Plant expression vector, recombinant transformant, artemisia for increasing artemisinin content in artemisia and application thereof
By overexpressing the transcription factor AabHLH39 in Artemisia annua, the problem of low artemisinin content was solved, and the artemisinin content was significantly increased to meet market demand.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHANGHAI JIAOTONG UNIV
- Filing Date
- 2023-03-02
- Publication Date
- 2026-05-12
AI Technical Summary
Artemisinin content in Artemisia annua is low, which limits its market supply and value. Existing chemical and microbial synthesis methods are difficult, costly, and require difficult equipment maintenance.
By constructing a plant expression vector containing the gene encoding the transcription factor AabHLH39 and transforming it into Artemisia annua, and then using Agrobacterium tumefaciens for gene transformation, the transcription factor AabHLH39 was overexpressed to regulate the synthesis of artemisinin.
It significantly increased the artemisinin content in Artemisia annua to over 14.6 mg/g, solving the problem of low artemisinin content and meeting market demand.
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Abstract
Description
Technical Field
[0001] This invention relates to a plant expression vector, recombinant transformant, Artemisia annua and its applications for increasing the artemisinin content in Artemisia annua, and relates to the field of genetic engineering technology. Background Technology
[0002] Artemisia annua L. is an annual herbaceous plant belonging to the genus Artemisia in the family Asteraceae. Artemisinin, a sesquiterpene lactone oxide containing a peroxy bridge extracted from its aerial parts, is currently the most widely used and most effective antimalarial drug, especially effective against cerebral malaria and chloroquine-resistant malaria.
[0003] Although microbial synthesis of artemisinin acid has been successful, it remains a semi-synthetic process of artemisinin production and is hampered by high upfront investment and difficult equipment maintenance. Meanwhile, the chemical synthesis of artemisinin itself is more challenging and cannot yet meet market demand. Currently, artemisinin is primarily derived from plant extracts, but its content in Artemisia annua is low, accounting for only 0.1-1% of the dry weight of Artemisia annua leaves, which limits its market supply and value. Therefore, how to increase the artemisinin content in Artemisia annua through genetic engineering is a continuously growing focus of research in this field. Summary of the Invention
[0004] This invention provides a plant expression vector comprising a gene sequence encoding transcription factor AabHLH39, which belongs to the bHLH class of transcription factors and can positively regulate the synthesis of artemisinin, thereby increasing the artemisinin content in Artemisia annua.
[0005] The present invention also provides a method for increasing the artemisinin content in Artemisia annua by including the above-mentioned plant expression vector, Artemisia annua, and transcription factor AabHLH39.
[0006] The first aspect of the present invention provides a plant expression vector comprising a gene encoding a transcription factor AabHLH39, the gene encoding the transcription factor AabHLH39 having a nucleotide sequence as shown in SEQ ID NO:1.
[0007] Furthermore, the plant expression vector is a pHB vector containing a gene encoding the transcription factor AabHLH39.
[0008] A second aspect of the present invention provides a recombinant transformant, the recombinant transformant comprising any of the plant expression vectors described above.
[0009] Furthermore, the recombinant transformant is Agrobacterium tumefaciens, which includes the plant expression vector.
[0010] A third aspect of the present invention provides an Artemisia annua that can overexpress transcription factor AabHLH39, wherein the gene encoding the transcription factor AabHLH39 has a nucleotide sequence as shown in SEQ ID NO:1.
[0011] The fourth aspect of the present invention provides the application of transcription factor AabHLH39 in increasing the artemisinin content in Artemisia annua, wherein the Artemisia annua overexpresses the transcription factor AabHLH39, and the gene encoding the transcription factor AabHLH39 has the nucleotide sequence shown in SEQ ID NO:1.
[0012] The fifth aspect of this invention provides a method for increasing the artemisinin content in Artemisia annua, comprising the following steps:
[0013] Construct a plant expression vector containing a gene encoding the transcription factor AabHLH39;
[0014] The plant expression vector was transformed into Agrobacterium tumefaciens to obtain recombinant transformants;
[0015] The recombinant transformants were transformed into Artemisia annua, and transgenic plants were obtained by screening.
[0016] Furthermore, a plant expression vector containing the gene encoding the transcription factor AabHLH39 was constructed, specifically including:
[0017] The gene encoding the transcription factor AabHLH39 was cloned from an Artemisia annua cDNA library;
[0018] The gene encoding transcription factor AabHLH39 was operatively ligated into the pHB vector to obtain a plant expression vector containing the gene encoding transcription factor AabHLH39.
