Magnolia biondii MbiPTS gene and its application in identification, synthesis and / or improvement of parthenolide in Magnolia biondii

Abstract

The present invention discloses a Magnolia bicolor MbiPTS gene and its application in identifying, synthesizing and / or increasing parthenolide in Magnolia bicolor. The CDS sequence of the Magnolia bicolor MbiPTS gene is shown in SEQ ID NO.1. The present invention first discovered the key gene MbiPTS for controlling the synthesis of parthenolide in the root bark of Magnolia bicolor. When there is a single base A deletion between the 1057bp to 1059bp sites of the MbiPTS gene ORF, the MbiPTS gene undergoes a frameshift mutation, a TAA stop codon appears at 1065bp, and protein translation is terminated prematurely, then the Magnolia bicolor does not contain parthenolide. The detection of this site can be used to identify whether Magnolia bicolor contains parthenolide. Injecting the Magnolia bicolor MbiPTS gene overexpression vector into the hypocotyl of Magnolia bicolor that does not contain parthenolide can restore the synthesis of parthenolide, providing a basis for increasing the content of parthenolide in the root bark of Magnolia bicolor.

Description

Technical Field

[0001] The invention relates to a Magnolia biondii MbiPTS gene and application thereof in identifying, synthesizing and / or increasing the amount of parthenolide in Magnolia biondii, and belongs to the field of plant molecular biology. Background Art

[0002] Parthenolide (PTL) was first extracted from chamomile flowers. Its molecular formula is C 15 H 20 O3, with a relative molecular mass of 248.3. In Western countries, parthenolide is mainly used to treat fever, headache, rheumatoid arthritis, etc., and has a history of more than 100 years of use. In recent years, Chinese scholars have used parthenolide as a skeleton and developed an original anti-glioma candidate new drug ACT001 through chemical modification, which has a significant effect on the treatment of glioblastoma (Ding et al. 2020). For example, GBM is rated as grade IV by the WHO, which is a highly malignant intracranial glioma with a median survival of less than 2 years and a long-term survival rate of about 4%. An Australian patient with recurrent GBM who was treated with ACT001 monotherapy was enrolled in a clinical trial in March 2017. The tumor size at the time of enrollment was close to 4 cm. After long-term treatment with ACT001 monotherapy, he achieved complete remission in October 2018. As of June 2021, the patient has been taking the drug for more than 50 months and is still in complete remission. The Phase II and Phase III clinical trials of ACT001 for the treatment of brain glioma were conducted at the University of Texas MD Anderson Cancer Center in the United States. The pharmaceutical company has also obtained orphan drug status for the treatment of glioblastoma in the United States and the European Union. The China National Medical Products Administration has also quickly approved the clinical trial of the drug in combination with radiotherapy for the treatment of brain metastases of solid tumors. Once the drug is launched on the market, there will be a huge demand for the raw material parthenolide.

[0003] Parthenolide was first extracted from chamomile flowers. Previous studies have shown that the content of parthenolide in chamomile flowers of different varieties and different ploidy is only 0.2% to 0.5% (Majdi et al., 2013). Liu et al. conducted a study on the metabolic pathway of parthenolide synthesis in chrysanthemums and identified that parthenolide is synthesized from FPP (farnesyl pyrophosphate) through genes such as GAS, GAO and COS, and oxygenated to costunolide, and finally synthesized under the action of PTS enzyme (Liu et al. 2020). Shi et al. optimized the above metabolic pathway genes and achieved the in vitro biosynthesis of parthenolide in yeast, but the conversion efficiency from costunolide to parthenolide in the last step was low, only 4.8% (Shi et al., 2022). The development of synthetic chemistry has provided new ways for the production of many drugs, but the chemical synthesis of parthenolide requires 13 chemical reactions, which is complicated and costly (Freund & Arndt, 2016). Summary of the invention

[0004] Purpose of the invention: The first purpose of the present invention is to provide a Magnolia bicolor MbiPTS gene and an overexpression vector. The second purpose of the present invention is to provide the use of the above gene in the synthesis of parthenolide. The third purpose of the present invention is to provide a method for identifying whether Magnolia bicolor contains parthenolide. The fourth purpose of the present invention is to provide a method for increasing the content of parthenolide in Magnolia bicolor.

