Baimuxiang aserfb3-4 gene and application thereof in regulating sesquiterpene synthesis

Abstract

This invention discloses white sandalwood. AsERFB3‑4 The gene and its application in regulating sesquiterpene synthesis are shown in SEQ ID NO:1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2. AsERFB3‑4 belong AP2 / ERF Family B3 subfamily, located in the cell nucleus, possesses transcriptional activation activity and can directly bind to and activate [various transcriptional activators]. AsHMGR1、AsHMGR2 , AsHMGR3、 AsMVK1 , AsMVD1 , AsTPS2、AsTPS3 , AsCYP71‑1, AsCYP71‑2 This gene is involved in the transcription of promoters for several key enzyme genes involved in the synthesis of sesquiterpenes. Overexpression of this gene in Aquilaria sinensis can increase the content of various characteristic sesquiterpenes, providing an important gene resource for regulating the quality of agarwood and cultivating plant varieties with high sesquiterpenes content.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to Aquilaria sinensis. AsERFB3-4 Genes and their application in regulating sesquiterpene synthesis. Background Technology

[0002] Agarwood belongs to the genus Aquilaria in the family Thymelaeaceae. Aquilaria ) or Aquilaria genus ( Gyrinops Agarwood, a resinous wood formed from damaged plant stems, is widely used in medicine, incense culture, and daily chemicals. Due to its high value and large international market demand, wild agarwood resources are on the verge of extinction due to over-logging. White agarwood (Aquilaria sinensis) Aquilaria sinensis Agarwood (Aquilaria sinensis) is a tropical and subtropical evergreen tree endemic to my country and the only source plant for Chinese agarwood. Under natural conditions, agarwood formation is extremely slow, requiring decades or even centuries to accumulate. To accelerate agarwood formation, researchers have successively developed methods such as mechanical damage, chemical induction, fungal inoculation, and whole-tree resin formation. However, the quality of artificially induced agarwood is still inferior to that of natural agarwood, one of the main reasons being the insufficient content of characteristic components. Sesquiterpenes are the main active components of agarwood and a key indicator for evaluating its quality. Their accumulation is considered a result of plant response to environmental stress, but the relevant regulatory mechanisms remain unclear. Therefore, elucidating the regulatory mechanisms of sesquiterpene biosynthesis is of great significance for revealing the formation mechanism of agarwood and developing efficient resin formation technologies.

[0003] The biosynthetic pathway of sesquiterpenes in agarwood has been largely elucidated. This pathway mainly involves the synthesis of the precursor isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) via the mevalonate (MVA) pathway in the cytoplasm and the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway in the plastids. Subsequently, under the sequential catalysis of geranyl diphosphate synthase (GPPS) and farnesyl diphosphate synthase (FPS), farnesyl pyrophosphate (FPP) is generated, which is then catalyzed by terpene synthase (TPS) to form a diverse sesquiterpene hydrocarbon skeleton. Finally, through hydroxylation and epoxidation by cytochrome P450 enzymes, it is converted into a rich variety of sesquiterpene compounds. The biosynthesis of plant terpenes is mainly regulated by key enzyme genes and transcription factors, among which transcription factors can activate the synergistic expression of multiple key enzyme genes, thereby effectively regulating the synthesis of terpenes. Currently, although preliminary explorations have been made into the regulatory mechanisms of sesquiterpene biosynthesis in Aquilaria species, and several transcription factors involved in the regulation have been identified, such as... AsMYC2 , AsERF1 , AsWRKY44 and AsZFP9However, existing research mainly focuses on the regulation of a single synthase gene by a single transcription factor, and there is a lack of exploration of core transcription factors that can simultaneously regulate the synergistic expression of multiple genes in the sesquiterpene synthesis pathway.

[0004] Therefore, identifying the core transcription factors of multiple genes that regulate the sesquiterpene synthesis pathway in Aquilaria species is of great significance for increasing the sesquiterpene content of Aquilaria through metabolic engineering and for cultivating Aquilaria malaccensis varieties with high sesquiterpene content through genetic engineering. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies, such as the inferior quality of artificially induced agarwood compared to natural agarwood, particularly the insufficient content of its characteristic component, sesquiterpenes, and the current lack of core transcription factors that can promote the expression of multiple key enzyme genes in the sesquiterpenes synthesis pathway. It provides a method for producing agarwood from white agarwood. AsERFB3-4 Genes and their application in promoting sesquiterpene synthesis. The transcription factor AsERFB3-4 identified in this invention can activate multiple key downstream synthetic genes, thereby effectively promoting the accumulation of sesquiterpene compounds.

