Gene Cloning, Functional Identification, Structural And Expression Analysis Of Sucrose Synthase From Cistanche Tubulosa Ⅲ

4 Expression analysis of CtSus in different parts of Cistanche tubulosa and cell culture system under drought stress

4.1 Expression analysis of CtSus in different parts of Cistanche tubulosa

In vitro, whole-cell transformation experiments and enzymatic catalytic reaction experiments confirmed that the protein encoded by the CtSus gene can catalyze the synthesis of UDP-glucose. To further explore the correlation between this gene and the biosynthesis of glycoside compounds in Cistanche tubulosa, the expression level of this gene in different parts of Cistanche tubulosa was analyzed.

HIGH QUALITY CISTANCHE HERBA WITH 10-98% ECHINACOSIDE

Echinacoside is the most representative glycoside compound in Cistanche tubulosa, and its content can reach more than 30% of the dry weight of Cistanche tubulosa plants [23]. The research group previously measured the content of echinacoside in different parts of Cistanche tubulosa plants. Specifically, the content of echinacoside in different tissues is as follows: haustoria>underground part>>aerial part; among them, the content of echinacoside in haustoria is the highest.

Real-time fluorescence quantitative PCR was performed using cDNA from different parts of Cistanche tubulosa as templates, and the results were analyzed by the 2–ΔΔCT method and differential analysis was performed. The results are shown in Figure 4A. The expression level of the CtSus gene in the haustoria was the highest, 1.5 times that of the aerial part, and the expression level in the underground part was significantly higher than that in the aerial part, which is consistent with the accumulation pattern of phenylethanoid glycosides represented by echinacoside in different parts of Cistanche tubulosa.

Figure 4 Relative expression levels of CtSus in different parts of C. tubulosa and suspension cells treated by PEG6000. A: Relative expression level of CtSus in different parts of C. tubulosa; B: Relative expression level of CtSus in C. tubulosa suspension cells treated by PEG6000 at different time points. n=3, 𝑥̅± s. *P ＜ 0.05, ***P ＜ 0.001

4.2 Expression analysis of CtSus in Cistanche deserticola suspension cells under drought stress conditions

Preliminary research on the project showed that drought stress induced by PEG6000 can significantly increase the accumulation of phenylethanol glycosides in Cistanche deserticola suspension cells. From 3 to 9 days after induction, the echinaceaside content increased significantly. From the 12th to the 15th day, the growth rate of echinacoside content slowed down and reached the maximum value on the 15th day. Then, as the culture time increased, the echinacoside content increased significantly. The content of fructoside gradually decreased [24]. Based on this research, this paper used the cDNA of untreated Cistanche deserticola suspension cells and PEG6000-induced Cistanche deserticola suspension cells as templates to conduct real-time fluorescence quantitative PCR detection to examine the CtSus gene in Cistanche deserticola suspension cells under drought stress conditions. Changes in expression levels. The results are shown in Figure 4B. In Cistanche deserticola suspension cells induced by PEG6000, the expression of CtSus increased significantly on the 6th day after induction, reached the highest value on the 9th day, and then fell back to the same level as the control. Groups of the same level. The above results show that drought stress can significantly increase the expression of the CtSus gene in the Cistanche deserticola suspension cell line, which is consistent with the accumulation pattern of echinaceaside under drought stress. However, the peak expression of the CtSus gene appears earlier than the peak of echinaceaside content, because the active glycosyl donor synthesized by CtSus catalysis is an important precursor required for the multi-step glycosylation reaction in the subsequent biosynthetic pathway of echinaceaside. , it is speculated that after being subjected to drought stress, organisms will preferentially mobilize genes related to primary metabolism to achieve the accumulation of active donors, and then achieve important secondary metabolism accumulation of metabolic products.

