Cadmium stress has significant effects on plant growth and development, yield, and quality (Ismael et al., 2019). When plants are subjected to stress, they will start the defense regulation of adverse environment by regulating the expression of related genes. Transcription factors can regulate the expression of downstream genes by binding to the cis-acting elements of specific genes, thus enhancing the tolerance of plants to stress (Chen et al., 2019). It has been found that NAC transcription factor (NAM, ATAF1/2, CUC2) is one of the plant-specific regulatory proteins (Zhu et al., 2014), which plays an important role in plant growth and development processes such as seed germination, lateral root formation, leaf senescence, cellulose synthesis, and secondary cell wall growth (Balazadeh et al., 2010; Yang et al., 2011a; Zhao et al., 2014). Overexpression of PopNAC122 gene in poplar (Populus L) reduced height growth and showed reductions in cell size and number (Grant et al., 2010). While the overexpression of MpSNAC67 gene in banana (Musa nana Lour) showed reductions in leaf area of transgenic banana lines (Tak et al., 2018). At the same time, some studies have shown that overexpression of TaNAC47 in Arabidopsis thaliana can improve the tolerance of transgenic plants to drought, salt and freezing stress (Zhang et al., 2015). Both SlNAC1 and SLNAM1 in tomato (Lycopersicon esculentum) were induced by salt stress (Yang et al., 2011b). Overexpression of CmNAC14 in melon (Cucumis melo L.) increased the sensitivity of melon seedlings to salt stress and inhibited the growth of melon seedlings under salt stress (Wei et al., 2016). By constructing HaNAC2 transgenic tobacco and studying its biological functions, it was found that the expression of HaNAC2 increased the resistance of transgenic tobacco to drought, salt and cold stress (Ren, 2016). The expression analysis of StNAC72 gene in potato after drought and rewatering showed that StNAC72 gene may be involved in the signal transduction process of drought and water stress (Gong et al., 2016). The analysis of NAC transcriptome data of potato by high-throughput sequencing showed that StNAC 072 and StNAC 101 were orthologs of stress response Arabidopsis thaliana to dehydration response 26 (RD26) (Singh et al., 2013). It has also been reported that the regulation of plants in osmotic stress was controlled by regulating the expression of StNAC262 gene in potato (Zhang et al., 2018). Although NAC transcription factor has been widely studied in different crops, the response of this gene to heavy metals is still unclear.

In this study, a NAC transcription factor gene StNAC2 was excavated and cloned from the previous transcriptome sequencing data of our group using potato ‘Yunshu 505’ as the material. The coding sequence characteristics of StNAC2 were analyzed, and the gene expression patterns in different tissues and different concentrations under Cd stress were studied, so as to provide a theoretical basis for revealing the molecular mechanism of StNAC2 response to stress in potato.

1 Results and Analysis

1.1 Cloning and sequence analysis of NAC2 gene of potato (Solanum tuberosum L)

Potato leaf cDNA was used as template for PCR amplification. The amplified products were detected by 1% agarose gel electrophoresis, and the segment of about 800 bp was obtained (Figure 1), which was consistent with the expected. The target segment was recovered and purified, ligated with pMD19-T vector, and transformed into competent cells of E. coil DH5α. The positive clones of the transformed products were screened by colony PCR and sent to Sangon Biotech (Shanghai) for sequencing. Sequencing analysis showed that there was a complete ORF with a length of 876 bp encoding 291 amino acids (Figure 2). There was a highly conserved NAM domain between the 22^nd and 390^th amino acids at the N-terminal, indicating that StNAC2 belonged to the NAC family transcription factor. ProtParam predicted the physical and chemical properties of StNAC2 protein were as follows: the relative molecular weight of StNAC2 protein was 33.58 512 kD, the theoretical isoelectric point (pI) was 7.04, the molecular formula was C₁₅₁₃H₂₂₉₇N₃₉₇O₄₄₃S₁₄, the fat coefficient was 63.68, and the instability coefficient was 50.33. StNAC2 was an unstable hydrophilic protein with the average hydrophilicity (GRAVY) of -0.660. Subcellular localization predicted that StNAC2 was mainly located in the cytoplasm and mitochondria, and there was no signal peptide and transmembrane domain. It was speculated that the protein was a non-secretory protein.

