Cloning of GsTPS9 Gene from Glycine soja and Study on Its Responses to Stresses

Ding Xiao-dong, Wang Di, and Xiao Jia-lei

Key Laboratory of Agricultural Biological Functional Genes, Northeast Agricultural University, Harbin 150030, China

Abstract: Trehalose synthase is an important functional enzyme in the synthesis of trehalose in organisms and also participates in plant stress-resistant physiological processes. The transcriptomic study showed that a trehalose-6-phosphate synthase gene was responsive to salt and alkaline stresses in Glycine soja. To dissect the molecular mechanisms of this enzyme in plant responses to stresses, the PCR technique was used to clone a trehalose-6-phosphate synthase gene from Glycine soja and it was designated as the GsTPS9. The full-length cDNA of this gene was 2 583bp which encoded 861 amino acids. The sequence and structure analyses indicated that the GsTPS9 had high homology with Glycine max GmTPS9. The qRT-PCR analysis revealed that the GsTPS9 gene was expressed in Glycine soja roots, stems and leaves, and the highest expression level was in roots; the GsTPS9 gene had different responses under the stresses of NaCl, NaHCO3, PEG6000, ABA, MeJA and SA. This study laid the foundation for revealing the mechanism of the TPS in plant signal transduction pathways.

Key words: Glycine soja, GsTPS9, gene cloning, stress

Introduction

Glycine maxhas been domesticated for thousands of years and is an important crop. Through longterm preferential selection, cultivatedGlycine maxhas lost key genes that are important for adapting to environmental challenges, and has lower genetic diversity than that ofGlycine soja(Songet al., 2019).Glycine sojais a highly adaptable herb that grows well in saline soil and cold regions, and is aGlycine maxrelative species with strong stresses tolerance (Moonet al., 2010).Glycine sojahas become a model species for studying the resistance of plants to stress, such as salt, alkali, cold and drought. It is a high level resource for mining the benefits of related genes for agricultural crop breeding (Davidet al., 2006). As a non-reducing disaccharide, trehalose has a very stable structure, which can help biological cells maintain the activity of nucleic acids, proteins and biofilms under unfavorable factors, and protect the structure of cells (Gabrielet al., 2009; Yanet al., 2015). Therefore, trehalose can increase the stress resistance of plants, and it is also used as an edsignal substance to regulate plant growth and development (Olivieret al., 2010). At present, people's researches on trehalose are mainly through functional analyses of genes related to the trehalose synthesis pathway, exploring how these genes regulate the metabolism of trehalose synthesis pathway, and then regulate the content of the trehalose in organisms (Avonceet al., 2006). Studies have shown that there are three main ways to synthesize trehalose in organisms (Yonghaket al., 2018). In other organisms, such as bacteria, the trehalose can be synthesized by a variety of pathways, but in higher plants, the TPS-TPP pathway is the only pathway for trehalose biosynthesis (Blazquezet al., 1998). This path is divided into two steps: first, trehalose-6-phosphate synthase (TPS) catalyzes glucose-6-phosphate and UDP-glucose to form trehalose-6-phosphate (T6P). Then, trehalose-6-phosphate phosphatase (TPP) dephosphorylates the T6P to form the final product trehalose (Vogelet al., 1998). The TPS-TPP pathway is first discovered inE. coli, and theOtsAgene encoding the TPS protein and theOtsBgene encoding the TPP proteins are cloned. Subsequently, inSaccharomyces cerevisiae, theTPS1 gene encoding the TPS proteins, and theTPS2 gene encoding the TPP protein are cloned (Walteret al., 1998). Trehalose in plants is first found inSelaginella(Zentellaet al., 1999).

As an important enzyme in trehalose biosynthesis, the TPS has a close relationship with the contents of trehalose that play an important role in regulating growth and development (Garget al., 2002). In the past, theTPSgene in bacteria and fungi was mainly studied, and theTPSgene in plants was seldom studied (Chenet al., 2018). In particular, theTPSgene inGlycine sojahas not been reported, and its function is largely unknown. Therefore, it is of great significance to explore the expression ofGlycine sojatrehalose-6-phosphate synthase gene and verify its function. In this study, theGsTPS9 gene was cloned fromGlycine sojaand analyzed by bioinformatics. Through the qRT-PCR, it was found that theGsTPS9 had a clear response to abiotic stress. This was useful for the indepth study of theTPSgene function and revealed the unknown new signal transduction pathway of the TPS. Laying a foundation was of extremely important theoretical and practical scientific significance for the development of high-quality breeding resources.