5 Study on the three-dimensional structure of CtSus protein and analysis of key active sites

Based on the function of CtSus in catalyzing the production of glycosyl donor UDP-glucose, the structural basis of CtSus catalytic activity was further studied. The online tool SOPMA was used to predict the secondary structure of the protein. The results showed that the secondary structure of CtSus contained 55.28% α-helices, 25.47% random coils, 12.80% extended strands, and 6.46% β-turns (Figure 5A), indicating that α-helices are the most important secondary structural units in CtSus protein, followed by random coils, which also account for a large proportion of the protein. Extended strands and β-turns are distributed throughout the protein. According to existing studies, sucrose synthase usually exists in the form of a tetramer, which is considered to be its active form. Therefore, this paper further used AlphaFold2 to predict the structure of CtSus protein and obtained its three-dimensional structure of protein tetramers. PDB (Protein Data Bank) database comparison found that the sequence similarity between Arabidopsis thaliana sucrose synthase AtSus1 (PDBID 3S28) and CtSus can reach 77.93%. The predicted CtSus structure was compared with the AtSus1 three-dimensional structure, and the root mean square deviation (RMSD) value after protein superposition was 1.11 Å, indicating that the spatial structures of the two are highly consistent (Figure 5B).

Figure 5 Structural investigation of CtSus. A: Predicted secondary structure of CtSus using SOPMA. Blue: α helix; Purple: Random coil; Red: Extended strand; Green: β sheet. B: Three-dimensional structure alignment of AtSus1 (in blue color) and CtSus (in green color). Both were shown as a tetramer. C: Key residues in the substrate binding pocket of AtSus1 (in blue color) and CtSus (in green color with labeled residues); D: Binding conformations alignment of UDP and fructose in AtSus1 (in blue color) and CtSus (in green color); E: Interactions between UDP and CtSus shown in 2D diagram analyzed by Discovery Studio Client

The reported protein-ligand crystal complex structure of Arabidopsis AtSus1 with UDP and fructose (PDBID3S29) was used as a template [16] to analyze the binding mode of CtSus with UDP and fructose. The molecular docking results are shown in Figure 5C. It can be observed that the substrate binding pockets of AtSus1 and CtSus are very similar in terms of amino acid type, spatial distribution, and configuration, and the overlap is high, proving that the sequence of sucrose synthase is highly conserved in plants. The conformations of the two ligands, UDP and fructose, in the protein substrate binding pocket, are shown in Figure 5D. The molecular docking most advantageous conformation of UDP and CtSus overlaps well with the conformation of UDP in the AtSus1-UDP crystal complex, proving the accuracy of the molecular docking results. The interaction between UDP and the key amino acid residues in the protein substrate binding pocket is shown in Figure 5E. UDP and CtSus are mainly bound together by hydrogen bonds and hydrophobic interactions. The key amino acid residues in the substrate binding pocket include Leu294, Gly301, Met576, Arg578, Lys583, Gln646, Asn652, Leu677, Thr678, and Glu681.

Discussion

Glycosylation modification is one of the important means to improve the physical properties and biological activities of natural products or drug precursors. Compared with traditional chemical methods, enzymatic glycosylation modification has the advantages of mild reaction conditions, strong selectivity, and environmental friendliness. However, the glycosylation reaction of glycosyltransferase requires a large amount of UDP-sugar donors, which are expensive and difficult to obtain, resulting in the fact that glycosyltransferases cannot be widely used in industrial production. Sucrose synthase can catalyze the reversible reaction: sucrose + UDP ⇌ UDP-glucose + fructose and can form a renewable UDP-glucose cycle through a coupling reaction with glycosyltransferase. Cistanche tubulosa is rich in a variety of glycoside compounds of different structural types, represented by phenylethanol glycoside compounds, suggesting that the active glycosyl donor synthesis pathway in its body involving sucrose synthase has a strong metabolism, but the relevant sucrose synthase from Cistanche plants has not been reported. In this study, a sucrose synthase gene CtSus was cloned from Cistanche tubulosa for the first time. The protein encoded by this gene contains the conserved domain of plant sucrose synthase. By comparing the sequence of sucrose synthases from other plants, it was found that the amino acid sequence similarity between it and sucrose synthases from plants of the same order was more than 90%, indicating a high degree of sequence conservation of sucrose synthases from plants. Molecular evolution analysis showed that CtSus belongs to the dicotyledonous plant sucrose synthase branch and is most closely related to the sucrose synthase PrSus from P. ramosa of the Orobanchaceae family.

