Stress is an important factor affecting plant growth and crop yield. As a sessile organism, plants must adapt to the external environment that is not conducive to their own growth and development, including biotic stress (pathogen infection and grazing of herbivores) and abiotic stress (drought, high temperature, chilling injury, salt injury, toxic metals in soil, etc.) (Fu et al., 2016; Zhu, 2016). In the long-term evolution process, plants have formed various molecular, cellular, physiological and biochemical mechanisms to resist and adapt to various environments that are not conducive to their own growth. When exposed to stress, plants can maintain growth and avoid injury by regulating the expression patterns of a series of genes. It is of great significance to explore stress resistance genes and study their stress resistance mechanisms to enhance the resistance of plants to stress. At present, several important regulatory genes of stress response have been identified and reported, including DRE/CRT binding transcription factors (DREBs/CBFs), mitogen-activated protein kinases (MAPKs), MYB, ABRE, bZIP and zinc finger proteins (Zhu, 2016; Haak et al., 2017; Dixit et al., 2018).

Stress associated protein (SAP) is a kind of zinc finger protein, which has the characteristics of A20/AN1 zinc finger domain. The conserved sequence of A20 zinc finger domain is Cx2-4Cx11Cx2C, which is located at the N-terminal of the protein and negatively regulates the central immune transcription factor NF-κB in humans, and the C-terminal is AN1 zinc finger domain (Giri et al., 2013). OsiSAP1 (indica rice) is the first reported SAP protein in plants. It is induced to express by low temperature, drought, high salt and ABA. The ability of tobacco overexpressing OsiSAP1 to resist abiotic stresses such as drought, high salt and low temperature is enhanced (Mukhopadhyay et al., 2004). At present, SAP gene has been identified in Arabidopsis (Vij and Tyagi, 2006; Dixit et al., 2018), rice (Vij and Tyagi, 2006), soybean (Zhang et al., 2019), cotton (Gao et al., 2016) and other plants. SAP gene plays an important role in abiotic stress. For example, Arabidopsis AtSAP5 has the function of E3 ubiquitin ligase, which is preferentially expressed in roots and induced by high salt, drought and low temperature. The drought tolerance of transgenic Arabidopsis is improved after overexpression of AtSAP5 (Kang et al., 2011); PtSAP13 enhances plant salt tolerance by up regulating the expression of stress-related genes and mediating a variety of biological pathways (Li et al., 2019). At the same time, SAP gene is also involved in biotic stress, and Arabidopsis overexpressing AtSAP9 is more sensitive to the non-host pathogen Pseudomonas syringae (Kang et al., 2017).

Soybean is an important food crop and cash crop. Drought, salinity, pest damage and other stresses have seriously affected the yield of soybean. Zhang et al. (2019) identified 27 soybean SAP genes. Only the function of GmSAP16 was deeply studied, and the function of other SAP genes was not clear. In this study, soybean GmSAP3 gene was cloned from Shangdou 1201 and analyzed by bioinformatics, tissue expression and temperature stress induced expression. The results of this study provide a certain reference for the further study of the biological function of GmSAP3.

1 Results and Analysis

1.1 Cloning of GmSAP3 gene and analysis of physicochemical properties of its coding protein

Using the cDNA of Shangdou 1201 leaves as a template, GmSAP3 gene was amplified by PCR, with a band of about 500 bp (Figure 1). The PCR product was recovered and connected to pMD19-T vector for sequencing. The sequencing results showed that the full length of GmSAP3 gene was 513 bp, which was completely consistent with the soybean Glyma.03G140500, and it was named as GmSAP3.

