Maugt79 gene and its application in regulating biosynthesis of scopoletin and response to drought stress

Abstract

The present application relates to the field of biotechnology, in particular to a white flower carum gene MaUGT79 and its application, the present application is based on white flower carum genome data and BSA resequencing data, cloning MaUGT79 gene, using real-time quantitative PCR technology to analyze the expression pattern under drought stress, and construct the recombinant prokaryotic expression vector to verify MaUGT79 gene function, it is found that transgenic escherichia coli can catalyze scopolin to produce scopolamine; construct plant overexpression vector and RNAi vector to transform the gene into white flower carum hairy root, verify MaUGT79 gene to participate in scopolin synthesis, has the function of resisting drought stress, the present application lays the foundation for revealing the drought tolerance mechanism of MaUGT79 gene, provides gene resources and theoretical basis for plant drought tolerance molecular breeding.

Description

[Technical Field]

[0001] This invention relates to the field of biotechnology, and in particular to the MaUGT79 gene and its application in the positive regulation of scopolamine biosynthesis and drought stress response. [Background Technology]

[0002] Drought stress is a significant abiotic factor affecting agricultural production. When plants are subjected to drought stress during their growth and development, they produce large amounts of reactive oxygen species (ROS), which are highly toxic to the plants themselves. This disrupts the dynamic balance between ROS production and scavenging within the plant, causing cell damage. After experiencing drought stress, the cell membrane is damaged, inhibiting plant growth.

[0003] Plants can synthesize a large number of secondary metabolites to regulate their adaptation to the environment. Under drought stress, plants exhibit stronger adaptability to extreme environments through the accumulation of phenylpropanoid compounds. Scopolamine and its aglycone scopolamine lactone are secondary metabolites synthesized via the phenylpropanoid pathway and have been shown to participate in plant responses to abiotic stresses such as drought. *Osmanthus fragrans* is rich in scopolamine and other secondary metabolites; however, the mechanism by which scopolamine metabolism responds to drought stress remains unclear, and its underlying molecular mechanisms are still ambiguous. Identifying genes related to scopolamine synthesis in *Osmanthus fragrans* is of great value in providing candidate genes for molecular breeding of drought-resistant plants.

[0004] UDP-glycosyltransferase (UGT) participates in the glycosylation of scopolamine to produce scopolamine glycosides. Currently, many UGT genes have been discovered; 122, 243, and 189 UGT genes are found in Arabidopsis thaliana, alfalfa, and sweet clover, respectively. Tobacco UDP-glucosyltransferase TOGT participates in the glycosylation of scopolamine glycosides and enhances antiviral activity after Tobacco mosaic virus (TMV) infection. Downregulation of TOGT expression leads to decreased UGT activity, reduced scopolamine and scopolamine glycoside content, accumulation of reactive oxygen intermediates (ROIs), and weakened antiviral activity. The tomato SlUGT75C1 gene plays an important role in ABA-mediated drought response. Overexpression of the UGT76E11 gene in Arabidopsis thaliana enhances the tolerance of transgenic lines to drought and salt stress by increasing flavonoid accumulation. In rice, the UGT83A1 (GSA1) gene regulates the redirection of lignin biosynthesis to flavonoid biosynthesis and flavonoid glycoside accumulation under abiotic stress, thereby protecting rice from abiotic stress damage. In contrast, research on the UGT gene in Osmanthus fragrans is relatively limited, and studies on its mediation of scopolamine accumulation to improve plant drought tolerance have not yet been reported. [Summary of the Invention]

[0005] In view of the above, it is necessary to provide a MaUGT79 gene and its application, which can positively regulate the biosynthesis of scopolamine and improve the drought resistance of sweet clover.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] The MaUGT79 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] This application also includes a protein encoded by the MaUGT79 gene, the amino acid sequence of which is shown in SEQ ID NO.2.

[0009] This application also includes primers for amplifying the MaUGT79 gene, wherein the primers are primer P1 / primer P2, the sequence of primer P1 is shown in SEQ ID NO.3, and the sequence of primer P2 is shown in SEQ ID NO.4.

[0010] This application also includes an expression vector containing the coding sequence of the MaUGT79 gene, SEQ ID NO.1.