Materials and Methods

Plant materials

Glycine sojaseeds (line G07256) with full grains were selected and relieved the dormant state of seed coat (Liet al., 2006).Glycine sojaseeds were treated with 98% concentrated sulfuric acid for 10 min, rinsed with distilled water five times, and placed on two layers. In a petri dish moistened with filter paper, incubated in an artificial climate box at 25℃. When the roots were about 1 cm, they were transferred to Hoagland liquid medium to keep the roots in a moist state.

Took 4-6 weeks oldGlycine sojaroots, stems and leaves, froze them in liquid nitrogen and placed in a refrigerator at -80℃ for gene cloning and tissue expression analysis. TookGlycine sojaplants with the same growth and soaked their roots in 100 mmol ? L-1NaCl, 10 mmol ? L-1NaHCO3, 20% PEG6000, 10 μmmol ? L-1ABA, 100 μmmol ? L-1MeJA and 100 μmmol ? L-1SA solutions, respectively. Samples were taken at 0, 3, 6 and 12 h after treatment for analysis of gene expression patterns under stresses.

Extraction of the total RNA and cloning of GsTPS9 gene

The total RNA ofGlycine sojawas extracted according to the steps in the plant RNA extraction kit instructions (TransGen, China). Then, the total RNA was reversed transcribed into cDNA. Used NCBI (https://www.ncbi.nlm.nih.gov/) to query the CDS full-length sequence information of theGsTPS9 gene. Using sequence as a template, PCR primers were designed with Primer 5.0 software: GsTPS9-F: 5'-ATGGCATCAAGATCATAT GTTAATCTAC-3'; GsTPS9-R: 5'-TCAAACCGTGC TCTCAAAAGAA-3'. The PCR reaction system was PrimeSTAR Max Premix (2×)* (TaKaRa, Japan) 25 μL,the upstream primer 2 μL, the downstream primer 2 μL, template ofGlycine sojacDNA 1 μL and ddH2O 20 μL. The PCR amplification program was as the followings: 98℃ denaturation 10 s, 55℃ annealing 5 s, extending at 72℃ for 15 s, 35 cycles, and carried out the PCR amplification of the target fragment. The PCR product reaction solution was subjected to agarose gel electrophoresis, and the target band was collected by gel recovery kit (TransGen, China). The recycled product was sent to the company (Jilin Kumei Biotechnology Ltd.) for sequencing.

Bioinformatics analysis

The predicted physicochemical properties of the GsTPS9 protein were used ProtParam (http://web.expasy.org/protparam/) online website. Utilized SMART: main page (http://smart.embl-heidelberg.de/smart/set_mode.cgi?GENOMIC=1) online website to analyze the conserved domains of the GsTPS9 protein. The SOPMA software was used to analyze and predict the secondary structure of the GsTPS9 protein. Made use of the Swiss model (http://swissmodel.expasy.org/) online website to predict the tertiary structure of the GsTPS9 protein. Used the TMpred (https://embnet.vital-it.ch/software/TMPRED_form.html) website to predict the transmembrane structure of GsTPS9. SignalP-5.0 (http://www.cbs.dtu.dk/services/SignalP/) online tool was used to predict the probability of signal peptide in the GsTPS9 protein. Utilized NetPhos3.1 Server (http://www.cbs.dtu.dk/services/NetPhos/) online tool to predict the phosphorylation site of the protein. Cell-PLoc2.0 package (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/) online website was used to predict the subcellular location of the GsTPS9 protein. NCBI (https://www.ncbi.nlm.nih.gov/) database was used to perform BLAST sequence alignment of the GsTPS9 protein, output the protein sequence in Fasta format, manually corrected the result, and made use of the obtained multiple sequence file for the construction of the phylogenetic tree. Utilized neighbor-joining in the MEGA7 software to generate a phylogenetic tree. DownloadedGlycine sojawhole genome protein sequence from Wildsoydb (http://www.wildsoydb.org/Gsoja_W05/) database, and usedArabidopsis thalianaAtKIN10 (NP-850488.1) sequence as a reference for members of the GsTPS family inGlycine soja. To retrieve all theGlycine sojagenomes potential GsTPS family members, the BLAST2.7.1 (ftp://ftp.ncbi.nlm.nih.gov/blast/executable/blast/2.7.1/) online website was performed a local search on the Wildsoydb database based on the BLASTP algorithm.