Figure 1 Detection of GmSAP3 PCR products by electrophoresis Note: M: DL2000 DNA Marker; 1: Target gene

The physical and chemical properties of GmSAP3 gene were analyzed by ProtParam. GmSAP3 gene has only one exon and no intron. The length of the encoded protein is 170 amino acids, the isoelectric point pI is 6.79, the molecular weight is 18.305 68 kD, the instability coefficient is 33.91, and the protein property is stable. The fat coefficient was 61.41, and the mean hydrophobic value (GRAVY) was -0.379. It was a hydrophilic protein. It was predicted that GmSAP3 protein was localized in the cytoplasm. Protein phosphorylation is an important post-translational modification of proteins and plays an important role in abiotic stress signaling pathways (Zhen et al., 2014). Use NetPhos 3. 1 Server predicted phosphorylation sites and found that there were 13 Serine, 5 Threonine and 3 Tyrosine sites in Gmsap3 protein (Figure 2). The secondary structure of GmSAP3 was predicted online using the SOPMA website (Figure 3). In the protein structure of GmSAP3, α-helix accounted for 18.24%, extended strand 27.06%, and random coil 54.71%.

Figure 2 Phosphorylation sites prediction of GmSAP3 protein

Figure 3 New ICT based fertility management model in private dairy farm India as well as abroad

1.2 Sequence alignment and evolution analysis of soybean GmSAP3 protein

NCBI website predicts that GmSAP3 protein has two conserved zinc finger protein domains, which are zf-A20 of 16-39AA and ZnF_AN1 domain of 118-148AA respectively. The amino acid sequences of soybean GmSAP3 were compared with those of Arabidopsis, kidney bean, rice, corn, cotton and sorghum (Figure 4). It was found that the amino acid sequences of SAP in different plants were relatively conservative in the two domains.

Figure 4 Multiple-sequence alignment of GmSAP3 protein sequence with SAP protein in other plant Note: ZmSAP: Zea mays (NP001358696.1); SbSAP4: Sorghum bicolor (XP021315499.1); OsSAP8: Oryza sativa (XP 015643189.1); GhSAP8: Gossypium hirsutum (XP_016671517.1); PvSAP: Phaseolus vulgaris (XP007145644.1); GmSAP3: Glycine max (Glyma.03G140500); GmSAP26: Glycine max (NP001235357.2); MtSAP8: Medicago truncatula (XP024632562.1); GmSAP13: Glycine max (NP001236983.1); AtSAP2: Arabidopsis thaliana (NP001077694.1)

In order to study the evolutionary relationship between soybean GmSAP3 and SAP of other plants, the sequence alignment of different plant SAP proteins from soybean GmSAP3 and NCBI databases was carried out by using MEGA X software, and the evolutionary tree was constructed. The results of evolutionary analysis showed that GmSAP3 and GmSAP26 are closely related, followed by Phaseolus vulgaris (PvSAP); SAP proteins of monocotyledonous plants such as maize, sorghum and rice clustered into one branch; Arabidopsis AtSAP2 has a long evolutionary distance from other SAP proteins (Figure 5).

Figure 5 Phylogenetic analysis of GmSAP3 and SAPs from other species

1.3 Tissue expression analysis of soybean GmSAP3

RT-PCR was used to analyze the expression patterns of GmSAP3 gene in soybean roots, stems, leaves, flowers and seeds at different developmental stages. GmSAP3 gene is expressed in different tissues of soybean (Figure 6), in which the expression is relatively high in roots and leaves, followed by flowers and stems (Figure 6A); The overall expression of GmSAP3 gene in seeds at different development stages was low. With the increase of seed development stage after anthesis, the expression of GmSAP3 showed an upward trend, with the highest expression in seeds 50 days after anthesis (Figure 6B).

Figure 6 Expression analysis of GmSAP3 in different tissues of soybean Note: DAF: Days after flowering

1.4 Expression analysis of soybean GmSAP3 gene under temperature stress

In order to study the response characteristics of GmSAP3 gene to temperature stress, the expression of GmSAP3 gene in soybean leaves under 40℃ high temperature treatment and 8℃ low temperature treatment for 0 h, 2 h, 4 h and 8 h was detected respectively, and the different treatment times of soybean leaves under 28℃ were used as the control (Figure 7). After high temperature (40℃) and low temperature (8℃), the expression of GmSAP3 gene was significantly higher than that of the control. When treated with high temperature (40℃) for 2 hours, the expression of GmSAP3 gene was weakly up-regulated. With the extension of treatment time, the expression of GmSAP3 showed an obvious upward trend, and reached the highest level after 8 hours of treatment; After low temperature (8℃), the expression of GmSAP3 was slightly higher than that of the control (28℃).