[0011] This application also includes the application of the MaUGT79 gene in catalyzing the conversion of scopolamine lactone to scopolamine glycoside.

[0012] This application also includes a method for catalyzing the conversion of scopolamine lactone to scopolamine glycoside using the MaUGT79 expression vector, the method being:

[0013] (1) Using the plasmid T-MaUGT79 as a template, PCR amplification was performed using primers P3 / P4 with a high-fidelity enzyme to obtain the MaUGT79 gene fragment;

[0014] (2) The plasmid pET32a was digested with BamHI and SacI to obtain the linearized vector of pET32a.

[0015] (3) The MaUGT79 gene fragment from step (1) and the linearized vector of pET32a are ligated to obtain the prokaryotic expression vector pET32a-MaUGT79;

[0016] (4) Transform the prokaryotic expression vector pET32a-MaUGT79 from step (3) into BL21 competent cells, pick single clones and inoculate them into LB liquid medium containing ampicillin for shaking culture, then add isopropyl-β-D-thiogalactoside, and after induction culture for 9 h, add the substrate scopolamine to obtain scopolamine.

[0017] The sequence of primer P3 is shown in SEQ ID NO.5, and the sequence of primer P4 is shown in SEQ ID NO.6.

[0018] This application also includes the application of the MaUGT79 gene in increasing the scopolamine content of Osmanthus fragrans and / or improving the drought resistance of Osmanthus fragrans.

[0019] This application also includes a method for increasing the scopolamine content of Osmanthus fragrans and / or improving the drought resistance of Osmanthus fragrans using the MaUGT79 expression vector, the method being:

[0020] (1) Using the plasmid of T-MaUGT79 as a template, PCR amplification was performed using primer P5 / primer P6 with high-fidelity enzyme to obtain the MaUGT79 gene fragment;

[0021] (2) The plasmid pBI121 was digested with BamHI and XbaI to obtain the linearized vector of pBI121.

[0022] (3) The MaUGT79 gene fragment from step (1) and the linearized vector of pBI121 are linked to obtain the prokaryotic expression vector pBI121-MaUGT79;

[0023] (4) The pBI121-MaUGT79 plasmid was transformed into Agrobacterium rhizogenes K599 by electroporation, and then Agrobacterium rhizogenes K599 was used to infect the hairy roots of the sweet osmanthus to obtain the product.

[0024] The sequence of primer P5 is shown in SEQ ID NO.7, and the sequence of primer P6 is shown in SEQ ID NO.8.

[0025] The present invention has the following beneficial effects:

[0026] Based on the genome data and BSA resequencing data of *Osmanthus fragrans*, this invention cloned the MaUGT79 gene, analyzed its expression pattern under drought stress using real-time quantitative PCR, and constructed a recombinant prokaryotic expression vector to verify the function of the MaUGT79 gene. It was found that transgenic *Escherichia coli* can catalyze the production of scopolamine from scopolamine lactone. Plant overexpression vectors and RNAi vectors were constructed to transform this gene into the hairy roots of *Osmanthus fragrans*, verifying that the MaUGT79 gene participates in scopolamine synthesis and has the function of resisting drought stress. This invention lays the foundation for revealing the drought tolerance mechanism of the MaUGT79 gene and provides gene resources and theoretical basis for molecular breeding of drought-resistant plants. [Attached Image Description]

[0027] Figure 1 This is the gene structure diagram of the MaUGT79 gene;

[0028] Figure 2 This is a phylogenetic tree of the amino acid sequence encoded by the MaUGT79 gene of Osmanthus fragrans and UGT in other plants;

[0029] Figure 3 This is a sequence alignment diagram of UGT amino acids related to coumarin synthesis;

[0030] Figure 4 This is a transcriptional response diagram of Osmanthus fragrans MaUGT79 to drought stress;

[0031] Figure 5 This is an SDS-PAGE image of MaUGT79 heterologously expressed in Escherichia coli BL21(DE3);

[0032] Figure 6 This is a high-performance liquid chromatography (HPLC) chromatogram of the products of the glycosylation reaction of scopolamine catalyzed by MaUGT79.

