Ginkgo terpene synthase gene GbTPS13 and application thereof in heterologous synthesis of ginkgolide precursor in tobacco

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a ginkgo terpene synthase gene GbTPS13 and application of the ginkgo terpene synthase gene GbTPS13 in heterologous synthesis of a bilobalide precursor in tobacco. The nucleotide sequence of the ginkgo terpene synthase gene GbTPS13 is as shown in SEQ ID NO. 1, and the coded amino acid sequence is as shown in SEQ ID NO. 2; ginkgo biloba diterpene synthase GbTPS13 is identified in tobacco tissues, L-pimaric diene can be catalytically generated through overexpression of GbTPS13 in tobacco, the diterpene compound is a common precursor of bilobalide, and the method is used for constructing a tobacco chassis system capable of being used for synthesizing bilobalide. And a potential starting platform and basic framework are provided for complete and large-scale biosynthesis of bilobalide.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to a ginkgo terpene synthase gene. GbTPS13 And its application in the heterologous synthesis of ginkgolide precursors from tobacco. Background Technology

[0002] ginkgo( Ginkgo biloba Ginkgo biloba, an ancient species that has survived on Earth for approximately 200 million years, is hailed as a "living fossil" of plants. It is also one of the world's most popular medicinal plants, and its extracts contain various secondary metabolites unique to ginkgo, such as flavonoids with potential therapeutic value for cardiovascular and cerebrovascular diseases. Due to its widespread application in pharmaceuticals, food, skincare products, and landscaping, ginkgo has been cultivated on a large scale. All parts of the ginkgo plant have significant research value. The leaves are rich in various active compounds, especially flavanol glycosides and terpene trilactones; research in traditional Chinese medicine has confirmed that these components can improve blood circulation and inhibit thrombus formation. Unique and medicinally active terpene lactones (TTLs) in ginkgo, mainly including ginkgolides A, B, C, and ginkgolides (BB), are primarily detected in its roots and leaves.

[0003] The precursor biosynthesis of TTLs mainly relies on the mevalonate (MVA) and methyl erythritol phosphate (MEP) pathways. First, terpene synthases (TPS) catalyze the formation of the terpene backbone, which is then modified by cytochrome P450 (CYP450) enzymes. Although more than ten genes involved in the TTL biosynthesis pathway have been successfully cloned, research on the key enzyme GbTPSs remains limited. To date, only a few terpene synthases involved in ginkgolide synthesis have been reported: one diterpene synthase—L-piperene synthase (GbLPS), two sesquiterpene synthases—farnesol synthase (GbTPS1) and (-)-α-bisabolene synthase (GbTPS2), and one GbLPS2 whose function is not yet clearly characterized. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention aims to provide a ginkgo terpene synthase gene. GbTPS13 and its use in tobacco ( Nicotiana tabacum Application in the heterologous synthesis of ginkgolide precursors and overexpression in tobacco GbTPS13 The catalytic generation of L-pinenediene, a diterpenoid compound that is a common precursor to ginkgo diterpenoid lactones, can be used to construct a tobacco chassis system for the synthesis of ginkgo lactones.

[0005] To solve the above problems, the present invention adopts the following technical solution: In a first aspect, the present invention provides a ginkgo terpene synthase gene. GbTPS13 The Ginkgo terpenoid synthase gene GbTPS13The nucleotide sequence is shown in SEQ ID NO.1.

[0006] Furthermore, the Ginkgo terpenoid synthase gene GbTPS13 The encoded amino acid sequence is shown in SEQ ID NO.2.

[0007] Furthermore, the Ginkgo terpenoid synthase gene GbTPS13 It is only expressed in ginkgo roots.

[0008] Secondly, the present invention provides a gene engineering vector comprising the aforementioned ginkgo terpene synthase gene. GbTPS13 .

[0009] Thirdly, the present invention provides a genetically engineered bacterium, characterized in that it includes the aforementioned genetic engineering vector.

[0010] Fourthly, the present invention provides the aforementioned Ginkgo terpene synthase gene. GbTPS13 Or the application of the genetically engineered vector or the genetically engineered bacteria in the heterologous synthesis of ginkgolide precursors in tobacco.

[0011] Furthermore, the ginkgolide precursor is a diterpenoid, L-pinediene.

[0012] Furthermore, the Ginkgo terpenoid synthase gene GbTPS13 Promotes the synthesis and accumulation of ginkgolide B and ginkgolide C.

[0013] Fifthly, the present invention provides a method for heterologous synthesis of ginkgolide precursors in tobacco, characterized in that the ginkgolide synthase gene is overexpressed in tobacco tissue. GbTPS13 .

[0014] Furthermore, this includes transferring the genetically engineered vector or the genetically engineered bacteria into the tobacco leaf disc.