[0019] Furthermore, the Agrobacterium tumefaciens is EH105.
[0020] Furthermore, based on the mass of the Artemisia annua leaves, the content of artemisinin is above 14.6 mg / g.
[0021] This invention constructs a plant expression vector containing a gene encoding the transcription factor AabHLH39 and transforms it into Artemisia annua, enabling Artemisia annua to overexpress the transcription factor AabHLH39 and increase the content of artemisinin in Artemisia annua. Attached Figure Description
[0022] Figure 1The expression pattern of transcription factor AabHLH39 was analyzed. Among them, a represents the relative expression level of transcription factor AabHLH39 in Artemisia annua stems, roots, old leaves, flower bud 1, flower bud 0, young leaves, terminal buds, and glandular hairs, and the expression level of transcription factor AabHLH39 in roots was set to 1; b represents the relative expression level of transcription factor AabHLH39 in leaves at different developmental stages of Artemisia annua, and the expression level of transcription factor AabHLH39 in leaf 0 (the youngest leaf) was set to 1. The expression of actin was used as an internal control. The data were obtained from three independent Artemisia annua plants.
[0023] Figure 2 The results of GUS staining analysis in proAabHLH39-GUS transgenic Artemisia annua are shown. GST represents secretory glandular trichomes, and TST represents T-type glandular trichomes.
[0024] Figure 3 To determine the subcellular localization of transcription factor AabHLH39, YFP expression driven by the 35S promoter was used as the control group. The scale bar was Bar = 50 μm.
[0025] Figure 4 To verify the interaction between AaWRKY9 and AabHLH39 proteins in a yeast two-hybrid experiment, transformed yeast cells were spotted in different selective media: SD / -Trp / -Leu, SD / -Trp / -Leu / -His, and SD / -Trp / -Leu / -His / -Ade. After incubation at 30°C for four days, the cells were photographed. The yeast two-hybrid experiment was repeated three times. This figure shows a representative experimental result.
[0026] Figure 5 To verify the protein interaction between AaWRKY9 and AabHLH39 in tobacco cells using a bimolecular fluorescence complementation assay, YFP fluorescent protein was fused to the N-terminus of AaWRKY9 (AaWRKY9-nYFP), and AabHLH39 protein was fused to the C-terminus of YFP fluorescent protein (AabHLH39-cYFP). This transformation experiment was repeated three times. The scale bar in the figure is 20 μm.
[0027] Figure 6 The expression of AabHLH39 in AabHLH39-overexpressing plants, empty vector transgenic plants (VE), and wild-type plants (CK) was analyzed, with actin as an internal control.
[0028] Figure 7 To determine the artemisinin content in AabHLH39 overexpressing plants, empty vector transgenic plants (VE), and wild-type plants (CK) by HPLC, all values were obtained from three replicates of each line of cuttings. *P<0.05, **P<0.01, and asterisks indicate t-test results. Detailed Implementation
[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0030] bHLH transcription factors, or basic helix-loop-helix (bHLH) proteins, are a superfamily of transcription factors widely found in plants, animals, and fungi. Members of the bHLH superfamily contain two highly conserved and functionally distinct domains: a basic region and a helix-loop-helix (HLH) region. The basic region, consisting of 10–20 amino acids, is located at the N-terminus of the bHLH domain and serves as a DNA-binding region, recognizing both the E-box (5'-CANNTG-3') and the G-box. The C-terminal HLH region relies on the interaction of hydrophobic amino acids to form homodimers or heterodimers of the two HLH proteins, which then regulate the expression of downstream target genes. Therefore, bHLH transcription factors often function in a dimer form.
[0031] bHLH transcription factors are a crucial class of transcription factors in plants, widely involved in plant growth and development, responses to biotic and abiotic stresses, and the transduction of hormone signals. In grapes, VvMYC1 regulates the accumulation of tannins and anthocyanins in the skin and seeds. VvMYC1 participates in the regulation of tannin and anthocyanin production in both tested grape varieties and is expressed in roots, stems, and leaves. Ectopic expression revealed that petunia JAF13 is a tissue-specific bHLH transcription factor gene that promotes pigment accumulation in petunias. Transient expression showed that anthocyanin synthesis regulatory genes are species-conserved, but the target gene promoter exhibits species specificity.