[0033] Figure 7This is an agarose gel electrophoresis image of hairy roots transgenic with OE-MaUGT79 and RNAi-MaUGT79 genes, identifying positive hairy roots by PCR. In the image, M is the marker, CK is the negative control (empty vector transgenic PCR product), (-) is the blank control (ddH2O PCR product), and (+) is the positive control (pBI121-MaUGT79 plasmid PCR product). Lanes 1-6 are OE-MaUGT79 transgenic hairy root PCR products, and lanes 7-12 are RNAi-MaUGT79 transgenic hairy root PCR products.

[0034] Figure 8 This is a graph showing the relative expression levels of MaUGT79 in the hairy roots of OE-MaUGT79 and RNAi-MaUGT79-positive white sweet clover and the hairy roots of the empty vector control.

[0035] Figure 9 This is a graph showing the scopolamine content in the hairy roots of OE-MaUGT79 and RNAi-MaUGT79 positive and empty vector control genes of white sweet clover.

[0036] Figure 10 This image shows the growth of hairy roots of *Sweet clover* transgenic with the OE-MaUGT79 and RNAi-MaUGT79 genes under drought stress. EV represents the empty-vector transgenic *Sweet clover*, OE-MaUGT79 represents the transgenic line overexpressing the MaUGT79 gene, and RNAi-MaUGT79 represents the transgenic line expressing the MaUGT79 gene via RNA interference. After culturing in 25% PEG6000 solution for 3 days, the growth of the overexpressing transgenic lines was superior to that of the empty-vector transgenic lines, and the growth of the empty-vector transgenic lines was superior to that of the RNAi transgenic lines.

[0037] Figure 11 This is a schematic diagram illustrating the determination of physiological parameters of the hairy root complex of *Osmanthus fragrans* overexpressing and RNAi-MaUGT79 transgenic plants. In diagram A, MDA content in the hairy roots of overexpressing and RNAi-MaUGT79 transgenic plants is measured; in diagram B, O... 2- C represents the detection results of H2O2 content in hairy roots of overexpressed and RNAi-MaUGT79 transgenic plants; D represents the detection results of scopolamine content in hairy roots of overexpressed and RNAi-MaUGT79 transgenic plants.

Detailed Implementation Methods

[0038] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0039] Example 1:

[0040] Cloning and sequence analysis of the MaUGT79 gene:

[0041] (1) Using the reference genome of Osmanthus fragrans as a reference sequence, primers (primer P1 and primer P2) were designed to amplify the MaUGT79 gene from the whole plant of Osmanthus fragrans.

[0042] (2) Total RNA was extracted from the whole plant of Osmanthus fragrans according to the instructions of the RNA extraction kit. The extracted RNA was reverse transcribed into cDNA using a reverse transcription kit. Using this cDNA as a template, PCR amplification was performed using primers P1: 5'-ATGTCTATAAAACCACAAACCCACC-3' (SEQ ID NO.3) and P2: 5'-TTAATCATTCCTAAGCTCATTAAGCTC-3' (SEQ ID NO.4) with a high-fidelity enzyme.

[0043] The reaction system for the above PCR amplification is as follows: 2× Max Buffer 12.5 μL, dNTP 0.5 μL, forward and reverse primers (10 μM) 1 μL each, Max Super-Fidelity DNA Polymerase (Novizan) 0.5 μL, ddH2O 8.5 μL.

[0044] The PCR program was as follows: 95℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min 30 s, 35 cycles; 72℃ for 5 min.

[0045] (3) After the reaction, 5 μL of the PCR product was subjected to 1.5% agarose gel electrophoresis. A band of approximately 1392 bp was observed in the UV gel imaging system, consistent with the expected size. The MaUGT79 gene fragment was recovered using a PCR product purification kit. The recovered and purified DNA fragment was ligated using the 5 min TA / Blunt-Zero Cloning Kit instructions. The obtained recombinant plasmid T-MaUGT79 was transformed into E. coli competent cells. Positive clones were screened using ampicillin (Amp) (100 mg / L) and identified by bacterial culture PCR. The bacterial culture of the positive recombinant plasmid was sequenced. The composition of introns and exons of the MaUGT79 gene was analyzed using an online gene structure analysis system (http: / / gsds.cbi.pku.edu.cn / index.php). A phylogenetic tree was constructed using MEGA 7.0. The characteristic motif of the glycosyltransferase UGT gene was searched using MEME software (meme-suite.org / tools / meme). The constructed phylogenetic tree is shown below. Figure 1 As shown.