To explore the catalytic activity of CtSus, this study combined the glycosyltransferase UGT71BD1, whose activity has been previously verified by the research group, to construct a dual-plasmid co-expression system. Through whole-cell catalytic experiments, the conditions were achieved without adding additional UDP-sugar donors. Glycosylation reaction of coumarin compound cinnamon and stilbene compound resveratrol. Compared with the control group, the addition of CtSus significantly increased the conversion rate of UGT71BD1-catalyzed glycosylation reactions. On this basis, this study further constructed a CtSus recombinant expression plasmid and achieved soluble expression of the recombinant protein in E. coli. In vitro, enzymatic catalytic reactions show that in the presence of sucrose and UDP, CtSus can catalyze the generation of UDP-glucose, and after the trigger factor affinity tag contained in the recombinant protein is removed, the product of CtSus catalyzing the generation of UDP-glucose is obtained. The rate has been significantly improved. Whole-cell transformation and in vitro enzymatic catalytic reaction results confirmed the activity of CtSus as a sucrose synthase catalytic active sugar donor UDP-glucose. To further explore the correlation between the CtSus gene and the biosynthesis of glycosides in Cistanche tuberosum, the expression of CtSus in different parts of Cistanche tuberosum was analyzed by real-time fluorescence quantitative PCR experiments. The results showed that the gene was expressed in the haustoria of Cistanche tuberosum. The highest expression level. Cistanche deserticola is a parasitic plant and cannot obtain the nutrients required for growth and development through photosynthesis. Therefore, it needs to be parasitic on the roots of the host plant and rely on the host plant to obtain nutrients to maintain growth. In plants, sucrose mostly provides energy and carbon source donors [12]. However, sucrose cannot be used directly by cells and needs to be further decomposed. The haustoria is the bridge connecting Cistanche deserticola and the host plant, and plays a vital role in its growth process. crucial

[25], therefore the high expression of sucrose synthase in the haustoria of Cistanche deserticola is reasonable. The high expression of CtSus in the haustoria is also consistent with the large accumulation pattern of phenylethanoid glycosides in the haustoria. In addition, through fluorescence quantitative PCR analysis of changes in CtSus gene expression levels in Cistanche deserticola suspension cells at different time points under drought stress, it was found that drought stress can significantly increase the expression of CtSus genes in the suspension cell line, which is consistent with echinaceaside. The accumulation pattern in suspension cell lines under drought stress is consistent. The above results suggest that CtSus is involved in the biosynthetic pathway of phenylethanoid glycosides represented by echinacea in Cistanche tulipis in vivo. It is one of the many biosynthetic pathways. The first-step glycosylation reaction provides the active glycosyl donor UDP-glucose. In short, this study

The study identified a new sucrose synthase gene in Cistanche deserticola, which enabled the in vitro enzymatic synthesis of active glycosyl donors and provided new genetic elements for the construction of engineering bacteria for the biosynthesis of Cistanche deserticola glycosides.

Author contributions: Tian Weisheng was responsible for bioinformatics analysis, expression analysis, enzyme activity analysis and writing of the first draft of the CtSus gene; Yan Yaru was responsible for gene screening and cloning; Cui Xiaoxue and Huang Wenqian participated in bioinformatics analysis and expression analysis; Wang Yingxia and Zhao Saijing participated in vector construction and enzyme Activity analysis; Li Jun and Shi Shepo mainly guided the enzyme activity analysis and expression analysis; Tu Pengfei and Liu Xiao were responsible for the paper idea design, guiding experiments, and writing and revising the paper. All authors participated in revising the paper.

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