1.2 Analysis of StNAC78 protein structure and evolution

It is predicted by SOPMA that the secondary structure of StNAC2 protein was mainly random coil structure (65.64%), followed by α-helix (16.84%) and extension strand (14.78%) (Figure 3A). SWISS-MODEL was used to construct the three-dimensional structural model, and the similarity with the database model was 74.53% (>50%), indicating that it can be used as a structural model for further analysis (Figure 3B).

The amino acid sequences of StNAC2 were compared with other plants on NCBI (Figure 4), and the protein sequences with high homology among species were selected to construct the phylogenetic tree (Figure 5). The results showed that the amino acid sequences of potato StNAC2 protein were highly consistent with those of Capsicum annuum L, Nicotiana tabacum L, Ricinus communis L, Petunia hybrida, Diospyros kaki Thunb, Camellia sinensis, Pistacia chinensis Bunge, Gossypium spp, Gossypium hirsutum Linn, Populus euphratica, Jatropha curcas L, and Hevea brasiliensis published on GenBank, and the similarity was more than 70%, indicating that NAC2 protein is highly conservative in the process of species evolution. Phylogenetic tree showed that potato StNAC2 had the closest relationship with pepper protein, with a similarity of 85.67%. It was also clustered with Nicotiana tabacum and belonged to Solanaceae plants, indicating that the protein source homology between the same family and genus was higher, and it was speculated that the protein function was the most similar.

1.3 Relative expression analysis of StNAC2 gene in response to Cd stress

Under Cd stress, the expression of StNAC2 gene in leaves was up-regulated with the increase of stress concentration and reached the maximum with the treatment of 50 mg/kg, which was 9.56 times higher than that of the control. But it was significantly down-regulated at high concentration of 100 mg/kg. The expression of StNAC2 gene in stem was the same as that in leaves. It was significantly up-regulated under 50 mg/kg treatment, which was 1.76 times higher than that in the control, and significantly down-regulated under 100 mg/kg treatment, indicating that the expression of StNAC2 gene increased gradually with the increase of stress concentration, but the expression of NAC2 gene was inhibited under high concentration of 100 mg/kg stress (Figure 6). It was speculated that StNAC2 has a certain response and regulation to Cd stress.