[0046] (4) Sequencing results showed that the nucleotide sequence of the MaUGT79 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the gene structure of the MaUGT79 gene is shown in... Figure 2 As shown.

[0047] from Figure 1 It is evident that this phylogenetic tree is clearly divided into two major branches, with the MaUGT79 gene of Osmanthus fragrans located on the branch related to coumarin biosynthesis.

[0048] from Figure 2 It is evident that the MaUGT79 gene of Osmanthus fragrans consists of one exon and has no introns.

[0049] Figure 3For the alignment of UGT amino acid sequences related to coumarin synthesis, the red arrow at the N-terminus points to the crucial histidine (His19) residue, which is conserved in the UGT of all plants; the green arrow points to the proline (Pro22) residue, which plays an important role in sugar donor recognition. The black box at the C-terminus points to the conserved PSPG box domain. *Osmanthus fragrans* MaUGT79 contains the above characteristic sequences. The sequences used in the figure are from: *Arabidopsis thaliana* AtUGT73B3 (AAL32831), AtUGT73B4 (BAE99671); tobacco NtGT3 (BAB88934), NtGT1a (BAB60720); strawberry FaGT6 (ABB92748), FaGT7 (ABB92749); and *Osmanthus fragrans* MaUGT79 (the amino acid sequence shown in SEQ ID No. 2).

[0050] Example 2:

[0051] Application of MaUGT79 gene in the positive regulation of scopolamine biosynthesis

[0052] I. Constructing the overexpression vector, the specific method is as follows:

[0053] (1) Using the plasmid of T-MaUGT79 as a template, PCR amplification was performed using upstream primer P3 and downstream primer P4 with high-fidelity enzyme. Primer P3: 5'-GCCATGGCTGATATCGGATCCATGTCTATAAAACCACAAACCC ACC-3' (SEQ ID NO.5), primer P4: 5'-GCAAGCTTGTCGACGGAGCTCTTAATCATTCCTAAG CTCATTAAGCTC-3' (SEQ ID NO.6);

[0054] The PCR amplification reaction system is as follows: 2× Max Buffer 12.5 μL, dNTP 0.5 μL, forward and reverse primers (10 μM) 1 μL each, Max Super-Fidelity DNA Polymerase (Novizan) 0.5 μL, ddH2O 8.5 μL.

[0055] The PCR program was as follows: 95℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min 30 s, 35 cycles; 72℃ for 5 min.

[0056] (2) Take 5 μL of PCR product for 1.5% agarose gel electrophoresis. Use a UV gel imager to see the fragment that is consistent with the expected size. Use a PCR product purification kit to recover the MaUGT79 gene fragment.

[0057] (3) Linearization of the vector: The plasmid pET32a was digested with BamHI and SacI enzymes. After digestion, the product was purified using a PCR product purification kit. The digestion reaction system was as follows: 1 μg of vector plasmid, 1 μL of BamHI enzyme, 1 μL of SacI enzyme, 2 μL of 10× buffer, and ddH2O to a final volume of 20 μL. The digestion reaction was carried out at 37℃ for 1 h.

[0058] (4) The MaUGT79 gene fragment from step (2) and the linearized pET32a vector from step (3) were cloned using a one-step cloning kit. The MultiS One Step Cloning Kit (Novizan) was used for homologous recombination. The system consisted of: 2.5 μL of linearized pET32a vector, 1 μL of MaUGT79 gene fragment, 1 μL of 5×CE MultiS Buffer, and 0.5 μL of ExnaseMultiS. The reaction was carried out at 37°C for 30 min; then cooled to 4°C or immediately placed on ice. E. coli DH5α was transformed using the heat shock method. Single clones were picked for colony PCR detection. Positive clones were sequenced. Clones with correct sequencing results were considered successfully constructed vectors. The colonies were then cultured, and plasmids were extracted using a plasmid extraction kit to obtain the prokaryotic expression vector: pET32a-MaUGT79, which was stored at -20°C.