[0015] The beneficial effects of this invention are: GbTPS A genome-wide systematic identification of the gene family was performed. Through heterologous and homologous overexpression comparisons, the GbTPS13 diterpenoid synthase from Ginkgo biloba was identified in tobacco and Ginkgo biloba callus. Functional studies showed that overexpression in tobacco... GbTPS13 It can catalyze the formation of L-pinediene, a diterpenoid compound that is a common precursor to ginkgo diterpenoid lactones. This provides a promising starting platform and basic framework for constructing a tobacco chassis system that can be used to synthesize ginkgo lactones, and for the complete and large-scale biosynthesis of ginkgo lactones. Furthermore, transient overexpression in ginkgo callus tissue... GbTPS13 It can effectively enhance the levels of ginkgolides B and C, providing key gene resources and a feasible engineering platform for the synthetic biology research of ginkgolides, and laying the foundation for further analysis and reconstruction of their biosynthetic pathways. Attached Figure Description

[0016] Figure 1 This is a chromosomal mapping of the genes in this invention.

[0017] Figure 2 This is the phylogenetic tree diagram of the present invention.

[0018] Figure 3 For the present invention GbTPS13 The organizational expression pattern diagram.

[0019] Figure 4 In the positive strains of this invention GbTPS13 The level of expression.

[0020] Figure 5 For the overexpression of this invention GbTPS13 Graph showing the analysis of enzyme-catalyzed products in the strain.

[0021] Figure 6 For the present invention GbTPS13 Diagram showing the process of transferring ginkgo callus tissue.

[0022] Figure 7 This is a quantitative comparison diagram of four terpene lactone substances in Ginkgo callus tissue of the present invention. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments.

[0024] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0025] Example 1 GbTPS Gene family identification and analysis ginkgo( Ginkgo biloba The genome and coding sequences (CDS), protein sequences, and location information of Ginkgo biloba were obtained from the National Genome Science Data Center and the Genome Sequence Archive Database (https: / / ngdc.cncb.ac.cn / gwh / Assembly / 18742 / show). Based on the Pfam accession numbers (PF0393 and PF01397) corresponding to the C-terminal and N-terminal characteristic domains of terpene synthase (TPS), the corresponding Hidden Markov Model (HMM) configuration files were obtained and downloaded from the Pfam database. Subsequently, HMMER 3.2 software was used to analyze the Ginkgo biloba genome using these HMM files. GbTPS Identification of candidate genes. Based on the Ginkgo genome GFF3 annotation file, through analysis... GbTPS The location of genes on chromosomes determines their chromosomal position, and they are named accordingly.Figure 1 ).

[0026] Selected from gymnosperm North American spruce ( Picea sitchensis ), white spruce ( Picea glauca North American fir ( Abies grandis Norway spruce ( Picea abies ), Taiwan fir ( Taiwania cryptomerioides ),ginkgo( Ginkgo biloba ) and small bowl moss ( Physcomitrella patens Arabidopsis thaliana ( ) Arabidopsis thaliana One hundred TPS protein sequences were obtained and multiple sequence alignment was performed using MAGA 12. The alignment results were imported into Jalview 2.11.1.4 for visualization and editing. A phylogenetic tree was constructed using maximum likelihood (ML) with 1000 bootstrap replicates and other parameters kept at default settings. Finally, the phylogenetic tree was visualized using R 4.5.0. Figure 2 ).

[0027] A total of 33 were identified. GbTPS Gene family members are distributed across 7 chromosomes, among which GbTPS13 Located on chromosome 5 and in the TPS-d-3 branch unique to gymnosperms, this subfamily is mainly composed of diterpenoid synthases (diTPSs). GbTPS13 The gene sequence and the specific information of the encoded amino acids are shown in Table 1.

[0028] Table 1 GbTPS13 The gene sequence and the encoded amino acid sequence

[0029] Example 2 GbTPS13 Expression patterns in different tissues of Ginkgo biloba Explore GbTPS13 The dynamic expression characteristics of genes were analyzed using 131 publicly available transcriptome datasets from the National Center for Biotechnology Information (NCBI), covering various tissues and developmental stages, including leaves, stems, roots, reproductive organs, and cambium. Roots are the main site of ginkgolide synthesis, and the study found... GbTPS13 The gene exhibits tissue-specific expression, being expressed only in the root. Figure 3 ). In the root tissue GbTPS13 The expression level was used as the normalization benchmark (set to 1). GbGAPDH The gene was used as an internal control, and the results were verified by qRT-PCR using the primer set shown in Table 2. GbTPS13 The organization expression pattern. The results indicate that the gene...GbTPS13 The expression pattern is consistent with the transcriptome analysis, specifically high expression in the root and very low or no expression in other parts. Figure 3 ).