2 Discussion

In this study, StNAC2 gene was cloned from potato leaves by homologous cloning technology. Analysis results showed that the open reading frame (ORF) was 876 bp, encoding 291 amino acids, including a conserved NAM domain, belonging to the NAM subfamily of NAC family members. Prediction of subcellular localization is helpful to infer protein function (Yu et al., 2006). In this study, we predicted that StNAC2 protein was mainly localized in cytoplasm and mitochondria through subcellular localization, and the protein did not contain signal peptide and no transmembrane domain. Through the comparison of similar amino acid sequences among species, it was found that the similarity of NAC2 protein with Capsicum annuum, Nicotiana tabacum, Petunia hybrida, Diospyros kaki Thun, Camellia sinensis, Pistacia chinensis Bunge, Gossypium spp, Gossypium hirsutum Linn, Populus euphratica, Ricinus communis, Jatropha curcas and Hevea brasiliensis was more than 70%, indicating that NAC2 protein was highly conserved. Phylogenetic tree analysis showed that StNAC2 had the highest similarity with pepper protein, with the similarity of 85.67%. StNAC2 was clustered with tobacco, which belonged to Solanaceae plants, indicating that the protein source homology was higher, and the protein function was the closest between the same family and genus. Tissue specificity analysis by fluorescence quantitative PCR showed that StNAC2 gene was expressed in stems and leaves under Cd stress, but there was significant different. The expressions of StNAC2 gene in stems and leaves were reached the maximum with the treatment of 50 mg/kg, which were 1.76 times and 9.56 times higher than those of the control, respectively. The expression of StNAC2 gene decreased rapidly under 100 mg/kg treatment in stems and leaves, indicating that the StNAC2 gene was induced by Cd stress, and the expression increased with the increase of concentration. But the gene expression was inhibited under high concentration of 100 mg/kg stress. It was speculated that StNAC2 has a certain response and regulation to Cd stress. The results of this study were similar to those of Du et al. (2020). The expression pattern of Secale cereale AmeNACs in stem tissue induced by Cd was more than 5 times higher than that of the control, and the expression was significantly inhibited when the final concentration was 100 µmol/L. As one of the largest transcription factors in plant family, NAC family members are mostly inducible transcription factors (Zong et al., 2019). In recent years, it has made some progress in identifying potential stress-related genes that can improve the tolerance of plants to abiotic stress. Transgenic rice plants overexpressing OsNAC6 showed slow growth and low reproductive yield, and good tolerance to dehydration and high salt stress, strong resistance to rice blast as well (Nakashima et al., 2007). The overexpression of tobacco SlNAC35 promoted root growth and development under drought and salt stress, and improved resistance to bacterial pathogens (Wang et ai., 2016). The overexpression of TaNAC2 enhanced the tolerance of Arabidopsis to drought, salt and freezing stresses (Mao et al., 2012). The expression level of CSNAC019 gene in cucumber was significantly up-regulated under 300 μmol/L Cd stress. Based on the current studies on NAC transcription factor response to abiotic stress, most studies focused on drought resistance and salt tolerance (Hong et al., 2016), cold resistance (Zhao et al., 2016; Yang et al., 2015), heat resistance (Shahnejatbushehri et al., 2012), germination rate (Li et al., 2016), proliferation lateral root growth, leaf relative water content, cell membrane stability, total chlorophyll content, proline and soluble sugar content etc. (Pandurangaiah et al., 2014). Few studies were conducted on resistance to heavy metal stress. Therefore, the research on NAC transcription factor response to Cd stress was also particularly important.

In this study, the potato StNAC2 gene was preliminarily analyzed, which provided basic theoretical knowledge for the follow-up development of gene function. The specific function and mechanism of action of StNAC2 gene are still unclear. It is necessary to further verify its function and clarify its mechanism through experiments. In the later stage, overexpression vector and gene silencing vector were constructed to further study the molecular mechanism of StNAC2 gene in response to Cd stress through genetic transformation, and further verify the regulatory mechanism and function of StNAC2 gene in response to Cd stress.

3 Materials and Methods

3.1 Experimental materials and treatments

The seeds of potato ‘Yunshu 505’ were collected from the Experimental Base in Weining County, Guizhou Province (103°36'~104°45'E, 26°36'~27°26'N) and carried out pot experiment. The concentrations of Cd stress were set as 0, 10, 20, 50 and 100 mg/kg, respectively, and three pots were planted in each treatment. The pots were evenly mixed into the soil a week to be deposited, the potato was planted. The daily water and fertilizer management was carried out, and the plants grew until the functional leaves were fully expanded for sampling. Then it was quickly frozen with liquid nitrogen and stored in an ultra-low temperature refrigerator (−80°C) for further use. Plant total RNA extraction kit, purification recovery kit, PCR amplification enzyme, DNA Marker, Loading Buffer, PCR amplification enzyme were purchased from TransGen Biotech. E.coli DH5α competent cells, cloning vector pMD19-T vector and Taq DNA polymerase were purchased from Takara Bio (Beijing). The primers were synthesized with the help of Sangon Biotech (Shanghai).