[0059] II. Heterologous expression of Escherichia coli and substrate feeding:

[0060] (1) The expression vector pET32a-MaUGT79 and the empty vector pET32a prepared in step one were transformed into BL21(DE3) competent cells, respectively. Single clones were picked and inoculated into 5 mL of LB liquid medium containing 100 μg / mL ampicillin. The cells were incubated overnight at 37°C and 220 rpm until OD500 was reached. 600 The concentration was between 0.4 and 0.6. Then, IPTG (isopropyl-β-D-thiogalactopyranoside) at a concentration of 0.1 mM was added, and the cells were cultured at 22°C with shaking for 18 h to induce the expression of the target protein. The cells were collected by centrifugation at 8000 rpm for 5 min at 4°C, and resuspended in 400 μL of 50 mM Tris-HCl (pH = 7.5). The cells were then sonicated on ice for 3 min at 60 W for 1.5 s, with 1.5 s intervals. The whole-cell lysate was added to 10 μL of 5× loading buffer and heated at 95°C for 10 min to denature the cells. 10 μL of the lysate was then subjected to SDS-PAGE electrophoresis, and the results are shown below. Figure 5As shown, the pET32a-MaUGT79 prokaryotic expression vector was transformed into Escherichia coli BL21(DE3) and induced at 22℃ with an IPTG concentration of 0.1 mM for 18 h. The induced pET32a-MaUGT79 recombinant bacteria expressed a fusion protein of approximately 51.87 kDa, which is consistent with the expected molecular weight of MaUGT79.

[0061] (2) The pET32a-MaUGT79 expression strain and the empty vector expression strain were induced and cultured under the same conditions. After induction with 0.1 mM IPTG (isopropyl-β-D-thiogalactoside) for 9 h, the substrate scopolamine (0.1 mM) was added, and the culture was continued for another 9 h. After the reaction, the mixture was extracted with an equal volume of ethyl acetate. The recovered ethyl acetate was evaporated to dryness under reduced pressure, dissolved in methanol, filtered through a 0.45 μM filter membrane, and analyzed by high performance liquid chromatography. The results are as follows: Figure 6 As shown, HPLC detection results indicated that the extract of *E. coli* BL21(DE3) / pET32a-MaUGT79 containing scopolamine substrate exhibited a peak with the same retention time as the scopolamine standard (approximately 20 min), while no new product was detected in the empty vector extract of the control *E. coli* BL21(DE3) / pET32a(+). This suggests that the MaUGT79 protein can catalyze the conversion of scopolamine lactone to scopolamine.

[0062] Example 3:

[0063] Application of MaUGT79 gene in drought stress response

[0064] Analysis of MaUGT79 expression patterns in the roots of Osmanthus fragrans under drought stress:

[0065] To identify whether MaUGT79 is involved in the drought stress response of *Osmanthus fragrans*, roots were subjected to drought stress to analyze the transcriptional level of MaUGT79 in the roots under drought stress. *Osmanthus fragrans* plants grown for 6 weeks under 30% PEG6000 stress were subjected to 24 h of stress. RNA was extracted from the underground parts of the stressed *Osmanthus fragrans* plants, and its expression level under drought stress was analyzed using real-time quantitative PCR. The results are as follows: Figure 4 As shown, the expression level of MaUGT79 in the roots of Osmanthus fragrans under drought stress for 24 h showed a significant upregulation trend compared with the control, indicating that MaUGT79 responds to drought stress in the roots of Osmanthus fragrans.

[0066] Construction and validation of plant overexpression vectors:

[0067] I. Construction of plant overexpression vectors:

[0068] (1) Using the T-MaUGT79 plasmid as a template, PCR amplification was performed using primers P5 and P6 via a high-fidelity enzyme. The PCR amplification reaction system was as follows: 2× Max Buffer 12.5 μL, dNTP 0.5 μL, forward and reverse primers (10 μM) 1 μL each, Max Super-Fidelity DNA Polymerase (Novizan) 0.5 μL, ddH2O 8.5 μL. PCR program: 95℃ 3 min; 95℃ 30 s, 55℃ 30 s, 72℃ 1 min 30 s, 35 cycles; 72℃ 5 min.