[0006] In a first aspect, the present invention provides a transcription factor gene that promotes the biosynthesis of sesquiterpenes in Aquilaria sinensis. AsERFB3- 4 The gene is characterized in that its nucleotide sequence is the nucleotide sequence shown in SEQ ID NO: 1.

[0007] Furthermore, the gene AsERFB3-4 The encoded protein is located in the cell nucleus and has transcriptional activation activity.

[0008] Furthermore, the protein is capable of binding to and activating sesquiterpene synthesis-related enzyme genes. AsHMGR1, AsHMGR2 , AsHMGR3, AsMVK1 , AsMVD1 , AsTPS2, AsTPS3 , AsCYP71-1, AsCYP71-2 Promoter transcriptional activity in the middle.

[0009] Secondly, the present invention provides a material made of white sandalwood. AsERFB3-4 A gene-encoded protein, characterized in that the amino acid sequence of the protein is the amino acid sequence shown in SEQ ID NO: 2.

[0010] Thirdly, the present invention provides a method comprising... AsERFB3-4 Gene recombinant vectors or expression cassettes.

[0011] Fourthly, the present invention provides a host cell comprising the above-described recombinant vector or expression cassette.

[0012] Fifthly, the present invention providesAsERFB3-4 The application of the gene, the protein, or the recombinant vector or expression cassette in promoting the biosynthesis of sesquiterpenoids from Aquilaria sinensis; the protein is capable of binding to and activating the promoters of the following key enzyme genes for sesquiterpene synthesis: AsHMGR1 (SEQ ID NO: 5) AsHMGR2 (SEQ ID NO: 6) AsHMGR3 (SEQ IDNO: 7) AsMVK1 (SEQ ID NO: 8) AsMVD1 (SEQ ID NO: 9) AsTPS2 (SEQ ID NO: 10) AsTPS3 (SEQ ID NO: 11) AsCYP71-1 (SEQ ID NO: 12) AsCYP71-2 (SEQ ID NO: 13).

[0013] Furthermore, the sesquiterpenoid compound is selected from one or more of the following: (-)-aristolone, humulene, styraxene, δ-elemene, dehydroacorene, δ-juniperene, cedrol, α-linolene, longleafene, longleafene, β-longiene, trans-nerolidol, α-cucurbitene, and α-didehydroacorene.

[0014] Sixthly, the present invention provides a method for increasing the sesquiterpene content of Aquilaria sinensis, comprising: The AsERFB3-4 Genes were introduced into Aquilaria sinensis and overexpressed therein; the introduction method included Agrobacterium-mediated transient transformation. Beneficial effects

[0015] The white sandalwood disclosed in this invention AsERFB3-4 As a core regulatory factor, the gene can activate multiple key enzyme genes in the sesquiterpene synthesis pathway (such as...). AsHMGR , AsTPS and AsCYP71 This invention utilizes family genes to promote the accumulation of various characteristic sesquiterpenes in Aquilaria sinensis. It provides important genetic resources for regulating the sesquiterpene content of agarwood and cultivating high-quality agarwood varieties through biotechnology. Attached Figure Description

[0016] Figure 1 for AsERFB3-4 Figure showing the results of subcellular localization and transcriptional activity analysis: Figure 1 A is AsERFB3- Subcellular localization of the GFP fusion protein in onion epidermal cells (scale bar: 200 μm). ​ B is in the yeast system ​ Results of transcription activation activity assay.

[0017] ​ Yeast one-hybrid verification ​ Results of binding with the promoter of the key enzyme gene for sesquiterpene synthesis.

[0018] ​ For detection by dual-luciferase reporter system ​ LUC fluorescence imaging results of the regulation of promoter activity of target genes.

[0019] ​ For detection by dual-luciferase reporter system ​ Quantitative results of LUC / REN relative activity in regulating target gene promoter activity (*) P < 0.05,** P < 0.01).

[0020] ​ For GC-MS detection ​ The effect of transient overexpression on the accumulation of sesquiterpenoids in the stems of Aquilaria sinensis.

