CN113293173B - Agrobacterium tumefaciens binary expression vector - Google Patents

Abstract

The invention discloses an agrobacterium tumefaciens binary expression vector, which comprises: the vector pCambia2301 skeleton of GUS screening marker gene, 35S promoter gene, P19 protein coding gene and GFP screening marker gene are deleted, the optimized agrobacterium binary expression vector can improve the expression quantity of heterologous gene and can monitor the expression condition of foreign protein in real time and in situ. The target genes TDC, SLS, STR, NPF2.9, SGD and HYS related to the ajmaline biosynthesis can be cloned into the agrobacterium binary expression vector, so that the heterologous biosynthesis of ajmaline in tobacco is promoted, and the application prospect is realized.

Description

A binary expression vector for Agrobacterium

technical field

The invention belongs to the technical field of genetic engineering and biosynthesis, and in particular relates to an Agrobacterium binary expression vector, in particular to an Agrobacterium binary expression vector which improves the detection effect of Agrobacterium-infected plants and improves the expression effect of target genes , and its use in promoting the heterologous biosynthesis of amoline in tobacco.

Background technique

Terpenoid indole alkaloids are an important class of plant secondary metabolites and important medicinal compounds. Some compounds have been used in the treatment and prevention of human diseases (Wu Shiwen et al., 2018): vinblastine, vinorelbine and vincristine can be used in the treatment of various tumors/cancers; Vindoline and catharanthine are also used in the treatment of diabetes; ajmalicine has anti-inflammatory and blood pressure-lowering effects. However, the content of terpenoid indole alkaloids in plants is extremely low (accounting for about 0.0002% of fresh weight), and most of the plant hosts are woody plants with long growth cycle, so it is difficult to meet the growing market demand. Synthetic biology is an effective means to solve this problem.

Based on metabolic engineering or synthetic biology strategies, heterologous synthesis of amoline is an effective means to meet the growing market demands. At present, the genes involved in amoxicillin and other heterohimbine alkaloids in Vinca have been identified (Miettinen et al., 2014; Stavrinides et al., 2016; Stavrinides et al., 2015.), which is the follow-up study of amohimbine It laid the foundation for the study of synthetic biology of alkali and isoyohimbine alkaloids.

Tobacco is an important model chassis system for synthetic biology research due to its large biomass and ease of expression of plant-derived genes. This system has been successfully used for the heterologous synthesis of artemisinin and its precursors, paclitaxel precursors, saponins and iridoids (Farhi et al., 2011; Fuentes et al., 2016; Li et al., 2019 ; Malhotra et al., 2016; Miettinen et al., 2014; Paddon et al., 2013; Reed et al., 2017.), an important plant model chassis system in synthetic biology research, for the terpene indole in tobacco The research on synthetic biology of alkaloids has laid a solid foundation.

Patent document CN202010092362.3 discloses a tobacco system for the production of amoline. The key gene cloned from periwinkle is constructed as an Agrobacterium expression vector, and heterologously expressed in tobacco through the tobacco transient expression system to participate in the synthesis of amoline The key gene for the biosynthesis of amoline in tobacco.

The Agrobacterium infection method is an effective method for stable or transient transformation of plants, and the Agrobacterium-mediated transient transformation system is an effective method for tobacco synthetic biology research. The pEAQ series vectors and pCambia2301 vectors are two general-purpose Agrobacterium binary expression vectors, which have been successfully used in Agrobacterium infection experiments. However, both types of vectors have certain defects: after the pEAQ series vectors infect tobacco, the expression of the target gene can only be determined by qRT-PCR or western-blot detection after sampling, and the expression of the target protein cannot be determined. Visualization: The GUS screening marker is introduced into the pCambia2301 vector, and the expression of the protein can be detected visually by GUS staining. However, this detection requires the sample to be removed and stained, and it is impossible to monitor the expression of the target protein in situ and in real time.

Green fluorescent protein (Green fluorescent protein, GFP) is a visual screening marker, which has been successfully used in the screening of transgenic plants in a variety of plants, or as an expression screening tag for foreign proteins (Estrada-Navarrete et al., 2007; Harper et al., 1999; Sparkes et al., 2006). However, traditional GFP needs to use a confocal laser microscope or under a specific filter to observe the generation of fluorescence. In order to detect the fluorescence of GFP more easily, Japanese scientists constructed a GFP protein visible to the naked eye and successfully used it in the construction of landscape plants (Chin et al., 2018). It laid the foundation for the real-time detection of foreign proteins in the process of synthetic biology research.

Contents of the invention

In order to improve the expression level of heterologous target genes in plants such as tobacco, and improve the effect of visual real-time detection for monitoring the expression of exogenous target proteins, the inventors systematically optimized the commonly used commercial Agrobacterium binary expression vector pCambia2301, and obtained expression At the same time, the green fluorescent protein GFP screening marker visible to the naked eye is introduced, which is easy to monitor the expression of heterologous proteins in plants such as tobacco in real time.

