Gene Cloning, Functional Identification, Structural And Expression Analysis Of Sucrose Synthase From Cistanche Tubulosa Ⅱ
Sep 06, 2024
Results and analysis
1 Mining and cloning of sucrose synthase genes in Cistanche tubulosa
The amino acid sequence of the identified Arabidopsis thaliana sucrose synthase AtSus1 was used as a template[16], and sequence alignment was performed in the Cistanche tubulosa transcriptome database by local Blastp. At the same time, two sucrose synthase gene sequences with FPKM values greater than 10 were screened out in combination with transcriptome gene annotation and gene expression abundance data, which were named CtSus and CtSus1, respectively. The comparative transcriptome data of different parts of Cistanche (haustoria, underground parts and aerial parts) were further analyzed. The distribution of chemical components in different parts of Cistanche plants has been studied in depth. The results showed that glycoside compounds represented by phenylethanoid glycosides in Cistanche are mainly present in its underground parts, with the highest content in the haustoria[22]. In the early stage of this study, the content of phenylethanoid glycosides in plant materials used for comparative transcriptome sequencing of different parts was also determined, confirming that the content of phenylethanoid glycosides in different parts of Cistanche tubulosa was different in the following manner: haustorium>underground part>>aerial part.
Therefore, the expression level FPKM values of the two candidate sucrose synthase genes obtained in the initial screening in the transcriptome data of different parts of Cistanche tubulosa were normalized by Z-Score and differential analysis was performed (Figure 1A). The results showed that the CtSus gene had the highest expression level in the haustorium, and the expression level in the underground part was higher than that in the aerial part, which was consistent with the accumulation pattern of glycoside compounds in different parts of Cistanche tubulosa, while the expression level of the CtSus1 gene in the haustorium was low. Therefore, based on the above results, the CtSus gene was selected for subsequent sequence amplification, exogenous expression and functional identification. Using Cistanche tubulosa cDNA as a template, a single band product was obtained at ~2500 bp after PCR amplification (Figure 1B). The PCR product was recovered from gel and ligated to the cloning vector, and the full-length coding region of the target gene was obtained by sequencing. The length of the CtSus sequence was 2418 bp.
Figure 1 Gene exploring and cloning of CtSus from C. tubulosa and bioinformatic analysis of its encoded protein. A: Expression levels of the CtSus and CtSus1 genes in different parts of C. tubulosa normalized as Z-Scores based on their FPKM values; B: Gene amplification of CtSus; M: DNA marker; C: Conservative domain of CtSus protein predicted by SMART; D: Multiple sequence alignment of CtSus and sucrose synthases identified from other plants including Arabidopsis thaliana, Solanum tuberosum and Albuca bracteata. The sucrose synthesis domain and sugar transfer domain are represented by red and blue boxes, respectively; E: Phylogenetic analysis of CtSus and sucrose synthases from other plants; F: SDS-PAGE analysis of the heterologous expression of CtSus using pCold™ I vector in E. coli. Lane 1: Protein marker; Lane 2: Supernatant fraction; Lane 3: Precipitate fraction. The red arrow shows the CtSus protein. G: Colony PCR of a single clone containing both two recombinant plasmids. M: DNA marker; 1: pET-28a-UGT71BD1; 2: pCold™ I-CtSus
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2 Bioinformatics analysis of CtSus gene and its encoded protein
2.1 Analysis of physicochemical properties of CtSus and prediction of transmembrane domain structure
The physicochemical properties of CtSus encoded protein were analyzed using ProtParam online software. The protein contains 805 amino acids, with a molecular formula of C4189H6548N1104O1187S28 and a relative molecular mass of 92266.44; the theoretical isoelectric point is 6.00; the instability coefficient II is 35.45, which is a stable protein; the overall average hydrophilicity (GRAVY) is −0.187, which is a hydrophilic protein. MHMM 2.0 was used to predict the transmembrane domain of sucrose synthase CtSus, and the results showed that the protein encoded by this gene has no transmembrane domain.