[0030] Table 2 Primer sequence listing

[0031] Example 3 Root expression genes GbTPS13 Heterogeneous transformation and metabolite detection of Tobacco monotypic leaf Cloning root expression genes GbTPS13 And it was constructed onto the pCAMBIA1300 vector, and the constructed pCAMBIA1300- GbTPS13 Agrobacterium tumefaciens GV3101 was transformed with the empty vector pCAMBIA-1300 using a freeze-thaw method. The transformed Agrobacterium was then inoculated into LB broth containing kanamycin and rifampin and cultured at 28°C with shaking at 200 rpm until OD200 was reached. 600 The value reached approximately 0.6. Tobacco leaves used for Agrobacterium infiltration were collected from sterile seedlings cultivated under laboratory conditions. Transgenic tobacco plants were obtained by co-culturing Agrobacterium carrying the target gene with tobacco leaf discs using the Agrobacterium-mediated leaf disc transformation method. Positive seedlings were preliminarily screened by PCR, and RT-qPCR was used to further analyze the positive lines. GbTPS13 Further verification of the expression level ( Figure 4 Finally, the confirmed transgenic seedlings were transferred to a culture room for adaptive cultivation.

[0032] Select three GbTPS13 Leaves of transgenic tobacco lines with high expression levels were analyzed by GC-MS to identify their enzyme-catalyzed products in the heterologous tobacco system. GC chromatograms showed that, in the overexpression... GbTPS13 The strain exhibited specific peaks not seen in the empty vector (EV) control. Subsequent mass spectrometry analysis confirmed that... GbTPS13 The product is a diterpenoid, L-pinediene, whose functional classification is highly consistent with its phylogenetic classification results. Figure 5 ).

[0033] Example 4 GbTPS13 Transient transformation and metabolite detection in Ginkgo callus Tender leaves from one-month-old ginkgo seedlings were surface-sterilized using 75% ethanol and 5% sodium hypochlorite solutions, respectively, and then rinsed three times with sterile water. The sterilized leaves were then cut into 1×1 cm pieces. 2 Fragments were inoculated into callus induction medium and cultured at 25°C in the dark for 30 days. The culture was performed using a pCAMBIA1300-GbTPS13 Ginkgo callus was infected using Agrobacterium-mediated transient transformation with the vector. The infected callus tissue was then transferred to MS medium containing 150 μM acetylsyringone (AS) and co-cultured in the dark at 25°C for 3 days. RT-qPCR was used for detection. GbTPS13 The expression levels in transiently transformed Ginkgo callus were investigated, and the results showed that, compared with the control group transformed with the empty vector, the expression levels in transiently transformed Ginkgo callus were significantly lower. GbTPS13 The expression of all genes was significantly increased, indicating that the gene was successfully transferred into Ginkgo callus tissue. Figure 6 ).

[0034] GC-MS was used to quantitatively analyze four terpenoid lactones (BB, GA, GB, and GC) in Ginkgo callus. The results showed that GB had the highest content in Ginkgo callus, making it the main component of Ginkgo terpenoid lactones, followed by BB and GA, while GC had the lowest content. Functional validation results indicated that, compared with callus transformed with an empty vector, GbTPS13 Overexpression of [a substance] can significantly promote the synthesis and accumulation of GB and GC in Ginkgo callus. Figure 7 ).

[0035] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A ginkgo terpene synthase gene GbTPS13 characterized in that, The ginkgo terpene synthase gene GbTPS13 The nucleotide sequence of the ginkgo terpene synthase gene is shown as SEQ ID NO.

1.

2. The ginkgo terpene synthase gene of claim 1 GbTPS13 characterized in that, The ginkgo terpene synthase gene GbTPS13 The encoded amino acid sequence is shown as SEQ ID NO.

2.

3. The ginkgo terpene synthase gene of claim 1 GbTPS13 characterized in that, The ginkgo terpene synthase gene GbTPS13 Only expressed in ginkgo roots.

4. A genetically engineered vector, characterized by, The ginkgo terpene synthase gene of claim 1 GbTPS13 .

5. A genetically engineered bacterium, characterized by, The genetically engineered vector of claim 3.

6. The ginkgo terpene synthase gene of claim 1 GbTPS13 or the use of the genetically engineered vector of claim 4 or the genetically engineered bacteria of claim 5 in the heterologous synthesis of ginkgolide precursors in tobacco.

7. Use according to claim 6, characterized in that, The ginkgolide precursor is diterpene levopimaradiene.

8. Use according to claim 6, characterized in that, The ginkgo terpene synthase gene GbTPS13 Promote the synthesis and accumulation of ginkgolide B and ginkgolide C.

9. A method for the heterologous synthesis of ginkgolide precursors in tobacco, characterized in that, Overexpression of the ginkgo terpene synthase gene of claim 1 in tobacco tissue GbTPS13 .

10. The method of claim 9, wherein, The genetically engineered vector of claim 4 or the genetically engineered bacteria of claim 5 is introduced into tobacco leaf disc.

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