[0005] Technical solution: The Magnolia biondii MbiPTS gene of the present invention, the CDS sequence of the Magnolia biondii MbiPTS gene is shown as SEQ ID NO.1.

[0006] Furthermore, the amino acid sequence of the protein encoded by the Magnolia biondii MbiPTS gene is shown in SEQ ID NO.2.

[0007] The Magnolia biondii MbiPTS gene overexpression vector of the present invention contains the Magnolia biondii MbiPTS gene mentioned above.

[0008] The Magnolia biondii MbiPTS gene and the Magnolia biondii MbiPTS gene overexpression vector of the present invention are used in identifying, synthesizing and / or increasing the content of parthenolide in Magnolia biondii.

[0009] The method for identifying whether Magnolia bicolor contains parthenolide adopts KASP gene analysis technology to detect the CDS sequence of MbiPTS gene in the Magnolia bicolor sample to be tested. If AAA is present at 1057-1059 bp, the root bark of the Magnolia bicolor sample contains parthenolide; if a single base A deletion occurs at the 1057-1059 bp site, the root bark of the Magnolia bicolor sample does not contain parthenolide.

[0010] Furthermore, the primer pair used for PCR includes a reverse primer whose nucleotide sequence is shown in SEQ ID NO.3 to 4, and a universal forward primer whose nucleotide sequence is shown in SEQ ID NO.5; if the 1057bp site in the amplified product is a homozygous T / T site or a heterozygous T / C site, the root bark of the Magnolia biondii sample contains parthenolide; if the 1057bp site in the amplified product is a homozygous C / C, the root bark of the Magnolia biondii sample does not contain parthenolide.

[0011] Furthermore, the PCR amplification program is pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing-extension at 62°C for 60 s, for a total of 10 cycles, with the annealing temperature lowered by 0.6°C after each cycle; denaturation at 94°C for 20 s, annealing-extension at 56°C for 60 s, for a total of 30 cycles; 37°C for 1 min, and collecting fluorescence signals.

[0012] The method for increasing the content of parthenolide in Magnolia bicolor comprises the following steps: constructing an MbiPTS gene overexpression vector of Magnolia bicolor, transforming the overexpression vector into Agrobacterium rhizogenes K599, and then injecting the overexpression vector into the hypocotyl of Magnolia bicolor.

[0013] Furthermore, the original vector used to construct the Magnolia biondii MbiPTS gene overexpression vector was pcambia2301.

[0014] Furthermore, the method for verifying whether the MbiPTS gene has been successfully introduced into Magnolia biondii is to use GUS staining solution to stain the successfully induced hairy roots. Magnolia biondii whose hairy roots are dyed blue means that the MbiPTS gene has been successfully introduced.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following outstanding and significant advantages: The present invention is the first to discover the key gene MbiPTS that controls the synthesis of parthenolide in the root bark of Magnolia bicolor. When there is a single base A deletion between the 1057bp to 1059bp sites of the MbiPTS gene ORF, a frameshift mutation occurs in the MbiPTS gene, a TAA stop codon appears at 1065bp, and protein translation is terminated prematurely, and the Magnolia bicolor does not contain parthenolide. The detection of this site can be used to identify whether Magnolia bicolor contains parthenolide. Injecting the Magnolia bicolor MbiPTS gene overexpression vector into the hypocotyl of Magnolia bicolor that does not contain parthenolide can restore the synthesis of parthenolide, providing a basis for increasing the content of parthenolide in the root bark of Magnolia bicolor. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the genome-wide association site for parthenolide content;

[0017] Figure 2 Schematic diagram of the MbiPTS overexpression vector structure;