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2.2 Prediction of CtSus conservative structure and sequence alignment
The online software SMART was used to predict the conservative domains of CtSus protein (Figure 1C). The protein contains two conservative domains. The N-terminus (amino acids 8 to 553) is the sucrose synthase domain, which can catalyze the reversible reaction of UDP and sucrose to generate UDP-glucose and D-fructose; the C-terminus (amino acids 557 to 739) belongs to the glycosyltransferase domain (Figure 1D). DNAMAN software was used to align the CtSus protein sequence with the sucrose synthase amino acid sequence in the NCBI database (Table 2). The results showed that the CtSus amino acid sequence showed high similarity with sucrose synthase sequences from other plants. The highest similarity between CtSus and the sucrose synthase sequence from sesame (Sesamum indicum) was 94.78%, and the similarity with the sucrose synthase sequences from other plants was also above 65%, indicating that the sucrose synthase from plants has a high degree of sequence conservation.
Table 2 Amino acid sequence similarities between CtSus and sucrose synthases identified from other plant
2.3 Phylogenetic analysis
The phylogenetic tree constructed by MEGA software was used to analyze the phylogenetic relationship between the sucrose synthase CtSus from Cistanche tubulosa and sucrose synthases from other plants. The results are shown in Figure 1E. Sucrose synthases from plants show distinct evolutionary branches in dicots (regions I and II) and monocots (region III). CtSus is in the dicot branch, and the sequences that are closely related to CtSus are all from Lamiaceae plants (Cistanche tubulosa is a Lamiaceae Orobanchaceae plant), while the other branch is mainly composed of Solanales plants, which further indicates the high conservation and homology of plant-derived sucrose synthase genes in the evolution of plants in the same order. Among them, the sucrose synthase PrSus (AEN79500.1) from Phelipanche ramosa of the Orobanchaceae plant is the closest to CtSus.
3 Exogenous expression and activity analysis of CtSus
3.1 Exogenous expression of CtSus gene
The pET-28a-CtSus, pET-24b-CtSus and pCold™ I-CtSus plasmids verified by sequencing were transferred into the expression competent E. coli Transetta (DE3), and the positive clones were screened by colony PCR for sequencing verification. The obtained recombinant expression strains were induced by IPTG low temperature for protein expression, and the bacteria were collected by centrifugation. The lysis buffer was added to break the bacteria to obtain the supernatant and precipitate, and SDS-PAGE was performed for detection. The results showed that when pET-28a and pET-24b were used as expression vectors, CtSus was not expressed in E. coli, while when pCold™ I was used as the expression vector, a clear CtSus recombinant protein band was detected in the ~100 kDa region, which was consistent with its theoretically predicted relative molecular mass of 92.2 kDa (Figure 1F).
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3.2 Whole-cell transformation
In order to preliminarily verify the catalytic function of CtSus, a co-expression strain of CtSus and the glycosyltransferase gene UGT71BD1 was constructed. UGT71BD1 was cloned and identified from Cistanche tubulosa and is a UDP-glucosyltransferase that can widely accept aromatic compounds such as phenylethanoid glycosides, different types of flavonoids, stilbene and coumarin as receptors and catalyze the glucosylation reaction of the substrate phenolic hydroxyl group using UDP-glucose as the sugar donor[18]. The introduction of CtSus can theoretically provide more sufficient glycosyl donor UDP-glucose for the glucosylation reaction catalyzed by UGT71BD1, thereby promoting the reaction equilibrium to move toward the formation of glycosylation products, thereby increasing the yield of glycosylation products. The pCold™ I-CtSus plasmid and the pET-28a-UGT71BD1 plasmid were simultaneously transformed into the expression competent E. coli Transetta (DE3). Single clones containing both recombinant plasmids were screened by colony PCR, and positive recombinant expression strains were obtained by sequencing verification (Figure 1G). The dual gene co-expression was induced in vitro, and the pET-28aUGT71BD1 single gene expression strain was used as the control group. The activity of CtSus in catalyzing the generation of UDP-glucose donor was preliminarily verified by whole cell transformation. The results of HPLC and high-resolution mass spectrometry showed that when whole cell transformation reaction was carried out with aesculetin 1 and resveratrol 2 as substrates, the generation of obvious glycosylation products was detected after 24 h of transformation, as shown in Figure 2.