[0069] The primer P5 sequence is as follows: 5'-GAGAACACGGGGGACTCTAGAATGTCTATAAAACCACAAA CCCACCT-3' (SEQ ID NO.7);

[0070] The primer P6 sequence is as follows: 5'-CCATGGTACCCCCGGGGATCCGATCATTCCTAAGCTCATTAA GCTC-3' (SEQ ID NO.8);

[0071] (2) Take 5 μL of PCR product for 1.5% agarose gel electrophoresis. Use a UV gel imager to see the fragment that is consistent with the expected size. Use a PCR product purification kit to recover the MaUGT79 gene fragment.

[0072] (3) Linearization of the vector: The plasmid pBI121 was digested with XbaI and BamHI enzymes. After digestion, the product was purified using a PCR product purification kit. The digestion reaction system was as follows: 1 μg pBI121 plasmid, 1 μL XbaI enzyme, 1 μL BamHI enzyme, 2 μL 10× buffer, and ddH2O to a final volume of 20 μL. The digestion reaction was carried out at 37℃ for 1 h.

[0073] (4) The MaUGT79 gene fragment from step (2) and the linearized pBI121 vector from step (3) were cloned using a one-step cloning kit. The MultiS One Step Cloning Kit (Novizan) was used for homologous recombination. The system consisted of: 2.5 μL of linearized pBI121 vector, 1 μL of MaUGT79 gene fragment, 1 μL of 5×CE MultiS Buffer, and 0.5 μL of ExnaseMultiS. The reaction was carried out at 37°C for 30 min; then cooled to 4°C or immediately placed on ice. E. coli DH5α was transformed using the heat shock method. Single clones were picked for colony PCR detection. Positive single clones were sent for sequencing. Single clones with correct sequencing results were considered successfully constructed vectors. The cells were then cultured, and plasmids were extracted using a plasmid extraction kit to obtain the plant expression vector: pBI121-MaUGT79, which was stored at -20°C.

[0074] II. Construction of RNAi Expression Vectors

[0075] (1) According to the Gateway instructions, the attB site was merged into the 5' ends of the gene-specific upstream and downstream primers, resulting in primers P7 and P8. Using P7 / P8 as the primer pair and the T-MaUGT79 plasmid as the template, the gene was... PCR amplification was performed using Max Super-Fidelity DNAPolymerase high-fidelity enzyme, and the PCR products were recovered using a gel recovery kit.

[0076] The P7 sequence is as follows: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAATGTCTATAAAACCACAAACCCACCT-3' (SEQ ID NO.9);

[0077] The P8 sequence is as follows: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTTTAATCATTCCTAAGCTCATTAAGCTC-3' (SEQ ID NO.10);

[0078] The BP reaction system consisted of: 0.4 μL Gateway BPⅡClonase mixture, 1 μL gel-recovered product (50 ng / μL), and 0.6 μL pDONR-Zeo (150 ng / μL). The reaction was carried out overnight at 25°C.

[0079] (2) Transform the ligation product into Escherichia coli DH5α. Spread 100 μL of the culture onto an LB agar plate containing bleomycin (Zeo) antibiotic at a final concentration of 33 mg / L. Use universal primers M13F and M13R to detect positive single clones and send them for sequencing. Shake the culture to obtain plasmids from the correctly sequenced single clones, extract the plasmids using a plasmid extraction kit, and store at -20°C.

[0080] The RNAi vector pK7GWIWG2(II)RR and the entry vector were used to construct the RNAi recombinant vector via a ligation reaction. The ligation reaction system was as follows: 0.4 μL Gateway LRⅡ Clonase mixture, 0.6 μL Entry Clone (150 ng / μL), 0.6 μL Target vector (150 ng / μL), and 0.4 μL ddH2O. The reaction was carried out overnight at 25°C. The ligation product was transformed into E. coli DH5α. After transformation, 100 μL of the culture was plated onto LB agar plates containing spectinomycin Spe antibiotic at a final concentration of 50 mg / L. After detection of positive monoclonal cultures, positive monoclonal cultures with the target band were shaken and the plasmid was extracted using a plasmid extraction kit. The successfully constructed RNAi-MaUGT79 recombinant plasmid was stored at -20°C.