Specifically, the present invention includes the following technical solutions.

An Agrobacterium binary expression vector, which comprises: the carrier pCambia2301 backbone in which the GUS selection marker gene has been deleted, a 35S promoter gene, a P19 protein coding gene and a GFP selection marker gene.

Preferably, in the above-mentioned Agrobacterium binary expression vector, the 35S promoter gene, the P19 protein coding gene and the GFP selection marker gene are fused by self-cleaving polypeptide 2A to form a fusion gene.

The above-mentioned fusion gene may include the following biological elements: 35S promoter, 5'UTR, P19 protein, 2A peptide, eyGFPuv, 3'UTR, NosT. In this case, the fusion gene structure is 35S promoter-5'UTR-P19-2A peptide-eyGFPuv-3'UTR-NosT.

Among them, P19 is derived from tomato bush dwarf virus, which can inhibit the host's RNA silencing effect on foreign genes, improve the stability of heterologous gene transcripts, and promote the expression of heterologous proteins. 5'UTR is the non-coding region (UntranslatedRegion), that is, the untranslated region, which is located downstream of the transcription initiation site and is transcribed but not translated. The 3' UTR extends from the stop codon at the end of the coding region to the end of the poly-A tail (Poly-A). eyGFPuv is a cPYGFP derivative that exhibits bright fluorescence under the excitation of invisible ultraviolet (UV) light. The protein not only has strong fluorescence, but the excitation light is located in the invisible ultraviolet spectral region. Nost is the terminator of the nopaline synthase gene (nos). The gene sequences of P19 protein, 5'UTR, 3'UTR and NosT can be, for example, in the literature (Frank Sainsbury, Eva C.Thuenemann and GeorgeP.Lomonossoff, pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants, Plant Sequence published in Biotechnology Journal (2009) 7, 682-693).

Self-cleaving 2A peptide (2A) or self-cleaving 2A peptide can be selected from P2A, E2A, T2A, F2A, but not limited thereto. When the 2A peptide is F2A, the fusion gene structure is 35S promoter-5'UTR-P19-F2A-eyGFPuv-3'UTR-NosT.

Preferably, the nucleotide sequence of the fusion gene is SEQ ID NO:1.

In this case, the nucleotide sequence of the constructed Agrobacterium binary expression vector can be SEQ ID NO:2.

In one embodiment, the above-mentioned Agrobacterium binary expression vector can be cloned into genes related to the synthesis of amoline and other terpenoid indole alkaloids. These genes related to the synthesis of amothine encode tryptophan decarboxylase (TDC), split loganin synthase (SLS), isososidine synthase (Strictosidine synthase, STR), Strictosidine beta-D-glucosidase (Strictosidine beta-D-glucosidase, SGD), nitrate transporter NPF2.9, isoyohimbine alkaloid synthase HYS. These enzymes and coding genes have been disclosed in the patent document CN202010092362.3, the entire contents of which are incorporated into the present invention.

When the Agrobacterium binary expression vector contains two or more of the above-mentioned genes, the two or more genes can be fused through the 2A peptide gene to form a fusion gene.

As a preferred embodiment, the above-mentioned tryptophan decarboxylase TDC is derived from Catharanthus roseus, and the nucleotide sequence of its coding gene is SEQ ID NO:3. The above-mentioned split loganin synthase SLS may be derived from Catharanthus roseus, or SLS1, and the nucleotide sequence of its coding gene is SEQ ID NO:4. The amino acid sequence of the above-mentioned isosidine synthase (STR) can be GenBank accession number: CAA43936, and its coding gene can be GenBank accession number: X61932. The aforementioned isossididine beta-D glucosidase SGD is derived from Catharanthus roseus, and the nucleotide sequence of its coding gene is SEQ ID NO:5. The amino acid sequence of the above-mentioned nitrate transporter NPF2.9 can be GenBank accession number: AQM73449, and its coding gene can be GenBank accession number: KX372303. The amino acid sequence of the above-mentioned heteroyohimbe alkaloid synthase HYS can be GenBank accession number: ANQ45225, and its coding gene can be GenBank accession number: KU865325.

When the coding genes (or biological elements) of the above-mentioned TDC, SLS, STR, SGD, NPF2.9, and HYS are cloned, the above-mentioned Agrobacterium binary expression vectors can be used for amoline synthesis and other terpenoid indole biological A dedicated carrier for base synthesis.

The second aspect of the present invention provides an Agrobacterium transformed with the above special vector.

A third aspect of the present invention provides the application of the above-mentioned Agrobacterium in the production of amoline, that is, using the above-mentioned Agrobacterium to infect crops suitable for expressing amoline or other terpene indole alkaloids, and the crops As a biosynthetic system for the production of amoline or other terpenoid indole alkaloids.

