. obovate on the 28th day of treatment were higher than those in R. stylosa and B. gymnorrhiza. III mRNA expression was 2.41 times higher than the control at C2. BgCHI III mRNA expression was 1.97 times higher than the control at C4. In addition, the maximum expression level was detected at C2 after 3 days in B. gymnorrhiza leaves ( Figure 8A). After 28 days of exposure, KoCHI III mRNA expression was 14.79 times higher than the control, and the maximum expression level was detected at C2 ( Figure 8B). Under the treatment of heavy metal C4 concentration, the expression levels of R. stylosa were relatively high, reaching a maximum value of 6.74 times that of the control group on the 28th day of treatment ( Figure 8C). Under treatment at C2 and C4 concentrations and with the increase in heavy metal stress time, the expression change had an upward trend in K. obovate. Under C1, C3, and C4 heavy metal treatment, the expression change had an upward trend in R. stylosa with the increase in heavy metal stress time. The expression levels of the CHI gene with C2 stress treatment in K. obovate on the 28th day of treatment were higher than those in R. stylosa and B. gymnorrhiza.  Data are the means ± standard of three separate individuals. Each bar with different lowercase letters was significantly different (p < 0.05).

Cloning and Structural Characterization Analysis of CHI III
In this study, chitinase genes were cloned for the first time from B. gymnorrhiza, K. obovate, and R. stylosa. These were predicted and analyzed for their secondary structure, subcellular localization, transmembrane domain, and functional domain of the cloned amino acid sequences. The amino acid sequences of chitinases from other plants also underwent multiple comparisons and were phylogenetically analyzed to demonstrate the homology between the cloned genes and chitinase genes from other plants. In the process of molecular evolution, amino acids with strong functions in the protein components evolved slowly, so the base evolution of amino acids with strong functions maintained relatively high conservation. Generally, the homology of sequences is closely related to the similarity of their functions, so the homology sequence of chitinase cloned via NCBI can be used to predict the function of the target gene according to its homology sequence. Constructing a phylogenetic tree can help us analyze protein evolution more clearly. The chitinases of glycoside hydrolase family 18 and glycoside hydrolase family 19 differ greatly in structure and distribution due to their different origin and evolution [34]. GH18 family chitinases include class III and V chitinases in plants [35,36]; class III and V chitinases have both lysozyme activity and chitinase activity [24]. In terms of structure, the 18 family only contains one catalytic region. In terms of distribution range, the glycosidase 18 family is widely distributed in microorganisms, animals, and plants. In our study, the cloned chitinase genes of mangrove plants were searched using Blast in NCBI, and it was found that the gene was highly similar to the chitinase gene of other plants, indicating that the three genes cloned were chitinase genes. Bioinformatics analysis showed that the chitinase gene encodes a typical type III chitinase that belongs to the glycoside hydrolase family 18. A conserved catalytic region means that it has a glycohydrolase function. Without CBD structure, it does not have anti-bacterial function in vitro. The chitinase protein is located outside the cell. When it encounters pathogens, it can directly hydrolyze new hyphae and resist pathogenic fungi. The sequences of type III chitinase genes from 11 other plants were selected for comparison in this chapter, and the three cloned type III chitinase genes were found to be the most evolutionarily similar (94.40% similarity) to the type III chitinase gene of R. apiculata. All three mangrove species belong to the family Mangroveaceae; the developmental tree branches were clustered together, and the kinship determination was based on amino acids. The results of kinship determination based on sequence analysis were the same as those based on traditional evolutionary kinship determination. The first basis for the investigation of gene function is to know the full-length sequence of the gene and the structural features of the protein space.