[0032] Research has shown that AabHLH39, a bHLH transcription factor, can increase the artemisinin content in Artemisia annua. Based on this, this invention was completed. In this invention, various vectors known in the art can be used, such as commercially available vectors, including plasmids and granules. In producing the Artemisia annua AabHLH39 protein polypeptide of this invention, the Artemisia annua AabHLH39 protein coding sequence can be operatively linked to an expression regulatory sequence, thereby forming an Artemisia annua AabHLH39 expression vector.
[0033] "Operationally linked to" refers to a situation where certain parts of a linear DNA sequence can influence the activity of other parts of the same linear DNA sequence. For example, if a signal peptide DNA is expressed as a precursor and participates in the secretion of a polypeptide, then the signal peptide (secretion leader sequence) DNA is operationally linked to the polypeptide DNA; if a promoter controls transcription of a sequence, then it is operationally linked to the coding sequence; if the ribosome binding site is positioned to enable translation, then it is operationally linked to the coding sequence. Generally, "operationally linked to" means adjacent, and for a secretion leader sequence, it means adjacent within the reading frame.
[0034] The Agrobacterium involved in this invention is Agrobacterium tumefaciens strain EH105, which can be purchased commercially (from CAMBIA, Australia, strain number Gambar1).
[0035] Experimental methods not specifically described in the following examples are generally performed under standard conditions, such as those described in Sambrook et al. Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.
[0036] Example 1: Cloning of the gene encoding transcription factor AabHLH39
[0037] 1. Extraction of total RNA from Artemisia annua genome
[0038] Artemisia annua leaf tissue was collected, ground in liquid nitrogen, and added to a 1.5 mL Eppendorf centrifuge tube containing lysis buffer according to the TIANGEN kit instructions. After thorough shaking, total RNA was extracted. The total RNA mass was identified by agarose gel electrophoresis, and the RNA content was then measured using a spectrophotometer.
[0039] 2. Cloning of the gene encoding transcription factor AabHLH39
[0040] Using the extracted total RNA as a template, cDNA was synthesized under the action of PowerScript reverse transcriptase; specific primers (SEQ ID NO: 3 and SEQ ID NO: 4) were designed, and the gene encoding AabHLH39 was amplified from the total cDNA by PCR. The PCR reaction system is shown in Table 1. Sequencing was performed after PCR.
[0041] Through the above steps, the full-length coding sequence of transcription factor AabHLH39 (SEQ ID NO:1) was obtained, and its protein coding sequence (SEQ ID NO:2) was deduced, wherein the start codon is ATG and the stop codon is TGA.
[0042] Table 1. PCR reaction system
[0043]
[0044]
[0045] Example 2: Analysis of the expression pattern of transcription factor AabHLH39
[0046] Using cDNA from Artemisia annua leaves at different tissues and developmental stages as templates, quantitative primers (SEQ ID NO: 5 and SEQ ID NO: 6) were used to detect the expression level of AabHLH39, and Artemisia annua β-Actin was selected as the internal reference gene for quantitative PCR.
[0047] Test results as follows Figure 1 As shown in Figure a, AabHLH39 was most highly expressed in the trichomes of Artemisia annua, followed by the shoots and flower buds (bud0, bud1), and least expressed in the roots, leaves (old leaf, young leaf), and stems; Figure 1 As shown in Figure b, AabHLH39 is expressed at a high level in the youngest leaves (leaf 1), and its expression level gradually decreases as the leaves develop.
[0048] Example 3: Cloning of the AabHLH39 gene promoter and construction of the pCAMBIA1391z vector
[0049] Based on the promoter sequence of the gene encoding AabHLH39 in the whole genome of Artemisia annua, the promoter sequence of the AabHLH39 gene was amplified from Artemisia annua DNA by PCR using specific primers (SEQ ID NO: 7 and SEQ ID NO: 8) and sequenced to obtain the sequence shown in SEQ ID NO: 11.
[0050] The promoter was constructed into the pCAMBIA1391z vector using homologous recombinase. The plant binary expression vector containing the AabHLH39 gene promoter fragment and the GUS gene fusion was transformed into Agrobacterium tumefaciens. Agrobacterium tumefaciens carrying the 1391Z-proAabHLH39 vector was transformed into Artemisia annua to study the expression of the GUS gene driven by this promoter in different tissues.
