Overexpression of the ThTPS gene enhanced T. hispida salt and drought stress tolerance

Background: Trehalose is a nonreducing disaccharide with high stability and strong water absorption properties that can improve the resistance of organisms to various abiotic stresses. Trehalose-6-phosphate synthase (TPS) plays important roles in trehalose metabolism and signaling. Results: A full-length cDNA of ThTPS was cloned from Tamarix hispida . The phylogenetic tree among ThTPS and 11 AtTPS in Arabidopsis indicates that the ThTPS protein had a close evolutionary relationship with AtTPS7. However, the function of AtTPS7 has not been determined. To analyze the abiotic stress tolerance function of ThTPS , the expression patterns of ThTPS were monitored under salt and drought stress and JA, ABA and GA3 hormone stimulation in T. hispida by qRT-PCR. The results showed that ThTPS expression was clearly induced by these 5 kinds of treatments at at least one studied point. Particularly under salt stress, ThTPS was highly induced in the roots of T. hispda . Furthermore, the ThTPS gene was transiently overexpressed in T. hispida . The results of physiological indexes and staining showed that overexpression of the ThTPS gene increased T. hispida salt and drought stress tolerance. Conclusion: The ThTPS gene can respond to abiotic stress such as salt and drought, and overexpression of ThTPS gene can significantly improve salt and drought tolerance. These findings establish a foundation to better understand the response of TPS genes to abiotic stress in plants. stress. This study will establish a theoretical foundation


Background
Trehalose is a nonreducing disaccharide composed of two glucose molecules linked by α,α 1-1 glycosidic bonds [1][2]. Trehalose was first discovered in bacteria by Wiggers in 1832, and the French chemist Berthelot subsequently discovered the sugar in molasses secreted 3 by weevils in the Asia Minor Desert and named it trehalose [3]. Currently, trehalose is widely found in various living organisms, such as bacteria, yeasts, molds, edible fungi, lower plants, insects and invertebrates, as well as some higher plants [3][4][5][6].
Trehalose in the living body can increase the resistance of organisms to adverse conditions. The resistance of many species to adverse environmental conditions is directly related to the concentration of trehalose in their bodies [7][8]. Trehalose is a typical stress metabolite. When the organism grows well, it does not accumulate trehalose in the body, while when the organism is in a stressful environment (such as starvation, dryness, high temperature and high salinity), trehalose can rapidly accumulate [9][10]. These trehaloses were degraded when the adverse environment was removed. In addition, the added trehalose also has clear protective effects on active substances, such as proteins, enzymes and cell membranes [11]. In plants, trehalose is an important substance in regulating diverse processes, such as development [12][13][14][15][16], response to biotic stresses [17][18][19][20][21] and abiotic stresses [22][23][24][25][26].
The synthesis of trehalose in plants was based on the synthesis of trehalose-6-phosphate (Tre6P) from UDP-glucose and glucose-6-phosphate catalyzed by trehalose-6-phosphate synthase (TPS) and then catalyzed by trehalose-6-phosphate phosphatase (TPP) to trehalose. However, when trehalose is synthesized in plants, there are many nonphosphatase enzymes that can catalyze Tre6P into trehalose, and it can directly catalyze the dephosphorylation of Tre6P to produce trehalose without TPP [4]. However, the TPS protein has an irreplaceable role in plant trehalose synthesis, and the successful transcription and expression of the TPS gene was decisive for the synthesis of trehalose in plants.
Some TPS transgenic plants can significantly improve abiotic stress tolerance. For example, the A. thaliana TPS1 gene enhanced the osmotic, drought, desiccation and temperature stress resistance of transgenic tobacco [35]. Transformation of the yeast TPS1 gene into potato significantly improved the drought resistance of transgenic plants [36][37]. Garg et al transferred the trehalose synthesis genes (otsA and otsB) of Escherichia coli into rice and improved salt, drought and low-temperature stress resistance [38]. Jang et al. showed that transformation of the E. coli trehalase synthase gene into rice can increase trehalose accumulation and drought, high salt and cold tolerance [39]. Overexpression of the TPS1 gene sorghum enhanced tolerance to salt stress [40]. Grifola frondosa Fr. TPS gene transformation in tobacco enhanced resistance to drought and salt [41].
Tamarix hispida is a woody halophyte with developed roots and strong sprouting ability. This species has strong drought, cold, salt and alkali resistance, making it a good plant to grow in sandy soil and various degrees of salinized soil. Therefore, this halophyte is an ideal material for anti-reverse gene cloning and the study of stress resistance mechanisms. In this study, the ThTPS gene was cloned from T. hispida, and the sequence characteristics of the gene and the expression pattern after abiotic stress were analyzed. Furthermore, ThTPS was transiently overexpressed in T. hispida. The related physiological index and staining analysis were carried out and compared between the overexpression and control T. hispida under salt stress. This study will establish a theoretical foundation to further analyze the stress tolerance function of the TPS gene and use genetic engineering to improve plant stress resistance. Class II subfamily members may have a regulatory function and are regulated transcriptionally by carbon status and stress [6]. In recent years, several approaches have found that A. thaliana TPS family proteins, class II subfamily member AtTPS6, regulate plant architecture, epidermal pavement cell shape and trichome branching [14]. AtTPS5 plays a role in thermo tolerance through its interaction with the transcriptional coactivator MBF1c [45]. However, there have been no reports describing AtTPS7 function to date.