[0018] Figure 3 Screening individuals with homozygous C / C mutation;

[0019] Figure 4 This is a schematic diagram of the injection site of K599 Agrobacterium in Magnolia grandiflora seedlings;

[0020] Figure 5 The results of the growth of hairy roots of Magnolia grandiflora after injection;

[0021] Figure 6 GUS staining results of positive roots after infection with Agrobacterium rhizogenes;

[0022] Figure 7 Results of determination of parthenolide content in positive roots and control roots after infection with Agrobacterium rhizogenes. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0024] Example 1 Development of molecular markers and primers

[0025] The invention conducts association analysis on the content of parthenolide in the root bark and the genotype through the natural population material of Magnolia biondii, and obtains the key site for the synthesis of parthenolide.

[0026] A key gene MbiPTS controlling the synthesis of parthenolide in the root bark of Magnolia bicolor was found. RNA was extracted from the root bark of Magnolia bicolor and converted into cDNA. The full-length sequence of MbiPTS was cloned, providing a basis for overexpressing the gene by genetic engineering to increase the content of parthenolide in the root bark of Magnolia bicolor.

[0027] After years of research, the project team found that parthenolide is present in high levels in the root bark of Magnolia grandiflora, and that there is abundant genetic variation in natural populations, ranging from 0.0% to 8.21%. Using phenotypic data and genotypes for association analysis is one of the main ways to screen for functional genes of important traits. First, referring to the determination method of parthenolide in the root bark of Magnolia grandiflora by Zhu Xinghua et al. (ZL201710854730.1), the content of parthenolide in the root bark of 500 Magnolia grandiflora samples from Nanzhao County, Henan Province was determined. Twenty-four individuals with content below 0.5% and above 3% were selected for whole genome resequencing, with an average sequencing depth of 30×. FastQC and Trimmomatic were used to filter the low-quality sequences and adapter sequences of the sequencing. Then, BWA (Li et al., 2010) was used to align the filtered sequences to the Magnolia grandiflora reference genome (https: / / doi.org / 10.5061 / dryad.s4mw6m947). Samtools (Li et al., 2009) was used to sort the alignment results and remove duplicates. GATK software was used for local realignment and base quality correction to ensure the accuracy of the obtained SNPs and Indels. The missing genotype information was inferred using the ProbABEL R package, and the genotype was associated with the phenotypic traits using the GEMMA software. Based on the degree of significant association, the key sites controlling the synthesis of parthenolide were identified. A total of 24,051,833 SNP sites were obtained through resequencing data analysis of 48 individuals. Through association analysis with the content of parthenolide in the root bark, it was found that 17 SNP sites located in the interval of 11,039,065 to 11,193,126 bp on chromosome 13 were closely associated with the content of parthenolide (the results of the genome-wide association analysis (GWAS) of parthenolide in Magnolia officinalis are shown in Figure 2). Figure 1 As shown in the figure, the parthenolide-related sequence signals are all distributed in the proximal telomeric region of chromosome 13).

[0028] A single base SNP site was found at 11105730 bp on chromosome 13 of Magnolia grandiflora, which is located on the MbiPTS gene. The CDS sequence of MbiPTS is shown in SEQ ID NO.1 ( ACATTCA GTTGTTGGATATTCTTCTCACAGACTGGGGTTATCCTACTAGTTATATAGCAGT TATGTTTTATAAGCAAATAAGGAGAATCTAGACATGAGTGAAGCTTCAGGTTTGGATGCTCATAAG ; Wherein, the underlined part is the UTR region, and the wavy line is the base deletion region). The amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2 (MAIFSSIFFFPIIILILFGLNRIQSKKHNQPPSPSKLPIIGNLHQLSSLPHRSLQALSRKYGPLMLLHLGSTPTLIVSTAEMAQEVMRTHDLAFSSRPTFTAAKELLYGCTDVAMAAYGEYWRQAKKISVTELLSPKRVQSFAYVREEEITSMIKEIKGSCSMSGTVDITKKFLHLTSSVVSRVALGKNYIERDSNGRNTFADMARELPLLLGSFSAADLFPLFGWFDVLTGLDGKLKRNSRGLDSFFDQVIEDHLNNKS GDVNDEHKDFVDILLHIQKDCTTGVQISKENIKAIALDMYVAATGLTNTLEWTMAELVKSPNAMMKAQEEVRRVVGRKMKVEEKDLLQMNYLKCVIKETLRLHPPAPLLIPRESISSVIL QGYVIPPKTRVLVNAFAIGRDPNSWENANEFLPERFVNNSIDFKGNDFQLIPFGSGRRICPGMTFANNFLEISLANVLYWFDWEFPGGANKDMDMSEDSGIGVHKKIPLHLIPTYHFS).