[0021] ​ For qRT-PCR detection ​ Results of the effect of expression on the expression of key enzyme genes in sesquiterpene synthesis (*) P < 0.05,** P < 0.01). Detailed Implementation

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; all materials and reagents used are commercially available; and all structures used are conventional structures unless otherwise specified.

[0023] Example 1: Aristolochia debilis ​ Cloning of genes This embodiment provides a transcription factor gene related to the synthesis of sesquiterpenes in Aristolochic acid. ​ The cloning method, with specific steps as follows: (1) Extraction of total RNA from plants The stem tissue of Aquilaria sinensis after injury treatment was selected, rapidly frozen in liquid nitrogen, and thoroughly ground. 50 mg of the ground tissue powder was accurately weighed, and total RNA was extracted from the stem tissue of Aquilaria sinensis using the Plant Total RNA Isolation Kit Plus from Chengdu Fuji Biotechnology Co., Ltd., according to the standard operating instructions of the kit.

[0024] (2) First-strand cDNA reverse transcription synthesis Using the total RNA extracted from the stem tissue of Aquilaria sinensis in step (1) as raw material, the MonScript™ RTIII All-in-One Mix with dsDNase reverse transcription kit from Mona Biotechnology Co., Ltd. was used. The kit was strictly followed to remove residual genomic DNA and reverse transcription was performed to synthesize the first-strand cDNA of Aquilaria sinensis stem tissue, which was used as the reaction template for subsequent gene amplification.

[0025] (3) PCR amplification of the target gene Based on the combined transcriptomic and metabolomic analysis of stem damage samples from Aquilaria sinensis, key genes of the AP2 / ERF transcription factor B3 subfamily involved in the regulation of sesquiterpene biosynthesis in Aquilaria sinensis were screened and named... ​ Design and synthesize a pair of specific amplification primers. The primer sequence information is as follows: Forward primer P1: 5′-ATGGATCCCTATCTTTTCCAGTC-3′, as shown in SEQ ID NO: 3; Reverse primer P2: 5′-TCACCAAGGCCGAGCGCCGTCGG-3′, as shown in SEQ ID NO: 4.

[0026] Using the first-strand cDNA of the stem of Aquilaria sinensis prepared in step (2) of this embodiment as an amplification template, PCR amplification was performed using the above-mentioned specific primers P1 and P2. The amplification reaction program was set as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ extension for 5 min to complete the specific amplification of the target gene fragment.

[0027] Obtained by PCR amplification ​ The complete coding region sequence of the gene is 633 bp, and its nucleotide sequence is shown in SEQ ID NO: 1. Based on the open reading frame of the gene, its encoded protein sequence is deduced. The protein encoded by this gene consists of 210 amino acid residues, and its amino acid sequence is shown in SEQ ID NO: 2.

[0028] (4) Construction of recombinant vectors and identification of positive clones The amplification obtained in step (3) ​ Gene-specific PCR products were ligated in vitro with the pMD19-T cloning vector; the ligation product was transformed into competent E. coli cells, and single-colony positive clones were obtained after plating culture and antibiotic selection; positive clones were selected for bacterial culture sequencing verification, and after confirmation of correct sequencing results, the cells containing Aristolochic acid were obtained. ​ The recombinant cloning vector for the target gene was named T-AsERFB3-4.

[0029] Example 2: Subcellular localization and transcriptional activity analysis of AsERFB3-4 To clarify ​ Subcellular localization was performed, and its coding region was ligated into the pNC-CAMBIA1304-SubC vector driven by the CaMV35S constitutive promoter. The recombinant vector was then... ​ GFP and the control empty vector pNC-CAMBIA1304-SubC were transformed into Agrobacterium GV3101, subsequently infecting onion epidermal cells. Fluorescence microscopy revealed that the control vector exhibited green fluorescence throughout the entire cell, while the recombinant vector... ​ -GFP's green fluorescence signal is confined to the cell nucleus ( ​ A).

[0030] To verify ​ To determine whether it possesses transcriptional activity, its coding sequence was ligated into the pGBKT7 vector, with pGBKT7-lam+AD as a negative control and pGBKT7-p53+AD as a positive control. ​ As shown in Figure B, the results indicate that both the control and experimental groups grew normally on the single-deficient (SD / -Trp) medium, indicating that the plasmid had been successfully transferred into yeast cells. However, on the triple-deficient medium (SD / -Trp-His-Ade-X-α-gal) containing X-α-gal, pGBKT7- ​ The positive control transformants grew normally and turned blue, while the negative control did not grow. This proves that... ​ It is a transcription factor with transcriptional activation function.