[0081] III. Transformation of the hairy roots of white sweet clover using the MaUGT79 gene

[0082] The pBI121-MaUGT79 plasmid, RNAi-MaUGT79, and empty vector were transformed into Agrobacterium rhizogenes K599 using an electroporation method. Single clones were picked for colony PCR detection, and the product fragment was consistent with the expected target fragment size, indicating that Agrobacterium rhizogenes with MaUGT79 gene overexpression and RNAi vector was successfully obtained.

[0083] 200 μL of the empty vector, MaUGT79 overexpression vector, and RNAi vector K599 bacterial suspension were plated. After 7 days of germination, the roots of *Osmanthus fragrans* were quickly cut 5 mm above the root tip using a scalpel. The cut seedlings were lightly dipped into *Agrobacterium* bacterial film and placed on antibiotic-free 1 / 2 MS medium, wrapped in aluminum foil, and incubated in the dark for 3 days. After 3 days of co-culture, the seedlings were removed from the dark conditions and placed on 1 / 2 MS solid medium in a filter paper-seedling-filter paper configuration. They were then placed upright in a 22℃ tissue culture room (16h light / 8h dark) and cultured for 14 days. Hairy roots began to grow. Subsequently, they were transferred to water and cultured for 15-20 days to observe growth and conduct subsequent experiments.

[0084] IV. Molecular-level identification of the hairy roots of transgenic white sweet clover

[0085] Genome and transcriptional levels of the hairy roots of transgenic Osmanthus fragrans were identified. Using a high-speed mixed assay, 1 mm of root tip was taken, 20 μL of a companion element was added, and the sample was treated at 95°C for 5 min. After centrifugation, 1 μL of the supernatant was used as a template. PCR was performed using upstream primers of the vector and the target bands. The identification results are as follows: Figure 7 As shown, this indicates that the target gene was successfully transformed into the hairy roots of Osmanthus fragrans. Furthermore, as... Figure 8As shown, the expression of MaUGT79 in transgenic hairy roots was detected by qRT-PCR. It was found that compared with the control plants, the expression level of MaUGT79 gene was higher in overexpressing hairy roots and lower in RNAi-repressed hairy roots.

[0086] V. Drought Tolerance Test of Transgenic Osmanthus fragrans Complex

[0087] After the control plants, plants overexpressing MaUGT79 vector, and plants using RNAi-MaUGT79 vector developed hairy roots for 60 days, the composite plants were transferred to a 25% PEG6000 solution for 3 days of simulated drought stress. Phenotypic observations were then performed on the control, MaUGT79 overexpressing, and RNAi lines after the stress. Results are as follows: Figure 10 As shown, after drought stress, the leaves of the overexpression complex showed a normal phenotype, while the leaves of the control complex wilted and turned yellow, and the leaves of the RNAi complex withered and curled. These results indicate that overexpression of the MaUGT79 gene improved the drought tolerance of the Osmanthus fragrans complex.

[0088] VI. Determination of Scopolamine Content in the Complex *Osmanthus fragrans* under Drought Stress

[0089] 100 mg of fresh hairy root sample cultured for 60 days was weighed and ground with liquid nitrogen. The mixture was then shaken and mixed with 5 mL of 80% ethanol, followed by ultrasonic extraction at room temperature for 30 min. The sample was centrifuged at 12000 rpm for 10 min, and the filtrate was collected. This extraction was repeated twice. The filtrates were then combined and concentrated under reduced pressure using a rotary evaporator at 50℃. After the sample was dried by rotary evaporation, it was dissolved in methanol and brought to a final volume of 10 mL to obtain the sample extract. HPLC was used for detection. Acetonitrile (elution A) and 0.1% phosphoric acid (elution B) were used as the mobile phase, and the detection wavelength was 346 nm. The flow rate was 1 mL / min, the column temperature was 30℃, and the injection volume was 10 μL. The detection results are as follows: Figure 9 As shown, compared with the control, the content of scopolamine in hairy roots of OE-MaUGT79 transgenic plants was significantly increased, while the content of scopolamine in hairy roots of RNAi-MaUGT79 transgenic plants was significantly decreased. This indicates that the MaUGT79 gene has a positive regulatory effect on the synthesis of scopolamine in Osmanthus fragrans.