Sequence and evolution analysis of the ThTPS gene in T. hispida
Other class II TPS proteins seem to lack significant enzymatic activity, many of which are extensively regulated by hormones, light and nutrient availability at the transcriptional level [46][47][48][49][50][51][52].

Bioinformatics analysis of the ThTPS gene in T. hispida
Through searching the transcriptome data of T. hispida using "trehalose-6-phosphate synthase" as a key word, a full-length ThTPS gene sequence was obtained. Through

RNA extraction and qRT-PCR analysis
The RNA of each sample was extracted using a plant RNA extraction kit (BioTeKe corporation), and the procedure was carried out according to the kit instructions. The RNA extraction concentrations and masses were measured using a Nanovue microphotometer and 0.8% agarose gel electrophoresis. Then, the total RNA of each sample was reverse transcribed into cDNA using TransScript One-step gDNA Remvoal and cDNA Synthesis SuperMix according the instruction manual. Then, qRT-PCR was carried out using the Actin (FJ618517), α-tubulin (FJ618518) and β-tubulin (FJ618519) genes as internal controls (reference genes). The internal control and ThTPS gene primer sequences are shown in Table 1. The reaction system of qRT-PCR was SYBR Green Mix 10 μl, 1 μl of each of the forward and reverse primers (10 μmol/L), cDNA 2.0 μl, and ddH 2 O supplemented to 20 μl.
The reaction procedure was 95°C for 3 min, 95°C for 20 s, 58°C for 15 s, and 72°C for 30 s for 45 cycles. Each sample was repeated three times. qRT-PCR experiments were performed using an Opticon Monitor 2 real-time PCR machine manufactured by Böhler.

ThTPS gene cloning and plant overexpression vector construction
According to the multiple cloning site of the plant overexpression vector pROKII and the gene sequence of ThTPS, Xba I and Kpn I restriction endonuclease sites were introduced at the 5' and 3' ends of the ThTPS gene, respectively. The primers TPS-CF (5'CTAGTCTAGAATGATGTCCAGATCTTATACC3') and TPS-CR (5'CGGGGTAC CCTAAGAGGGGCTGCCGCTAC3') were used to obtain the ThTPS gene by RT-PCR amplification. Then, the digested gene and the vector fragment were ligated and transformed into E. coli competent Top10 cells by the heat shock method. After culturing at 37°C for 8-12 h, single colonies were picked for PCR verification using vector primers and gene primers. The strains with the correct fragment sizes were sent for sequencing analysis. The sequenced correct overexpression vector strain was designated pROKII-ThTPS, and the plasmid was transformed into Agrobacterium tumefaciens EHA105 to obtain an overexpression strain.

ThTPS gene transient transform into T. hispida and stress resistance analysis
According to the method of Ji et al. [43], the pROKII-ThTPS overexpression strain (OE) and the pROKII empty vector (Con) strain were transiently transformed into T. hispida. After Evans blue staining analyses were carried out after the seedlings were treated for 12 h.
Each experiment was repeated at least three times.

Declarations 13
Ethics approval and consent to participate Tamarix hispida is a woody halophyte. Therefore, no voucher specimens were prepared.
No specific permits were needed for the described experiments, and this study did not involve any endangered or protected species.

Consent for publication
Yes.

Availability of data and material
Relevant data analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no conflict of interest.  Table   Table 1 The