[0031] In conclusion, ​ It is located in the cell nucleus and plays its regulatory role as a transcriptional activation regulator in the cell nucleus.

[0032] Example 3 ​ Transcriptional regulation of sesquiterpene synthesis genes To clarify ​ The downstream targeting regulatory mechanism of transcription factors was investigated, and their direct target genes were identified. This embodiment employed DAP-Seq technology for whole-genome binding site analysis: AsERFB3-4 recombinant protein was incubated with genomic DNA fragments from *Aristolochia debilis*, and the bound DNA fragments were enriched before high-throughput sequencing. Data processing and motif identification were completed using the standard analytical procedures of Lanjing Kexin (Hebei) Biotechnology Co., Ltd. Through whole-genome screening and sequence analysis, the conserved binding motif of the AsERFB3-4 protein was identified as the GGC-box. The downstream target genes identified included key functional genes in the biosynthesis of sesquiterpenes from *Aristolochia debilis*, including... ​ (SEQ ID NO: 5) ​(SEQ ID NO: 6) ​ (SEQ ID NO: 7) ​ (SEQ ID NO: 8) ​ (SEQ ID NO: 9) ​ (SEQ ID NO: 10) ​ (SEQ ID NO: 11) ​ (SEQ ID NO: 12) ​ (SEQ ID NO: 13).

[0033] To verify ​ The transcriptional regulatory functions of the aforementioned target genes related to sesquiterpene synthesis were detected in vitro and in vivo using a yeast one-hybrid assay and a dual-luciferase reporter system, respectively. ​ The binding affinity to the promoters of various target genes and the regulatory effect on the transcriptional activity of the target gene promoters were determined through the following experimental procedures: (1) Carrier construction Genomic DNA was extracted from *Aquilaria sinensis* using the plant genomic DNA extraction kit (DE-06111) from Chengdu Fujie Biotechnology Co., Ltd., as amplification template. Based on the *Aquilaria sinensis* genome sequence information, specific amplification primers were designed targeting the region approximately 650 bp to 1650 bp upstream of the start codon (ATG) of the nine target genes, and EcoRI, SpeI, or MluI restriction endonuclease sites were introduced at the 5′ end of the primers, respectively. PCR amplification was performed using high-fidelity DNA polymerase to obtain the promoter fragments of each target gene. The amplification products were separated by agarose gel electrophoresis and then purified by gel extraction. The purified promoter fragments and the pHis2.1 vector were double-digested with the corresponding restriction endonucleases (EcoRI and SpeI, or EcoRI and MluI), and the digestion products were purified again. The purified target fragments and vector fragments were ligated overnight at 16℃ using T4 DNA ligase to directionally construct the Pros-pHis2.1 series of bait recombinant vectors. Simultaneously, amplification... ​ The complete coding region sequence of the gene was ligated into the pGADT7 vector to construct the capture recombination vector pGADT7-AsERFB3-4. Specific promoter amplification information is shown in Table 1.

[0034] Table 1 Promoter amplification information

[0035] The promoter sequences of each target gene were cloned and integrated into the pGreen0800-LUC reporter vector, and simultaneously... ​ The gene coding sequence was inserted into the pGreenII62SK effector vector to complete the construction of the reporter vector and effector vector used in the dual luciferase assay.

[0036] (2) Validation of yeast single-heteromorphic interaction The bait plasmid and the capture plasmid constructed in step (1) were co-transformed into the competent strain of Saccharomyces cerevisiae Y187; the transformation products were plated on SD / –Leu / –Trp (SD / -TL) deficient solid medium for preliminary screening, and co-transformed positive colonies were obtained.

[0037] Positive single colonies obtained from the initial screening were picked and transferred to SD / –Leu / –Trp / –His (SD / -TLH) triple-deficiency selection medium supplemented with 3-amino-1,2,4-triazole (3-AT). The 3-AT concentrations corresponding to each promoter bait plasmid were as follows: ​ (2 mM) ​ (150 mM) ​ (150 mM) ​ (2 mM) ​ (50 mM) ​ (1 mM) ​ (2 mM) ​ (4 mM) ​ (150 mM). The strain was cultured at a constant temperature of 28℃ for 3–5 days, and the growth status of the strain was observed to determine the in vitro binding interaction between the protein and the promoter sequence.