[0090] VII. Determination of physiological indicators of the white-flowered sweet clover complex under drought stress

[0091] After simulating drought treatment for 3 days, control plants, hairy roots of *Sweet clover* overexpressing MaUGT79, and plants transformed with RNAi vectors were subjected to MDA (malondialdehyde), H2O2, and O2. 2- The content was determined according to the kit instructions. Physiological indicators related to the MaUGT79 complex of Osmanthus fragrans were detected, and the results are as follows: Figure 11As shown, the MDA, H2O2, and O content of the complex *Osmanthus fragrans* and the control *Osmanthus fragrans* before drought stress were... 2- There was no significant difference in content, but after drought stress, compared with the control plants, the plants overexpressing MaUGT79 had higher levels of MDA, H2O2, and O2. 2- The content was lower than that of the control plant. The RNAi-MaUGT79 plant had MDA, H2O2, and O content lower than that of the control plant. 2- The control plants had a higher content of MaUGT79. This indicates that overexpression of MaUGT79 improves the drought resistance of the Osmanthus fragrans complex.

[0092] In summary, the MaUGT79 gene isolated from Osmanthus fragrans in this application can promote the synthesis of scopolamine and enhance the drought resistance of Osmanthus fragrans.

[0093] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. MaUGT79 Genes, characterized by, The MaUGT79 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. MaUGT79 Gene-encoded proteins are characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Amplification as described in claim 1 MaUGT79 The primers for the gene are characterized by, The primers are primer P1 and primer P2, with the primer P1 sequence shown in SEQ ID NO.3 and the primer P2 sequence shown in SEQ ID NO.

4.

4. An expression carrier, characterized in that, The expression vector contains the content as described in claim 1. MaUGT79 The coding sequence of the gene is SEQ ID NO.

1.

5. As described in claim 1 MaUGT79 Application of genes in catalyzing the conversion of scopolamine lactone to scopolamine glycoside.

6. A method for catalyzing the conversion of scopolamine lactone to scopolamine glycoside using the expression vector as described in claim 4, characterized in that, The method is as follows: (1) With T- MaUGT79 Using the plasmid as a template, PCR amplification was performed using primers P3 / P4 with a high-fidelity enzyme; [The desired result was obtained]. MaUGT79 Gene fragments; (2) Use BamHI and SacI The pET32a plasmid was digested with enzymes to obtain the linearized pET32a vector; (3) Take the steps from (1) MaUGT79 The gene fragment and the linearized pET32a vector were ligated to obtain the prokaryotic expression vector pET32a- MaUGT79 ; (4) The prokaryotic expression vector pET32a- from step (3) MaUGT79 Transform BL21 competent cells, select single clones and inoculate them into LB liquid medium containing ampicillin for shake culture, then add isopropyl-β-D-thiogalactoside, induce culture for 9 h, and then add the substrate scopolamine to obtain scopolamine. The sequence of primer P3 is shown in SEQ ID NO.5, and the sequence of primer P4 is shown in SEQ ID NO.

6.

7. As described in claim 1 MaUGT79 Application of genes in increasing the scopolamine content and / or improving the drought resistance of Osmanthus fragrans.

8. A method for increasing the scopolamine content and / or improving the drought resistance of *Syzygium buergerianum* using the expression vector as described in claim 4, characterized in that, The method is as follows: (1) With T- MaUGT79 Using the plasmid as a template, PCR amplification was performed using primers P5 / P6 with a high-fidelity enzyme; [The desired result was obtained]. MaUGT79 Gene fragments; (2) Use BamHI and XbaI The plasmid pBI121 was digested with enzymes to obtain the linearized vector of pBI121; (3) Take the steps from (1) MaUGT79 The gene fragment and the linearized vector of pBI121 were ligated to obtain the prokaryotic expression vector pBI121- MaUGT79 ; (4) Using electric shock, pBI121- MaUGT79 The plasmid was transformed into Agrobacterium rhizogenes K599, and then Agrobacterium rhizogenes K599 was used to infect the hairy roots of the white sweet clover to obtain the product. The sequence of primer P5 is shown in SEQ ID NO.7, and the sequence of primer P6 is shown in SEQ ID NO.8.

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