[0051] (1) Pre-culture of explants
[0052] Artemisia annua seeds were soaked in 75% ethanol for 1 min; then soaked in 20% (w / v) NaClO for 20 min; rinsed 3-4 times with sterile water; the surface moisture was blotted dry with sterile absorbent paper; and inoculated onto hormone-free MS medium, which was a solid medium invented by Murashige and Skoog in 1962 and was commercially available; sterile seedlings of Artemisia annua were obtained by culturing at 25°C for 16 hours in sunlight and 8 hours in darkness. When the seedlings grew to about 5 cm, the explants from the leaves of the sterile seedlings were cut for transformation.
[0053] (2) Co-culture of Agrobacterium and explants
[0054] The above leaf explants were transferred to a co-culture medium consisting of 1 / 2 MS and 100 μmol / L AS. A 1 / 2 MS suspension containing activated Agrobacterium tumefaciens engineered bacteria with plant expression vector was added dropwise to ensure full contact between the explants and the bacterial suspension. The cultures were then incubated in the dark at 28°C for 3 days. Leaf explants in a 1 / 2 MS liquid culture medium suspension without the target gene were used as a control.
[0055] (3) Screening of resistant regenerated plants
[0056] Artemisia annua explants co-cultured for 3 days were transferred to a germination selection medium consisting of MS, 0.5 mg / L 6-BA, 0.05 mg / L NAA, 50 mg / L Hyg, and 500 mg / L Cb. The explants were cultured at 25°C in light for 16 hours and in darkness for 8 hours. Subculture was performed every two weeks. After 2-3 subcultures, Hyg-resistant shoot clusters were obtained. The well-grown resistant shoot clusters were cut off and transferred to a rooting medium consisting of 1 / 2 MSO and 125 mg / L Cb until rooting, thus obtaining Hyg-resistant regenerated Artemisia annua plants.
[0057] (4) PCR detection of transgenic plants
[0058] Based on the AabHLH39 promoter-GUS sequence of the expression cassette containing the target gene, forward and reverse primers were designed for the detection of the GUS gene (SEQ ID NO: 9 and SEQ ID NO: 10).
[0059] (5) Determination of the expression site of the promoter-guided GUS reporter gene in plants: Leaves from Artemisia annua plants that tested positive by PCR were subjected to GUS tissue staining. The results are as follows: Figure 2 As shown, AabHLH39 is predominantly expressed in the secretory glandular trichomes of Artemisia annua.
[0060] Example 4: Subcellular localization of AabHLH39
[0061] An AabHLH39 fusion YFP expression vector was constructed. The AabHLH39 gene (with its stop codon removed) was incorporated into the pHB-YFP vector using homologous recombinase and transformed into *Agrobacterium tumefaciens*. *Agrobacterium tumefaciens* carrying the pHB-AabHLH39-YFP vector was transiently transformed into tobacco. The subcellular localization of AabHLH39 was observed using laser confocal microscopy. Figure 3 As shown, the AabHLH39-YFP fusion protein is located in the nucleus of tobacco cells, while the control YFP protein (35S:YFP) is distributed throughout the entire tobacco cell (cell membrane, cytoplasm, and nucleus).
[0062] Example 5: Interaction between AabHLH39 and AaWRKY9 regulates artemisinin synthesis
[0063] (1) Construct pGADT7-AaWKRY9 and pGBKT7-AabHLH39 vectors. Verify the interaction between AaWKRY9 and AabHLH39 in yeast AH109 according to the Clontech yeast two-hybrid instructions. The AaWKRY9 sequence can be obtained from NCBI.
[0064] The construction of yeast two-hybrid vectors pGADT7-AaWKRY9 and pGBKT7-AabHLH39 involved: transforming yeast strain AH109 with plasmids pGADT7-AaWKRY9, pGBKT7-AabHLH39, pGADT7-AaWKRY9 and pGBKT7, and pGADT7 and pGBKT7-AabHLH39 respectively using chemical methods, plating them onto SD-TL medium, and incubating them at 30 degrees Celsius for three days; diluting the single colonies grown from SD-TL medium with 100 μL of sterile water, spotting them onto SD-LT, SD-LTH, and SD-LTHA medium, and incubating them at 30 degrees Celsius for three days, observing the yeast growth, and determining whether there was interaction.
[0065] The verification results are as follows Figure 4 As shown, this indicates that AabHLH39 interacts with AaWRKY9.
[0066] (2) Construct pxy104-AaWKRY9, pxy106-AaWKRY9, pxy104-AabHLH39 and pxy106-AabHLH39 vectors respectively, and use bimolecular fluorescence complementation (BiFC) to verify the interaction between AaWRKY9 and AabHLH39 proteins in plant cells.