[0038] (3) Agrobacterium transformation and transient infection of tobacco The recombinant reporter vectors and recombinant effector vectors of the dual-luciferase assay were introduced into Agrobacterium tumefaciens GV3101 competent cells, respectively; Agrobacterium strains containing different vectors were cultured, and the OD of the bacterial culture was adjusted. 600 At a concentration of 0.6, equal volumes of Agrobacterium tumefaciens bacterial suspension carrying the reporter vector and effector vector were mixed and co-infected into four-week-old Tobacco Benzovia leaves to complete the construction of the transient expression system.

[0039] (4) Detection of luciferase activity After infection treatment, tobacco leaves were cultured normally for 3 days. Fluorescence imaging of the leaves was observed using the domestically produced Rocel multifunctional plant in vivo imaging system. At the same time, the activity of firefly luciferase (LUC) in the samples was quantitatively detected using the commercial dual-luciferase reporter gene assay kit from Promega, USA, in accordance with the kit's standard operating procedure, to complete the quantitative analysis of transcriptional activity.

[0040] The results of the yeast one-hybrid assay showed that the AsERFB3-4 protein can specifically bind in vitro. ​ ​ , ​ , ​ , ​ , ​The promoter regions of key genes for sesquiterpene synthesis are detailed in the attached diagram. ​ .

[0041] Dual-luciferase imaging and quantitative fluorescence activity detection in tobacco plants confirmed that AsERFB3-4 can significantly activate the transcriptional expression activity of the promoters of the above-mentioned target genes. ​ ), positively promoting the transcriptional level of key genes in the sesquiterpene synthesis pathway ( ​ ).

[0042] Example 4 ​ Identification of its function through transient overexpression in Aristolochic acid To verify ​ The function of genes in plants was studied using Agrobacterium-mediated transformation. ​ Genes were transformed into Aquilaria sinensis stems for transient overexpression, and the synthesis of the product was detected using GC-MS to verify the function of AsERFB3-4. The specific steps are as follows: (1) ​ The coding region sequence was ligated into the plant expression vector pBI121, with an empty vector used as a control. Both plasmids were introduced into Agrobacterium tumefaciens strain GV3101 via a freeze-thaw method. Agrobacterium tumefaciens GV3101 containing the corresponding plasmid was resuspended in a buffer solution (10 mM MgCl2, 0.15 mM acetylsyl syringone, and 10 mM MES, pH 5.6) to achieve the final optical density (OD). 600 The value reached 0.60.

[0043] (2) Cut the stem tissue of Aquilaria sinensis into 1 cm long segments, sterilize with 0.1% mercuric chloride solution for 8 minutes, and then rinse thoroughly three times with sterile distilled water. Soak the sterilized stem segments in Agrobacterium tumefaciens solution at 28°C and 160 rpm for 8 hours, and then inoculate them on MS solid medium and culture in the dark for 3 days.

[0044] (3) Volatile sesquiterpenes were analyzed using gas chromatography-mass spectrometry (GC-MS). OE- ​Stem tissue samples, as well as control samples, were flash-frozen in liquid nitrogen and ground into a fine powder. Immediately, 500 mg of the powder was transferred to a 20 mL headspace vial containing a saturated NaCl solution (to inhibit enzyme reaction) and sealed with a PTFE-silicone septum cap. For solid-phase microextraction (SPME), the headspace vial was first equilibrated at 60°C for 5 minutes, followed by headspace extraction at 60°C using a 120 μm DVB / CWR / PDMS fiber head for 15 minutes. Subsequently, thermal desorption was performed at 250°C for 5 minutes at the GC inlet. The GC column oven program was as follows: hold at 40°C for 3.5 minutes, increase to 100°C at 10°C / min, then increase to 180°C at 7°C / min, and finally increase to 280°C at 12°C / min, holding at 250°C for 5 minutes. Mass spectra were obtained and compared with a reference library to determine the relative abundance of compounds. Partial least squares discriminant analysis (PLS-DA) was used to assess differential metabolite accumulation (VIP ≥ 1.0, |log2FC| ≥ 1 and...). P ≤ 0.05).