[0029] When the 1057bp to 1059bp site of the MbiPTS gene ORF is a single base A deletion, the MbiPTS gene undergoes a frameshift mutation, a TAA stop codon appears at 1065bp, and protein translation terminates prematurely, and the Magnolia bicolor does not contain parthenolide. When the MbiPTS gene has no single base deletion mutation between 1057bp and 1059bp of the ORF, the gene can be normally translated into protein, and the protein sequence length is 498aa, and parthenolide can be normally synthesized in the root bark of Magnolia bicolor. KASP primers were developed for this SNP site to detect single base deletions at the 1057bp to 1059bp site of the MbiPTS gene ORF. They are Mbi13-11105730 (Mbi13-11105730R-C reverse primer: 5′-gaaggtgaccaagttcatgctGCAAAGTCTCCTTAATTACGCATTC-3′ (SEQ ID NO. 3); Mbi13-11105730R-T reverse primer: 5′-gaaggtcggagtcaacggattGCAAAGTCTCCT TAATTACGCATTT-3′ (SEQ ID NO. 4); Mbi13-11105730F universal forward primer: 5′-AGTGGAA GAGAAGGACCTTCTTC-3′ (SEQ ID NO. 5).

[0030] Among them, if the primer Mbi13-11105730 amplifies the homozygous T / T or heterozygous T / C site of Magnolia biondii, the MbiPTS gene can normally translate into a protein sequence of 498aa, that is, it can synthesize parthenolide normally; if the primer Mbi13-11105730 amplifies the homozygous C / C site of Magnolia biondii, the MbiPTS gene has a single base A deletion at 1057bp~1059bp of ORF, and this single base deletion causes a frameshift mutation, and the TAA stop codon appears prematurely at 1065bp, and the protein translation is terminated prematurely at 355aa, resulting in the loss of protein function, and therefore the parthenolide cannot be synthesized normally.

[0031] Example 2 Primer Mbi13-11105730 detects half-sib families

[0032] Primer Mbi13-11105730 was used to perform fluorescence quantitative PCR amplification on DNA from different half-sib families of Magnolia grandiflora. 24 individuals of each of the four half-sib families No. 2, No. 4, No. 22 and No. 29 were amplified from seeds of 30 trees collected from Nanzhao, Henan Province (these Magnolia grandiflora were open-pollinated, so the seeds on each tree were from the same half-sib family). Genomic DNA from Magnolia grandiflora leaves was extracted using the Tiangen New Plant Genome Extraction Kit (DP320), and the extracted DNA was diluted to 30 ng / μL and stored at -20℃ for later use.

[0033] The PCR reaction system was as follows: 10.0 μL of 2×castPCR Genotyping Master Mix, 2 μL of 30 ng / μL DNA template, 0.3 μL of 10 μM Allele1 primer (Mbi13-11105730R-C) and Allele2 primer (M bi13-11105730R-T), 0.8 μL of 10 μM universal forward primer, and 6.6 μL of dd H2O in each 20 μL system. The PCR amplification program was as follows: 94°C pre-denaturation for 15 min; 94°C denaturation for 20 s, 62°C annealing-extension for 60 s, for a total of 10 cycles (annealing temperature was reduced by 0.6°C after each cycle); 94°C denaturation for 20 s, 56°C annealing-extension for 60 s, for a total of 30 cycles; 37°C for 1 min, and fluorescence signal collection. The experimental results showed that only half-sib family No. 4 had a homozygous C / C genotype. Further testing of 120 individuals in half-sib family No. 4 revealed that 42 individuals were homozygous C / C genotypes ( Figure 3 ), 30 seedlings were injected with the overexpression vector and the rest were used as blank controls.