[0067] Specifically, this includes constructing bimolecular fluorescent complementary BiFC vectors: pxy104-AaWKRY9, pxy106-AaWKRY9, pxy104-AabHLH39, and pxy106-AabHLH39. These vectors were then transformed into Agrobacterium GV3101, and tobacco leaves were transiently transformed using the Agrobacterium injection method, as follows:
[0068] 1. Tobacco that has been grown in an artificial climate chamber for about 3 weeks was selected for Agrobacterium infection.
[0069] 2. Select Agrobacterium colony clones and incubate them overnight at 28°C in 5 mL LB medium.
[0070] 3. After determining the OD600 value of the Agrobacterium tumefaciens bacterial suspension, centrifuge at 3000 rpm for 10 min to collect the bacterial suspension and discard the supernatant. Resuspend the bacterial suspension in MS liquid medium, adjust the OD600 to approximately 0.6, add 10 mM MES (pH 5.6) and 200 u Macetosyringone, and let stand at room temperature for 3 h.
[0071] 4. Agrobacterium containing pxy104-AaWKRY9 and pxy106-AaWKRY9, pxy104-AabHLH39 and pxy106-AabHLH39 and an empty vector control plasmid are mixed in a 1:1 ratio and can then be used for injection into tobacco leaves.
[0072] 5. Using a 1mL syringe (without the needle), inject the solution into the back of the tobacco leaf and mark the injected leaf.
[0073] 6. The injected tobacco plants were returned to the artificial climate chamber for 2 days of cultivation, and the fluorescence was observed using a laser confocal microscope.
[0074] The verification results are as follows Figure 5 As shown, when AaWRKY9-cYFP and AabHLH39-nYFP are co-expressed in tobacco cells, recombinant YFP fluorescent signals can be observed in the cell nucleus. In contrast, no YFP fluorescent signals were observed when AabHLH39-nYFP and cYFP, and AaWRKY9-cYFP and nYFP, were co-expressed, indicating that AaWKRY9 and AabHLH39 interact in tobacco. AaWKRY9 directly binds to the promoter of the artemisinin synthase gene, promoting artemisinin synthesis, and AabHLH39 interacts with it, enhancing the activation of artemisinin synthesis by AaWKRY9.
[0075] Example 6: Obtaining AabHLH39 overexpressing transgenic Artemisia annua plants
[0076] The gene sequence encoding AabHLH39 was constructed on the pHB vector to obtain a plant overexpression vector. The plant overexpression vector was transformed into Agrobacterium tumefaciens strain and Artemisia annua was transformed according to the method in Example 3 to obtain transgenic Artemisia annua plants.
[0077] Example 7: Determination of artemisinin content in transgenic Artemisia annua using HPLC-ELSD
[0078] (1) HPLC-ELSD conditions, system suitability, and preparation of standard solutions
[0079] HPLC: A Water Alliance 2695 system was used with a C-18 reversed-phase silica column (SymmetryShield™ C18, 5μm, 250*4.6mm, Waters). The mobile phase was methanol:water with a volume ratio of 70:30. The column temperature was 30℃, the flow rate was 1.0mL / min, the injection volume was 10μL, the sensitivity (AUFS = 1.0), and the theoretical plate number calculated based on the artemisinin peak was not less than 2000.
[0080] ELSD: Water Alliance 2420 system was used, with an evaporative light scattering detector drift tube temperature of 40°C, a gain of 7, and a carrier gas pressure of 5 bar.
[0081] Accurately weigh 2.0 mg of artemisinin standard (Sigma) and dissolve it completely in 1 mL of methanol to obtain a 2 mg / mL artemisinin standard solution, which is stored at -20°C for later use.
[0082] In this invention, when the mobile phase is methanol:water in a ratio of 70%:30%, the retention time of artemisinin is 5.1 min, with good peak shape. The theoretical plate number, calculated based on artemisinin, is not less than 2000.
[0083] (2) Construction of standard curve
[0084] The reference solution was injected at concentrations of 2 μl, 4 μl, 6 μl, 8 μl, and 10 μl under the corresponding chromatographic conditions, and the chromatograms and chromatographic parameters were recorded. Regression analysis was performed using peak area (Y) against the standard content (X, μg). The study showed that artemisinin in this invention exhibited a good log-log linear relationship in the range of 4-20 μg. The log-log linear regression equation for the artemisinin reference standard was: Y = 1.28e+000X + 4.71e+000, R = 0.979546.