3.2 Total RNA extraction and cDNA synthesis

The total RNA, extracted by Trizol method, was used as a template for reverse transcription into cDNA. The following components were successively added to the DEPC water-treated tubules: Total RNA 5 μL, 5 x TransScript All in-one Super Mix for PCR 4 µL, RNase-free Water 11 µL, with a total volume of 20 μL. After gently mixing and incubating at 25℃ for 10 min, 42℃ for 30 min, and 85℃ for 5 s by PCR, respectively, and the first strand cDNA was synthesized.

3.3 Cloning of StNAC2 gene

The full-length gene and ORF sequence were found in NCBI using the NAC transcription factor coding gene ID number (LOC102585728) in transcriptome sequencing data. Primer 5.0 was used to design specific primers, fluorescent quantitative primers and Actin primers for amplifying the gene ORF (Table 1). Potato cDNA was used as template for PCR amplification with 25 μL PCR reaction system: cDNA 1 μL, each primer 1 μL, 2 x TransTaqPCR Super Mix Ⅱ 12.5 µL, ddH₂O 9.5 μL. PCR reaction procedure was as follows: Pre-denaturation at 94℃ for 3 min, denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72℃ for 1.5 min, 35 cycles, and finally preservation at 72℃ for 5 min, preservation at 4℃. After 1% agarose gel electrophoresis, the target band was recovered, and the PCR product was ligated to the cloning vector pMD19-T vector and transformed into E.coli DH5 α competent cells. After PCR identification, the positive clones were selected and sent to Sangon Biotech (Shanghai) for sequencing.

3.4 Analysis of sequence biological information

The open reading frame of the cloned sequence was analyzed with the help of the ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder) provided by NCBI and the amino acid sequence of StNAC2 gene was deduced. The conserved domain of the gene was predicted with the help of the software (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) in NCBI. ProtParam (http://web.expasy.org/protparam/) was used to analyze the number of amino acids, relative molecular weight, theoretical PI value, amino acid composition, the number of positively and negatively charged residues, atomic composition, molecular formula, total atomic number, extinction coefficient, half-life, fat coefficient, instability coefficient and total average hydrophilicity of the protein. Software (http://web.expasy.org/cgi-bin/protscale/protscale.pl) was used to analyze the hydrophilicity and hydrophobicity of the StNAC2 protein. Software (https://npsa-prabi.ibcp.fr/cgi-bin/secpred_sopma.pl) was used to perform the prediction of protein secondary structure. SWISS-MODEL (https://swissmodel.expasy.org/) was used to predict and draw protein tertiary structure model. DNAMAN7.0 was used for multi-sequence alignment, and MFGA7 (Neighbor-Joiningling proximity method) was used to construct the phylogenetic tree.

3.5 Expression analysis of NAC2 gene in potato

StNAC2 fluorescent primers were designed, and Actin was used as reference gene (Table 1) for semi-quantitative analysis by SYBR Green method. Using 25 μL reaction system: cDNA 2 μL, forward and reverse primers 1 μL, respectively, qPCR MasterMix 12.5 μL, ddH₂O 8.5 μL. The amplification procedure was as follows: 95°C for 3 min, 95°C for 15 s, 40 cycles, 60°C for 40 s. Three biological replicates were designed, and the expression levels of the target gene under different treatments and tissues were calculated with the help of 2^-(ΔΔct) (Bai et al., 2015). Fluorescence quantitative data were analyzed by Excel and SPSS software.

Authors’ Contributions

MLL is the experimental designer and executor of this study. HGD participated in data analysis and the writing of the first draft. TWJ, HY, and LDD participated in the experimental design and the analysis of experimental results. HTB is the designer and director of the project, guiding experimental design, data analysis, paper writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was supported by the National Natural Science Foundation of China-Karst Science Research Center of the People’s Government of Guizhou Province (U1612442) and the Project of Top Disciplines Biology Construction in Guizhou Province (GNYL［2017］009).

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