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 in 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 has the ability to catalyze the synthesis of UDP-glucose. In order 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.

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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, and the specific performance was: the content of echinacoside in different tissues was haustorium>underground part>>aerial part; among them, the content of echinacoside in haustorium was 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 haustorium was the highest, 1.5 times that of the aboveground part, and the expression level in the underground part was significantly higher than that in the aboveground part, which is consistent with the accumulation pattern of phenylethanol glycosides represented by echinacoside in different parts of Cistanche tubulosa.

Figure 4 Relative expression levels of CtSus in different parts of C. tubulosa and in 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

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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 suspension cells. From 3 to 9 days after induction, the echinaceaside content increased significantly. From 12th to 15th day, the growth rate of echinaceaside content slowed down and reached the maximum value on the 15th day. Then, as the culture time increased, the echinaceaside content increased significantly. The content of fructoside gradually decreased [24]. On the basis of 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, and 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 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. , therefore 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 the accumulation of important secondary metabolites.

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5 Three-dimensional structure study 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).

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 has a good overlap 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: eu294, Gly301, Met576, Arg578, Lys583, Gln646, Asn652, Leu677, Thr678, and Glu681.

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