Analysis of cis-acting elements of promoter

The promoter region of the GsTPS9 was analyzed by the NCBI, and the base 1 681 bp upstream of the start codon of the GsTPS9 was selected as its promoter. Plant CARE (http://bioinformatics.psb.ugent.be/web tools/plantcare/html/?tdsourcetag=s_pcqq_aiomsg) online website was used to conduct a predictive analysis of thecis-acting elements of its promoter.

Fluorescence quantitative qRT-PCR analysis

Glycine sojaGAPDH was as the internal reference, the primers qRT-GsTPS9-F: 5'-AGGAACTGCACAGAT GGATAAA-3'; qRT-GsTPS9-R: 5'-CCACTTAGAGA AGGAGAACAACC-3' were designed. SYBR Green method was used for the qRT-PCR amplification. The reaction system was TransStart Top Green qPCR SuperMix 10 μL, the upstream and downstream primers each 0.4 μL, the 10-fold diluted cDNA 1 μL and ddH2O 8.2 μL. The amplification program was 95℃ pre-denaturation 30 s; denaturation at 95℃ for 5 s, annealing at 60℃ for30 s, a total of 35 cycles.

Statistical analysis

All the experiments in this study were repeated at least three times, and the data were expressed as Mean±SD. The relative quantitative analysis of gene expression was carried out using the method of 2-ΔΔCTrelative quantification. The GraphPad Prism 8.00 software was used for graphing. The IBMSPSS software was used to analyze the significance of the difference.

Results

GsTPS9 target fragment amplification

TakingGlycine sojaas the material, the total RNA was extracted by plant RNA extraction kit. Then, the total RNA was reversed transcribed into cDNA. Used theGlycine sojacDNA as the template and designed specific primers to amplify target fragments by the PCR technology. The PCR product reaction solution was subjected to agarose gel electrophoresis. The target fragment of 2 583 bp was obtained (Fig. 1).Collected the target band, according to the gel recovery purification kit, and named it as the GsTPS9 after the sequencing and comparison were correct.

Fig. 1 PCR amplification results of GsTPS9 geneM, DNA Marker DL2000 Plus; 1, PCR product of GsTPS9 gene.

Prediction of GsTPS9 physical and chemical properties

Utilized ProtParam online tool analysis, the relative molecular mass of the GsTPS9 protein was 97 434.98 ku, the total number of atoms was 13 698, the isoelectric point was 7.47, the molecular formula was C4369H6845N1175O1268S41, and the total number of positively charged residues (Arg+Lys) was 109. The total number of negatively charged residues (Asp+Glu) was 103, the aliphatic amino acid index was 90.74, the instability coefficient was 46.85, and the total average hydrophilicity was -0.216. It was speculated that the GsTPS9 protein might be an unstable hydrophilic protein.

Prediction of GsTPS9 structure

Used the SMART: Main page online website to analyze the conserved domain of the GsTPS9 protein, and it was found that there might be a low-complexity region at amino acids 42-57. The secondary structure of the GsTPS9 protein was analyzed by the SOPMA software. The GsTPS9 protein had 371α-helix, accounting for 43.14% of the secondary structure; 143 extended strands, accounting for 16.63% of the secondary structure;β-turn, accounting for 5.23% of the secondary structure; and 301 random regions, accounting for 35% of the secondary structure (Fig. 2). The tertiary structure of the GsTPS9 was predicted by the Swiss model online tool (Fig. 3).

Fig. 2 Prediction of GsTPS9 secondary structure

Fig. 3 Prediction of GsTPS9 tertiary structure

Prediction of GsTPS9 transmembrane structure, phosphorylation site and subcellular localization

The TMpred online website analysis was used to predict the transmembrane region and direction of the GsTPS9 protein. The GsTPS9 protein didn't have a transmembrane structure (Fig. 4), and it was inferred that the GsTPS9 might be a non-transmembrane protein. The probability of the GsTPS9 protein with signal peptide was predicted to be 0.0037, and it was speculated that the GsTPS9 was a non-secreted protein. The online tool of the NetPhos3.1 Server was used to predict the phosphorylation sites of the GsTPS9 protein. It showed that there were 79 phosphorylation sites, of which 50 sites might be phosphorylated by serine, 17 sites phosphorylated by Threonine, and 12 sites phosphorylated by tyrosine (Fig. 5). It was speculated that the GsTPS9 protein underwent phosphorylation modification mainly with serine. Using an online website to predict the subcellular location of the GsTPS9 protein, it was found that it might be located in the chloroplast of the cytoplasm.