[0085] (3) Take the youngest leaves from both wild-type and transgenic plants and immediately place them in liquid nitrogen. Extract RNA using the Tiangen plant total RNA extraction kit, reverse transcribe it into cDNA, and use the cDNA as a template. Quantitative primers (SEQ ID NO: 5 and SEQ ID NO: 6) are used to detect the expression level of AabHLH39. Artemisia annua β-Actin is selected as the internal reference gene for quantitative PCR. The detection results are as follows: Figure 6 As shown.
[0086] (4) Sample preparation and determination of artemisinin content
[0087] Take 2g of fresh Artemisia annua leaves from the upper, middle, and lower parts of the plant and dry them in an oven at 45℃ until constant weight. Then, knock off the dried branches and flower buds, and grind them into powder. Weigh about 0.1g of the dry powder into a 2mL Eppendorf tube, add 2mL of ethanol, sonicate at 40W for 30min, centrifuge at 5000rpm for 10min, and filter the supernatant through a 0.22μm filter membrane. The resulting solution can then be used for HPLC-ELSD determination of artemisinin content.
[0088] Artemisinin content was determined by HPLC-ELSD with a sample injection volume of 20 μl. The artemisinin content (mg) in the sample was calculated by substituting the peak area into the linear regression equation, and then divided by the dry weight of Artemisia annua leaves (g) to calculate the artemisinin content in the Artemisia annua plant.
[0089] The calculation results are as follows Figure 7 As shown, in this invention, transgenic plants overexpressing AabHLH39 significantly increased the artemisinin content in Artemisia annua. When the artemisinin content in untransformed Artemisia annua was 12 mg / g DW, the artemisinin content in Artemisia annua overexpressing the AabHLH39 vector reached 24 mg / g DW during the same period, which was twice that of untransformed Artemisia annua. This provides strong experimental evidence for using this gene for overexpression studies to increase the artemisinin content in Artemisia annua. Figure 6-7 In the diagram, 13, 18, and 29 represent different transgenic material numbers, which are three independent genetic transformation materials.
[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.
Claims
1. A plant expression vector, characterized in that, The plant expression vector includes encoding transcription factors. AabHLH39 The gene that encodes the transcription factor AabHLH39 The gene has a nucleotide sequence as shown in SEQ ID NO:
1.
2. The plant expression vector according to claim 1, characterized in that, The plant expression vector contains encoding transcription factors. AabHLH39 The pHB vector of the gene.
3. A recombinant transformant, characterized in that, The recombinant transformant includes the plant expression vector as described in claim 1 or 2.
4. The recombinant transformant according to claim 3, characterized in that, The recombinant transformant is Agrobacterium tumefaciens, which includes the plant expression vector.
5. An artemisia annua, characterized in that, Artemisia annua overexpression transcription factor AabHLH39 Encoding the transcription factor AabHLH39 The gene has a nucleotide sequence as shown in SEQ ID NO:
1.
6. Transcription factors AabHLH39 Its application in increasing the artemisinin content in Artemisia annua is characterized by... Artemisia annua overexpresses the transcription factor AabHLH39 Encoding the transcription factor AabHLH39 The gene has a nucleotide sequence as shown in SEQ ID NO:
1.
7. A method for increasing the artemisinin content in Artemisia annua, characterized in that, Includes the following steps: Constructing a structure containing encoding transcription factors AabHLH39 Plant expression vectors for the genes; the transcription factors AabHLH39 The gene has the nucleotide sequence shown in SEQ ID NO:1; The plant expression vector was transformed into Agrobacterium tumefaciens to obtain recombinant transformants; The recombinant transformants were transformed into Artemisia annua, and transgenic plants were obtained by screening.
8. The method according to claim 7, characterized in that, Constructing a structure containing encoding transcription factors AabHLH39 Plant expression vectors for genes, specifically including: Cloning transcription factors from Artemisia annua cDNA library AabHLH39 The genes; The encoding transcription factor AabHLH39 The gene can be operatively linked into the pHB vector to obtain a vector encoding a transcription factor. AabHLH39 Plant expression vectors for genes.
9. The method according to claim 7, characterized in that, The Agrobacterium tumefaciens is EH105.
10. The method according to claim 7, characterized in that, Based on the mass of Artemisia annua leaves, the content of artemisinin is above 14.6 mg / g.