Figure 2 Whole-cell biotransformation of substrate 1 or 2 by recombinant strain harboring UGT71BD1 or recombinant strain harboring UGT71BD1 coupling with CtSus, respectively. A: HPLC chromatograms of the whole-cell biotransformation of substrate 1. The detection wavelength was 360 nm; B: HRESI-MS and MS2 spectra of product 1a; C: HPLC chromatograms of the whole-cell biotransformation of substrate 2; The detection wavelength was 330 nm. D: HRESI-MS spectra of products 2a and 2b
When 1 (m/z 179.034 4 [M+H]+, molecular formula C9H6O4) was used as substrate, the product chromatographic peak 1a (m/z 41.087 2 [M+H]+, predicted molecular formula C15H16O9) was detected at 5.39 min, which was consistent with the UV absorption of the substrate and the control product. At the same time, the fragment peak of m/z 179.033 4 [M+H]+ was detected in the MS2 spectrum of 1a, which was produced by 1a losing a molecule of glucose (162 Da), indicating that 1a was the product of substrate 1 connected to a molecule of glucose. The conversion rate was calculated by integrating the peak area. It was found that the conversion rate of UGT71BD1 whole cells catalyzing substrate 1 to generate glycosylated product 1a was 71.87%, while the addition of CtSus increased the conversion rate of the reaction to 95.84%, which was 1.3 times that of the control group. When the substrate was compound 2 (m/z 227.072 5 [M-H]-, molecular formula C14H12O3), the product chromatographic peaks 2a (m/z 389.123 4 [M-H]-, predicted molecular formula C20H22O8) and 2b (m/z 575.175 9 [M+Na]+, predicted molecular formula 26H32O13) were detected at 10.32 and 6.52 min, respectively, which were consistent with the characteristic UV absorption of the substrate and the control product. A fragment peak was detected at 227.071 3 [M-H]-, which was produced by the loss of one glucose molecule (162 Da), indicating that 2a was the product of substrate 2 connected to one glucose molecule. By comparing with the data in the literature [18] and the mass spectrometry prediction information, it was determined that product 2b was the product of substrate 2 connected to two glucose molecules. The conversion rate was calculated using the integrated peak area. It was found that the addition of CtSus could increase the conversion rate of the glycosylation reaction of substrate 2 from 64.01% to 78.51%, among which the yield of monosaccharide product 2a increased from 45.09% to 63.58%, and the yield of disaccharide product 2b changed from 18.92% to 14.93%.
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3.3 CtSus
Recombinant protein purification and in vitro enzyme catalytic activity identification The results of the whole cell conversion experiment suggested that CtSus has the activity of catalyzing the production of active glucose donor UDP-glucose. In order to further verify the catalytic activity through in vitro enzymatic catalysis, the fusion protein of CtSus gene in pCold™ expression system was separated and purified by affinity chromatography. The results showed that when pCold™ I was used as the expression vector, the recombinant protein was expressed in large quantities, but mostly in the precipitate. When pCold™ TF was used as the expression vector, CtSus gene was expressed in large quantities in E. coli (Figure 3A). The fusion protein was purified by HisTrap FF affinity chromatography for in vitro enzymatic reaction. The results are shown in Figure 3B. When sucrose and UDP were used as substrates, compared with the negative control group, a new product peak was detected at 10.32 min. Its retention time and UV absorption were consistent with UDP-glucose. The product was proved to be UDP-glucose by comparison with the standard. However, since the fusion protein expressed by the pCold™ TF expression vector contains a large trigger factor soluble tag (the tag protein size is 48 kDa), it will have a great impact on the catalytic activity of the protein. Therefore, the tag of the fusion protein was further cut by Factor Xa enzyme, and the CtSus protein without exogenous tags was purified (Figure 3A). The protein was subjected to in vitro enzymatic catalysis, and the results showed that the yield of UDP-glucose increased from 3.53% to 10.66% (Figure 3B). The above results confirmed the activity of CtSus in catalyzing the production of UDP-glucose through in vitro enzymatic catalysis, and also showed that the presence of a large affinity tag is conducive to the soluble expression of CtSus, but it will also significantly affect the in vitro catalytic activity of the enzyme.
Figure 3 Heterologous expression and functional identification of CtSus. A: SDS-PAGE analysis of CtSus proteins. Lane 1: Protein marker; Lane 2: Recombinant CtSus with trigger factor; Lane 3: Trigger 13 factor tag cleavage using Factor Xa protease to give the CtSus protein labeled by the red arrow. B: In vitro enzymatic assays using fusion protein and trigger factor-free CtSus proteins to catalyze the synthesis of UDP-glucose in the presence of sucrose and UDP, respectively, with reference standard UDP-glucose. Boiled protein was used as a control group under the same conditions. The detection wavelength was 260 nm