Fig. 4 Prediction of GsTPS9 transmembrane structure

Fig. 5 Prediction of GsTPS9 phosphorylation site

Phylogenetic analysis of GsTPS9

Through the NCBI online website BLAST GsTPS9 homologous protein, the results showed that the GsTPS9 had high homology with the TPS protein in GmTPS9 (Glycine max, XP_003519705.1), CcTPS9 (Cajanus cajan, XP_020216429.1), ApTPS9 (Abrus precatorius, XP_027359678.1), MpTPS (MucunapruriRDX94536.1), AtTPS9 (Arabidopsis thaliana, NP_173799.1), SoTPS9 (Smyzygium oleosu, XP_030468899.1), StTPS9 (Senna tora, KAF7829374.1) and SlTPS (Solanum lycopersicum, BAF98176.1). Their protein sequences were used to construct a phylogenetic tree (Fig. 6). Analyzing the genetic relationship of the TPS protein among different species, it was found that the GsTPS9 was most closely the related to the GmTPS9, and was closely related to the CcTPS9 and the ApTPS9.

Analysis of GsTPS9 family members

Through BLASTP search onGlycine sojagenome, all the potential GsTPS family members inGlycine sojagenome were retrieved. Their protein sequences were used to construct a phylogenetic tree (Fig. 7). A total of 27 members of the trehalose-6-phosphate synthase (TPS) family were identified inGlycine soja. The analysis found that the functions of most TPS family members inGlycine sojawere still unknown, which provided new possibilities for the research direction.

Analysis of promoter cis-acting elements

To explore the regulatory molecular mechanism of theGsTPS9 gene expression level, thecis-acting elements in its promoter region were predicted and analyzed (Table 1). All the components obtained from the analysis mainly included: CAAT-box (majority), TATA-box (majority), environmental stresses transcription factor MYB (majority), multiple light-responsive elements (GA-motif, GT1-motif and TCT-motif), stresses response element (TGACG-motif), hormone response element (CGTCA-motif) and other elements. Analysis of theGsTPS9 gene expression might be regulated by photoperiod and light intensity, and might also be affected by the external environment and change.

Fig. 6 Phylogenetic analysis of GsTPS9 protein and other plants TPS proteins

Fig. 7 Evolutionary analysis of GsTPS family members

Table 1 Analysis of cis-acting elements of GsTPS9 gene promoter

Analysis of temporal and spatial expression pattern of GsTPS9 gene in Glycine soja

To study the spatiotemporal expression pattern of theGsTPS9 gene inGlycine soja, the relative expression levels of theGsTPS9 gene in the three different organs of roots, stems and leaves were detected (Fig. 8). Realtime fluorescent quantitative PCR showed that theGsTPS9 gene was expressed in the roots, stems and leaves ofGlycine soja, but the expression level was the highest in the roots and the lowest in the leaves. It was speculated that theGsTPS9 gene might respond to abiotic stresses.

Response of GsTPS9 to stresses

Through previous predictions ofcis-acting elements in the promoter of theGsTPS9 gene, it was found that there had a variety of response elements. It indicated that theGsTPS9 gene might respond to conditions, such as abiotic stresses. Therefore, tookGlycine sojaseedlings that had grown in Hoagland nutrient solution for 21 days and grew consistently. They were treated in Hoagland containing NaCl, NaHCO3, PEG6000, ABA, SA and MeJA. Samples were taken at 0, 3, 6, 9 and 12 h, and the relative expression of theGsTPS9 gene was detected by qRT-PCR (Fig. 9).

Fig. 8 Relative expression levels of GsTPS9 gene in different tissues of Glycine sojaExperiment is repeated three times, the data are expressed as Mean±SD, and error bar indicates SE (t-test, different lowercase letters are used to indicate significant difference P＜0.05).

Fig. 9 Relative expression of GsTPS9 gene in response to stressesExperiment is repeated three times, data are expressed as Mean±SD, and error bar indicates SE (t-test, different lowercase letters are used to indicate significant difference P＜0.05).

It was found that the treatments of NaCl, NaHCO3, PEG6000, ABA, SA and MeJA all caused changes in the transcription level of theGsTPS9 gene. It was worth mentioning that after NaHCO3and PEG6000 treatment, the relative expression of theGsTPS9 gene was significantly increased. It was speculated that theGsTPS9 gene might be played an important role in plant response to alkali and drought stresses.