Expression of CHI III in Leaves in Response to Heavy Metal
Plant chitinases are disease-course-associated proteins that can disrupt the cell walls of pathogenic bacteria and the exoskeletons of insects and are also regulated by other disease-resistance-related molecules such as salicylic acid (SA), which play an important role in plant defense processes and are thus widely used in the genetic engineering of plant disease resistance [37][38][39][40]. In addition, chitinases are involved in plant responses to abiotic stresses such as drought, metal ions, and high temperatures. When plants were subjected to environmental stress, type III chitinase showed strong induction and expression [28,40]. The cotton type III chitinase gene is GhaChi, which is induced by ABA [41]. The sugarcane type III chitinase gene plays a positive role in response to biotic and abiotic stresses [26]. When Arabidopsis plants were subjected to environmental stress, especially salt stress and mechanical damage, only class III chitinase genes were induced and expressed [37]. Zhou's study shows that SlChi is a gene responsive to salt stress in Salix. When Salix was treated with 100 mmol/L NaCl stress, the expression level of endogenous SlChi increased with time, and the gene expression level after 48 h of continuous high salt treatment was 40 times that of non-treatment [42]. Furthermore, it has now been shown that overexpression of chitinase in transgenic plants does improve resistance to heavy metals [43]. Previous studies have focused on a single phenotype of the gene and studied the differences in its expression in different tissues, lacking the analysis of multiple phenotypes of the gene in multiple species. Among them, the mechanism of action of chitinase in mangrove plants under heavy metal stress is less studied, and one study cloned and compared the differences between different tissues of the type I chitinase of A. corniculatum [31]. In this study, when K. obovate was treated with C2 heavy metal stress, the expression level of endogenous KoCHI increased with treatment time, and the expression level of the gene after 28 days of heavy metal treatment was 14.49 times that of non-treatment. Additionally, the expression level of the gene after 28 days of heavy metal treatment was 6.47 times higher in R. stylosa treated with C4 heavy metal stress. The CHI III expression in leaves was up-regulated with heavy metals, which was in good agreement with previous results in other plants [20,44]. In nature, mangrove plants are stressed by various heavy metals. When the concentration of heavy metal is too high, it causes a series of injuries and cells produce a series of stress responses [45,46]. At present, it is generally believed that plants have an innate "immune system", oxidative burst (OXB), meaning that plant cells rapidly produce reactive oxygen species (ROS) under stress conditions that initiate other signal cascades in the body [47,48]. In most cases, H 2 O 2 is the main accumulation product of OXB [49,50]. In the present study, the H 2 O 2 content accumulated more after heavy metal stress treatment, and according to previous studies, many heavy metals entering plant cells would cause the accumulation of ROS, and the antioxidant system in plants would act as a regulator to convert ROS into H 2 O 2 , which would be a signaling factor to trigger the expression of some genes when accumulated in excess [51,52]. Our experiments to understand the structural features of the chitinase gene properties and protein space revealed that type III chitinase acts mainly outside the cell and has metal binding sites that may bind directly to heavy metals. The possible intrinsic molecular mechanism is that heavy metal stress leads to high levels of H 2 O 2 in vivo, and H 2 O 2 becomes a signaling factor triggering the expression of chitinase, which may bind to heavy metals and attenuate heavy metal damage to plant leaves ( Figure 9). There are different phenotypes of chitinases, and the functional roles they have can vary, as other reports say that chitinases are considered components of the second line of defense under metal stress [53]. They may alter the kinetics and permeability of the cell wall to metals, as well as affect the metal binding and fixation capacity of the cell wall [54]. They seem to be stable components of the plant metal stress defense [27]. Indeed, transgenic plants overexpressing chitinase have shown increased tolerance to metal [43]. in the body [47,48]. In most cases, H2O2 is the main accumulation product of OXB [49,50].
In the present study, the H2O2 content accumulated more after heavy metal stress treatment, and according to previous studies, many heavy metals entering plant cells would cause the accumulation of ROS, and the antioxidant system in plants would act as a regulator to convert ROS into H2O2, which would be a signaling factor to trigger the expression of some genes when accumulated in excess [51,52]. Our experiments to understand the structural features of the chitinase gene properties and protein space revealed that type III chitinase acts mainly outside the cell and has metal binding sites that may bind directly to heavy metals. The possible intrinsic molecular mechanism is that heavy metal stress leads to high levels of H2O2 in vivo, and H2O2 becomes a signaling factor triggering the expression of chitinase, which may bind to heavy metals and attenuate heavy metal damage to plant leaves ( Figure 9). There are different phenotypes of chitinases, and the functional roles they have can vary, as other reports say that chitinases are considered components of the second line of defense under metal stress [53]. They may alter the kinetics and permeability of the cell wall to metals, as well as affect the metal binding and fixation capacity of the cell wall [54]. They seem to be stable components of the plant metal stress defense [27]. Indeed, transgenic plants overexpressing chitinase have shown increased tolerance to metal [43]. Overall, expression of the chitinase gene may have improved the tolerance and accumulation of heavy metals in mangrove plants. In coastal wetlands, mangrove plants can be used as remediation plants for heavy metal pollution. Therefore, it is speculated that CHI may play a role in the resistance of mangrove plants to heavy metal stress.