Figure 7 Express analysis of GmSAP3 in soybean leaves under temperature stresses Note: ** indicates that p<0.01

2 Discussion

SAP gene widely exists in plants and participates in a variety of stress responses such as high temperature, cold injury, drought and metal damage (Dixit et al., 2018; Lai et al., 2020). In this study, the soybean GmSAP3 gene was cloned, which is similar to the sap gene family members of Arabidopsis and rice (such as OsSAP1 and ATSAP4) (Vij and Tyagi, 2006). The gene has only one exon and no intron. Ubiquitin pathway is widely involved in plant hormone signal transduction and plays an important role in embryonic development, hormone response and aging. The A20 domain of human A20 protein and Arabidopsis atsap5 protein has E3 ligase and ubiquitin binding activity (Kang et al., 2011). GmSAP3 protein has zf-A20 and ZnF_AN1 conservative domain (Figure 4) is speculated to have similar functions. The expression analysis of soybean SAP tissues showed that GmSAP3 gene was mainly expressed in soybean roots and leaves, but relatively low in flowers, stems and seeds. With the gradual increase of GmSAP3 gene expression during seed development (Figure 6), it is speculated that GmSAP3 gene may have important functions in different soybean tissues.

The growth and development of plants depend on the appropriate ambient temperature. Under high or low temperature and other stress environments, plants will show characteristic stress responses, such as changes in cell membrane structure, stomatal conductance, photosynthesis and expression of related genes, so as to produce a protective mechanism for themselves (Yuan et al., 2018, Jiang et al., 2020). It was found that some SAP genes were induced by temperature stress. For example, OsiSAP8 (Kanneganti and Gupta, 2008) was up-regulated after high and low temperature treatment. This study found that the gene expression of GmSAP3 was slightly up-regulated after 2 h of high temperature treatment, and then the expression was significantly higher than that of the control (Figure 7), indicating that the expression of GmSAP3 was induced by high temperature stress, which was consistent with the results of previous studies. After 2 h, 4 h and 8 h of low temperature treatment, the expression level of GmSAP3 was slightly higher than that of the control, suggesting that GmSAP3 may be induced by low temperature. In addition, there were some changes in the expression of GmSAP3 in soybean leaves at different time points of 0 h, 2 h, 4 h and 8 h under the normal temperature of 28℃. Previous studies have reported that there are circadian rhythm changes in grape SAP gene (Ding et al., 2019). The author analyzed the 1 500 bp promoter sequence upstream of the start codon of GmSAP3 gene with the help of plantcare website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), and found that there was a biological clock element, suggesting that there may be circadian rhythm changes in GmSAP3 gene. In this study, soybean GmSAP3 gene was cloned and analyzed for its expression. It was found that it responded to high temperature stress. This result provides a theoretical support for the subsequent study of gene function.

3 Materials and Methods

3.1 Test materials

The test material was Shangdou 1201, which was provided by the Soybean Research Institute of Shangqiu Academy of Agricultural and Forestry Sciences. Shangdou 1201 was planted in the field. The roots, stems, leaves and flowers of normal soybean plants were taken at the full flowering stage, and the seeds of different development stages were taken at the pod and grain filling stage. The samples were frozen with liquid nitrogen and stored in a -80℃ refrigerator for soybean RNA extraction, gene cloning and tissue expression pattern analysis.