[0034] Primers were designed in the UTR region of the MbiPTS gene, the forward fragment cloning primer MbiPTS_F_Re (SEQ ID NO.6, sequence 5′-gagaacacgggggactctagaACATTCAATGGCTATCTTTTCATCT-3′) and the reverse fragment cloning primer MbiPTS_R_Re (SEQ ID NO.7, 5′-gagctcggtacccggggatccCTTATGAGCATCCAAACCTGAAG-3′), where lowercase letters are homologous sequences of vector restriction sites and uppercase letters are specific sequences of cloned genes. The cDNA reversed from the RNA extracted from the root bark was used as a template to complete the cloning of the target fragment gene using PCR. The PCR amplification system was 50 μL: 5×GXL buffer 10 μL, 2.5 μM NTP 4 μL, cDNA 1 μL, GXL Taq 2.5U, 10mM forward and reverse primers 3 μL each, and dd H2O was supplemented to 50 μL. The PCR amplification procedure was as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 15 s, primer annealing and extension at 68°C for 3 min, a total of 35 cycles; final extension at 72°C for 10 min, and storage at 15°C. After the PCR was completed, 1% agarose gel was electrophoresed at 120V for 30 min, and then the target fragment was recovered by gel excision using the Tiangen Agarose Gel DNA Recovery Kit (Enhanced) (DP219). 150 ng of the pCambia2301 plasmid (purchased from Cambia) after double digestion with BamHI and XbaI was taken, and 60 ng of the MbiPTS gene cloned fragment was recovered by gel excision. The forward sequence of the MbiPTS gene fragment was recombined into the pcambia2301 plasmid using the Seamless Cloning and Assembly Kit (CU201-02), and then transformed into the competent E. coli DH5α cells. The plates were streaked and single clones were picked for expansion culture. Then, the transformed positive clones were selected for sequencing by PCR amplification and identification. The recombinant plasmid of pcambia2301 connected to the forward fragment was obtained, and the MbiPTS overexpression vector pcambia2301-35s-plus (vector structure as shown in the figure) was constructed. Figure 2 The plasmid for constructing the vector was extracted and transformed into Agrobacterium rhizogenes K599. The vector was introduced into the individuals of Magnolia grandiflora Mb iPTS gene whose translation was terminated prematurely by injection into the hypocotyl (the injection site was as shown in Figure 4 Each individual was injected with 100 μL of the solution. After the injection, the seedlings were planted in a 10 cm × 10 cm nutrient pot and then incubated at 25°C with a light condition of 16h / 8h. After 60 days of cultivation, the infection efficiency of Agrobacterium rhizogenes was tested. Of the 30 seedlings injected, 26 seedlings successfully grew hairy roots from the site of Agrobacterium rhizogenes injection ( Figure 5 ), the induction rate of hairy roots was 86.7%. The hairy roots that were successfully induced were stained with GUS chromosomes, and the hairy roots of 14 seedlings were stained blue ( Figure 6 ), indicating that the GUS gene on the vector was expressed in the hairy roots, and also proved that the MbiPTS gene was successfully transferred into the hairy roots of Magnolia biondii.