Plant Materials and Treatments
The hypocotyls of mature B. gymnorrhiza, K. obovate, and R. stylosa were collected from the Leizhou Peninsula, Guangdong Province, China, with salinity ranging from 22‰ to Overall, expression of the chitinase gene may have improved the tolerance and accumulation of heavy metals in mangrove plants. In coastal wetlands, mangrove plants can be used as remediation plants for heavy metal pollution. Therefore, it is speculated that CHI may play a role in the resistance of mangrove plants to heavy metal stress.

Plant Materials and Treatments
The hypocotyls of mature B. gymnorrhiza, K. obovate, and R. stylosa were collected from the Leizhou Peninsula, Guangdong Province, China, with salinity ranging from 22‰ to 30‰. Each pot was irrigated with 500 mL of 1/2 Hoagland's nutrient solution (containing 10‰ NaCl) every 3 days.

Extraction of the Total RNA
The total RNA of mangrove leaves was extracted using the TIANGEN polysaccharide polyphenol plant total RNA extraction kit centrifugal column method. The extracted RNA was analyzed for purity and integrity. The RNA was treated with DNase I, and Oligo(dt) 15 was used as the universal primer. The treated RNA was reverse transcribed with reverse transcriptase to synthesize first-strand cDNA for RT-PCR amplification.

Primer Designs and Sequence Comparison Analysis
The amino acid sequences of chitinase genes from five closely related plants were downloaded from NCBI, and the conserved sequences were found using BioEdit sequence software. The primers F1 and R1 were designed with Primer Premier 5.0 using the closest sequence as a template.

Cloning the Full-Length cDNA of the Chitinase Gene
The intermediate fragment was cloned via PCR with the first-strand cDNA as the template and the primers F1, R1 ( Table 2). The intermediate fragment sequence of the chitinase gene was obtained. Primers GSP-3 , GSP-5 , NGSP-3 , and NGSP-5 were designed according to the obtained intermediate fragment sequence (Table 2). The specific primers were designed using the software Primer Premier 5.0. The full-length cloning was carried out via the RACE method according to the instructions of the "SMARTTM RACE cDNA Amplification Kit" from CLONTECH. During RACE, the reverse transcription product was used as a template. The outer primer was used for amplification, and the inner primer was used for nested PCR amplification. Additionally, single primer control was set. The PCR products were recovered using a GenStar kit and ligated with pMD18-T vector. The ligation product was transformed into E. coli DH5α competent cells, and two-way sequencing was completed by the HuaDa Gene Sequencing Company. We obtained the full-length cDNA using Contig Express software. The sequencing results were searched and analyzed in GenBank.

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

Analysis of Gene Expression using Real-Time Quantitative PCR
The preparation method of first-strand cDNA is shown above. The specific primers for the type III chitinase gene used in semi-quantitative RT-PCR were F and R ( Table 2). The primers Bg18S (F), Bg18S (R), Ko18S (F), Ko18S (R), Rs18S (F), and Rs18S (R) ( Table 2) were designed according to the RNA18S histone of mangrove as the cDNA content reference and for DNA contamination detection. Methods: NTC control was carried out on each plate of each gene, and qPCR96Well was taken to prepare the following reaction system (Table 4). Fold induction in CHI III mRNA expression relative to the control was determined by the standard 2 -DDCT method of Livak and Schmittgen [56].

Statistics Analysis
Using statistical analysis software, significant differences between controls and treatments were determined via one-way ANOVA and analyzed using SPSS V.22.0 software (Dunnett's multiple comparison test). All experiment data are presented as mean ± standard values of three independent sets.

Conclusions
Three chitinase genes (CHI) were cloned for the first time from B. gymnorrhiza, K. obovate, and R. stylosa; these belong to the 18th glycosidase family and are class III chitinases. Our results indicate that leaf CHI III expression was regulated by Cu, Cd, and Pb. After exposure to heavy metal, CHI III mRNA was strongly activated. Moreover, CHI III consistently presented the highest transcript level after 28 days of exposure. There was a higher expression level in K. obovate compared with others. This study will open a window on the function of the chitinase gene and provide a solid basis for the mechanism of heavy metal resistance in mangroves.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.