3.2 Cloning of GmSAP3 gene

On the phytozome website (https://phytozome.jgi.doe.gov/pz/portal.html#) download the soybean GmSAP3 (Glyma.03G140500) gene sequence, and use Primer Premier 5.0 software to design primers. Using soybean leaf cDNA as template, PCR amplification was carried out with upstream and downstream primers ((F: 5’-CGCGGATCCATGGAGTCTCACGATGAGAC-3’, R: 5’-CCGGAGCTCCTAGATTTTGTCAAGCTTATCTG-3’, the underline represents the digestion sites Bam HI and Sac I respectively). The high fidelity enzyme Phanta^@ Max Super-Fidelity DNA Polymerase (P505) from Vazyme Biotech Co., Ltd. was used, and the reaction system was 50 μL, including 25 μL 2×Phanta Max Buffer, 1 μL 10 mmol/L dNTP Mix, 10 μmol/L of upstream and downstream primers 2 μL、cDNA 2 μL. Finally, ddH₂O is supplemented to 50 μL. PCR amplification procedure: 95℃ for 5 min; 95℃ for 30 s, 50℃ for 30 s, 72℃ for 1 min, 32 cycles; 72℃ for 5 min. After the PCR product was cut and recovered, it was connected with pMD19-T vector, and then transformed into E. coli DH5α competent cells were screened and sequenced.

3.3 Bioinformatics analysis

Physical and chemical properties of GmSAP3 protein was analyzed by ProtParam tool (https://web.expasy.org/protparam/); Using NetPhos 3.1 Server (http://www.cbs.dtu.dk/services/NetPhos/) to analyze the protein phosphorylation site; At SOPMA website (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_gor4.html), the secondary structure of GmSAP3 protein was analyzed; Using ProtComp 9.0 (http://www.softberry.com/berry.phtml) to predict the protein subcellular localization. MEGA X (Kumar et al., 2018) was used to conduct multiple alignment of protein sequences, draw the evolution tree, and use the adjacency method for analysis. The main parameters are as follows: p-distance, Bootstrap, 1000 repetitions, Pairwise deletion.

3.4 Temperature stress treatment

Full soybean seeds were seeded in a pot, cultured in an artificial climate chamber with vermiculite as the substrate, exposed to light for 16 h, dark for 8 hours, 28℃ in the day, 24℃ at night, and 70% humidity. When the first pair of true leaves are fully expanded, carry out temperature stress treatment, select soybean seedlings with the same growth, and transfer them to the pre-set temperature of 40℃ (high temperature stress), 28℃ (control) and 8℃ (low temperature stress) light incubators respectively. After treatment for 0 h, 2 h, 4 h and 8 h, respectively, take soybean leaves and put them in 2 mL cryopreservation tubes. After quick freezing with liquid nitrogen, store them in -80℃ ultra-low temperature refrigerator for standby.

3.5 Analysis of GmSAP3 gene expression in Soybean

Total RNA was extracted from different tissues of soybean and treated with temperature stress. CDNA was synthesized by reverse transcription and RT-PCR. A specific primer for GmSAP3 fluorescent quantitative PCR was designed, RT-F: 5’-CAGCATCGTCGGTTGAAAA-3’, RT-R: 5’-ACAGTCTTGACCTCCACAG-3’, Tubulin gene (GenBank: AY907703.1) was used as internal parameter, and the primer sequence was: Tubulin-F: 5’-GGAGTTCACAGAGGCAGAG-3’, Tubulin-R: 5’-CACTTACGCATCACATAGC-3’ reaction system, including 10 μL 2×Master mix, 0.5 mmol/L upstream and downstream primers 2.5 μL，5 μL cDNA (the cDNA obtained by reverse transcription was diluted 10 times with ddH₂O). The instrument used for RT-PCR is QuantStudio 3, and the reaction procedure was as follows: 95℃ for 5 min, 95℃ for 10 sec, 60℃ for 1 min, 40 cycles; 3 biological repetitions, 3 technical repetitions, the expression of GmSAP3 gene was calculated by 2^-ΔΔCt method (Livak and Schmittgen, 2001).

Authors’ contributions

ZJY and ZSL are the executors of the experimental design and research; ZJY, ZSL, CMN and XXJ completed the data analysis and the writing of the first draft of the paper; WPX participated in the experimental design; HZW is the designer and principal of the project, directing the writing and revision of the thesis. All authors read and approved the final manuscript.

Acknowledgements

This research is jointly funded by Henan Science and Technology Research Project (192102110024), Henan Graduate Education Reform and Quality Improvement Project (YDH[2018]No.23), Henan Institute of Science and Technology High-level Talent Scientific Research Launch Project (201010617004) and Henan University Key Scientific Research Project Plan Support (18B210004).

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