[0035] The hairy roots of all GUS-stained Magnolia dahurica individuals were collected as the experimental group, and the roots of Magnolia dahurica that were not injected with Agrobacterium rhizogenes and whose MbiPTS gene translation was terminated prematurely were used as the control. The root bark was dried at 60°C, and then 0.500g of the sample was accurately weighed, soaked in 50mL of ethanol for 24h, and filtered with a 0.22μm filter membrane. The content of root parthenolide was determined by liquid chromatography (Liu Jingsheng et al., 2024). The chromatographic column was Inertsil ODS-3 (4.6×250mm, 5μm), octadecylsilane bonded silica gel was used as the filler, acetonitrile: water = 60:40 was used as the mobile phase, the flow rate was 1.0mL / min, the column temperature was 30°C, the separation time was 15min, the detection wavelength was 220nm, and 20.0μL of the standard was loaded. The results showed that the root bark of Magnolia dahurica that was not injected with Agrobacterium rhizogenes and whose MbiPTS gene translation was terminated prematurely did not contain parthenolide ( Figure 7 B), while the average content of parthenolide in GUS-positive hairy roots was 0.29% ( Figure 7 C). It was demonstrated that overexpression of the MbiPTS gene can restore the anabolism of feverfew, and therefore it is believed that the MbiPTS gene is the only (key) gene in Magnolia biondii that converts costunolide into parthenolide.

Claims

1. A Magnolia grandiflora MbiPTS A gene characterized by Magnolia grandiflora MbiPTS The sequence of the gene is shown in SEQ ID NO.

1.

2. Magnolia biondii according to claim 1 MbiPTS A gene characterized by Magnolia grandiflora MbiPTS The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. A Magnolia MbiPTS A gene overexpression vector, characterized in that: The overexpression vector contains the Magnolia biondii described in claim 1 MbiPTS Gene.

4. The Magnolia biondii described in any one of claims 1 to 2 MbiPTS Gene, Magnolia biondii according to claim 3 MbiPTS Application of gene overexpression vector in identifying, synthesizing and / or increasing the content of parthenolide in Magnolia biondii.

5. A method for identifying whether Magnolia biondii contains parthenolide, characterized in that: KASP gene analysis technology was used to detect the MbiPTS For the ORF sequence of the gene, if AAA is present at 1057-1059 bp, the root bark of the Magnolia biondii sample contains parthenolide; if a single base A deletion occurs at the 1057-1059 bp site, the root bark of the Magnolia biondii sample does not contain parthenolide.

6. The method according to claim 5, characterized in that The primer pair used for PCR includes a reverse primer whose nucleotide sequence is shown in SEQID NO. 3~4, and a universal forward primer whose nucleotide sequence is shown in SEQ ID NO. 5; if the 1057 bp site in the amplified product is a homozygous T / T site or a heterozygous T / C site, the root bark of the Magnolia biondii sample contains parthenolide; if the 1057 bp site in the amplified product is a homozygous C / C, the root bark of the Magnolia biondii sample does not contain parthenolide.

7. The method according to claim 6, characterized in that The PCR amplification program was as follows: pre-denaturation at 94°C for 15 min; denaturation at 94°C for 20 s, annealing-extension at 62°C for 60 s, for a total of 10 cycles, with the annealing temperature lowered by 0.6°C after each cycle; denaturation at 94°C for 20 s, annealing-extension at 56°C for 60 s, for a total of 30 cycles; and incubation at 37°C for 1 min, and collecting fluorescence signals.

8. A method for increasing the content of parthenolide in Magnolia biondii, characterized in that: The following steps are involved: Construct the Magnolia grandiflora according to claim 1 MbiPTS The gene overexpression vector is transformed into Agrobacterium rhizogenes K599 and then injected into the hypocotyl of Magnolia biondii.

9. The method according to claim 8, characterized in that Magnolia grandiflora MbiPTS The original vector used for gene overexpression vector is pCambia2301.

10. The method according to claim 8, characterized in that verify MbiPTS The method for determining whether the gene has been successfully transferred into Magnolia grandiflora is as follows: the hairy roots that have been successfully induced are stained with GUS staining solution, and the Magnolia grandiflora whose hairy roots are stained blue is MbiPTS Gene transfer was successful.

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