Discussion

Unfavorable environmental factors seriously affect the growth and development of crops and cause huge economic losses. Therefore, understanding the molecular and physiological mechanisms of plant response to stresses and mining key regulatory genes in stress response is of great significance for improving crop resistance. Trehalose plays an important role in maintaining plant osmotic pressure, biofilm structure and regulating signal transduction. Therefore, trehalose can play an important role in plant response to biotic and abiotic stresses and in regulating plant development. The TPS is an important functional enzyme in the trehalose biosynthesis pathway. In this paper, theGsTPS9 gene was cloned for the first time, and thecis-acting elements in the catalytic region of theGsTPS9 gene were predicted. All the elements obtained by the analysis mainly included some and a variety of light-responsive elements (GA-motif, GT1-motif and TCT-motif), environmental stress-related transcription factor MYB, stresses response element (TGACG-motif), hormone response element (CGTCAmotif) and other elements. Thesecis-acting elements might be involved in light response, hormone response and environmental stress response. TheGsTPS9 gene expression might be regulated by light, hormones and environmental stresses. After theGlycine sojaseedlings were treated with abiotic stresses, for example, salt, alkali, drought, ABA, SA and MeJA, the relative expression of theGsTPS9 gene all responded to varying degrees.

Earlier studies found that the genes for trehalose synthaseOtsAandOtsBinE. coliwere co-expressed, and then successfully transferred intoOryza sativaL. to obtain transgenic plants (Olivieret al., 2010). The trehalose synthaseTPS1 gene in yeast was transferred intoSolanum tuberosumunder the drive of a specific promoter, and it was found that the drought resistance ofSolanum tuberosumwas improved. The trehalose synthaseTPSgene of yeast and the trehalose synthaseOstgene ofE. coliwere transformed intoNicotiana tabacumL. for genetic expression, and it was found that the ability of transgenic plants to tolerate drought was improved (Olivieret al., 2010). TheTPSgene of the basidiomyceteGrifola frondosawas transformed into sugarcane by theAgrobacterium-mediated method, and it was found that the resistance of transgenicSaccharum officinarumL. to osmotic stresses was improved (Zhanget al., 2006). Overexpression of trehalose-6-phosphate synthaseOtsAin bacteria could also increase the accumulation of oil in plant seeds and tissues (Zhaiet al., 2021). In recent years, a large number of exploratory studies had been conducted on theTPSgenes in plants. TheTPSgenes had been cloned fromArabidopsis thaliana,Oryza sativaL.,Manihot esculenta, Maize andPrunus mume(Delorgeet al., 2015; Liet al., 2015; Dinget al., 2017; Henryet al., 2014). Studies had found that theTPSin plants could affect the content of trehalose and play an important role in regulating stresses, thereby affecting plant growth and development. For example, the content of trehalose in theNtTPSplant of transgenicNicotiana tabacumunder salt stress was significantly higher than that of the experimental group and had a certain tolerance to salt stress (Matthewet al., 2021). Under dark conditions, the expression of the maizeTPSgene also occurred significantly (Henryet al., 2014); under drought stress, the trehalose content in the roots, stems and leaf tissues of theTPSgene of thePrunus mumechanged significantly (Yanget al., 2020). These studies had shown that theTPSgene could respond to biotic and abiotic stresses and improve the stress resistance of plants. These studies were of great significance for the in-depth study of theGsTPSgene and its influence on plant growth and development.

Sucrose non-fermenting 1-related protein kinase (SnRK1) is a type of Ser/Thr protein kinase that is widely found in plants. It participated in a variety of signal transduction pathways and played a role in plant resistance (Liuet al., 2019). The T6P was a signal molecule proposed in plants and acted as an inhibitor of the SnRK1 kinase (Zhanget al., 2020). Studies on wheat, potatoes and sugar beets also showed that the T6P acted as an inhibitor of the SnRK1 (Zhanget al., 2009; Debastet al., 2011; Wuet al., 2010). Other studies had shown that the AtTPS5 could be phosphorylated by the SnRK1 and further affected the T6P synthase activity in plants, and that the AtTPS5 shared 63.9% sequence identity with the GsTPS (Tianet al., 2019). The ABA was a very important plant hormone, which played a role when plants were under environmental stress (Zhanget al., 2005). The ABA signaling pathway was one of the main regulatory pathways for plants to respond to stresses. Studies had shown that the TPS5 negatively regulated the ABA signaling by mediating genes in the process ofArabidopsisseed germination and stomata closure (Tianet al., 2019). The GsTPS that interacted with the GsSnRK1 had been identified from theGlycine sojacDNA library (Songet al., 2019). What is the specific relationship between the SnRK1 and the TPS and whether they respond to stresses through the ABA pathway is still unclear. Many of the molecular mechanisms still need to be further understood and revealed.