[0045] (4) The expression of sesquiterpene synthesis-related genes in transgenic and control plants was detected by quantitative real-time PCR (qRT-PCR). cDNA from transgenic and control plants was obtained using the RNA extraction and cDNA synthesis methods described in Example 1. qRT-PCR was performed on a real-time quantitative PCR instrument using FastFire qPCR premix (Tiangen, Beijing, China). The qRT-PCR amplification conditions were as follows: initial denaturation at 95°C for 3 minutes, followed by 40 cycles (95°C for 10 seconds, 58°C for 15 seconds, 72°C for 20 seconds). All reactions were performed in triplicate using specific primers. 2 −ΔΔCT The method calculates relative gene expression levels.

[0046] GC-MS analysis showed transient overexpression in the stems of Aquilaria sinensis. ​ Subsequently, the content of several sesquiterpenoid compounds increased significantly, including (-)-aristolene, humulene, styraxene, δ-elemene, dehydroacrifen, δ-juniperene, cedrol, α-linolene, longleafene, lentinanene, β-longiene, trans-nerolidol, α-cucurbitene, α-didehydroacrifen, etc. ​ qRT-PCR analysis results showed that ​ Transient overexpression of [the substance] can activate the expression of genes related to sesquiterpene synthesis, including [genes such as...]. ​ , ​ , ​ , ​ , ​ ​ wait( ​ ).In summary, ​By directly activating the expression of genes related to the synthesis of the aforementioned sesquiterpenoids, the accumulation of sesquiterpenoids can be promoted.

Claims

1. A transcription factor gene that promotes biosynthesis of a baccatin in a white camphor tree. AsERFB3-4 characterized in that, The nucleotide sequence of the gene is the nucleotide sequence shown in SEQ ID NO:

1.

2. A protein encoded by the gene of claim 1, characterized in that, The amino acid sequence of the protein is the amino acid sequence shown in SEQ ID NO:

2.

3. A recombinant vector or expression cassette, characterized in that, Contains the gene as described in claim 1 AsERFB3-4 .

4. A host cell, characterized in that, It includes the recombinant vector or expression cassette as described in claim 3.

5. The application of the gene of claim 1, the protein of claim 2, or the recombinant vector or expression cassette of claim 3 in regulating the biosynthesis of sesquiterpenoids in Aquilaria sinensis, characterized in that, The protein can bind to and activate the promoter of the key enzyme gene for sesquiterpene synthesis; the gene of claim 1 or the recombinant vector or expression cassette of claim 3 generates the protein of claim 2 by expression, and the protein binds to and activates the promoter of the key enzyme gene for sesquiterpene synthesis, thereby regulating the biosynthesis of sesquiterpenoids in Aquilaria sinensis. The key enzyme gene for sesquiterpene synthesis was selected from... AsHMGR1 , AsHMGR2 , AsHMGR3 , AsMVK1 , AsMVD1 , AsTPS2 , AsTPS3 , AsCYP71-1 , AsCYP71-2 One or more of the following; among which AsHMGR The nucleotide sequence of 1 is shown in SEQ ID NO:

5. AsHMGR2 The nucleotide sequence is shown in SEQ ID NO:

6. AsHMGR3 The nucleotide sequence is shown in SEQ ID NO:

7. AsMVK1 The nucleotide sequence is shown in SEQ ID NO:

8. AsMVD1 The nucleotide sequence is shown in SEQ ID NO:

9. AsTPS2 The nucleotide sequence is shown in SEQ ID NO:

10. AsTPS3 The nucleotide sequence is shown in SEQ ID NO:

11. AsCYP71-1 The nucleotide sequence is shown in SEQ ID NO:

12. AsCYP71-2 The nucleotide sequence is shown in SEQ ID NO:

13.

6. The application according to claim 5, characterized in that, The sesquiterpenoid compounds in *Aristolochia debilis* are selected from one or more of the following: (-)-Aristolochne, Humulene, Aristolochne, δ-Elemonene, Dehydroacorene, δ-Juniperene, Cedrol, α-Ilanolene, Longleane, Calcium oleene, β-Longiene, trans-Norbutol, α-Cupressene, and α-Didehydroacorene.

7. A method for increasing the sesquiterpene content of Aquilaria sinensis, characterized in that, The gene described in claim 1 AsERFB3- 4 Import Aristolochic acid and overexpress it.

8. The method according to claim 7, characterized in that, The introduction method is Agrobacterium-mediated transient transformation.

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