Preferably, the aforementioned agricultural crop is tobacco.

For example, after infecting tobacco with the above-mentioned Agrobacterium, feeding tryptophan and loganin (Loganin), harvesting tobacco leaves and extracting amoline.

The present invention uses the commercialized Agrobacterium binary expression vector pCambia2301 as the backbone, and deletes the GUS screening marker so as to reduce the size of the entire vector; The GFP screening marker visible to the naked eye was used to construct the fusion gene; then the optimized Agrobacterium binary expression vector pF08 (pCambia2301-Optimized) was constructed. The optimized Agrobacterium binary expression vector can not only increase the expression of heterologous genes, but also monitor the expression of foreign proteins in real time and in situ. By cloning genes related to the synthesis of amoline and other terpenoid indole alkaloids into the optimized vector and performing transient expression in tobacco, the expression system of amoline-producing tobacco can be constructed, and the expression of related genes can be increased. It promotes the heterologous biosynthesis of amoline and other terpenoid indole alkaloids, and has development and application potential.

Description of drawings

Fig. 1 shows a schematic structural diagram of the optimized vector Agrobacterium binary expression vector pCambia2301-Optimized (pF08) constructed in the present invention.

Fig. 2 shows the expression situation of GFP (365nm ultraviolet irradiation ). Photo A is part of the injection infection of tobacco leaves, the green part (light part) is the injected area, indicating that the GFP tag is expressed smoothly; the red part (dark part) is the non-injected area, indicating that there is no expression of the GFP tag. Photo B is the injection infection of the whole leaf. The green (light color) part indicates that the GFP tag is successfully expressed; the red (dark color) part indicates that the GFP tag in the injected part is not expressed or the injection fails to cover the entire leaf. Photo A and Photo B verify the function of the optimized vector pF08 constructed by the present invention.

Figure 3 shows that the original vector pCambia2301 (TDC-2A-SLS, STR-2A-NPF2.9, SGD-2A-HYS) with TDC, SLS, STR, SGD, NPF2.9, HYS gene cloned infects tobacco in Arabidopsis The mensuration of moline content and the optimization carrier (pF08-TDC-2A-SLS, pF08-STR-2A-NPF2.9, pF08-SGD-2A-HYS) constructed by the present invention compare the mensuration of moline content in tobacco Condition. Among them, Figure A is the qualitative analysis result of amoline in infected tobacco, ajm: amoline standard; Figure B is the result of the content detection (μg/g fresh weight) of amoline in infected tobacco.

Detailed ways

The present invention systematically optimizes the commercial original Agrobacterium binary expression vector pCambia2301, that is, the original pCambia2301 vector is used as the backbone, the GUS screening marker (2922 nucleotides) in the original backbone is deleted, and the 35S promoter is cloned into A vector system pCambia2301-Optimized (pF08) with higher expression effect was obtained from the silenced P19 protein coding gene and the GFP screening marker gene eyGFPuv (or called eYGFPuv) visible to the naked eye under ultraviolet excitation. The introduction of the GFP screening marker facilitates the real-time monitoring of the expression of the heterologous protein.

After the heterologous protein gene is loaded in the optimized vector, the expression level of the heterologous protein is increased. The expression effect of the optimized vector pF08 was verified by amoline biosynthesis in tobacco, and the target genes TDC, SLS, STR, NPF2.9, SGD, HYS related to amoline biosynthesis were cloned in the vector pF08 , Agrobacterium was constructed, and after infecting tobacco, the production of amoline was increased, which proved that the expression of these heterologous proteins was significantly increased.

In this paper, sometimes for the sake of simplicity of description, the name of a certain protein such as TDC protein and the name of its coding gene (DNA) are mixed, and those skilled in the art should understand that they represent different substances in different description occasions. For example, TDC (gene), when used to describe the function or class of tryptophan decarboxylase, refers to the protein; when described as a gene, refers to the gene encoding that tryptophan decarboxylase, and so on , which is easily understood by those skilled in the art.

The present invention will be described in further detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention but not to limit the scope of the present invention.

The examples herein refer to the addition amount, content and concentration of various substances, and the percentage content mentioned therein refers to the mass percentage content unless otherwise specified.

In the examples, if there is no specific statement about the operating temperature, the temperature usually refers to room temperature (15-30° C.).

Example

Materials and methods

The whole gene synthesis, primer synthesis and sequencing in the examples were all completed by Nanjing GenScript Biotechnology Co., Ltd.

The molecular biology experiments in the examples include plasmid construction, enzyme digestion, connection, competent cell preparation, transformation, medium preparation, etc., mainly referring to "Molecular Cloning Experiment Guide" (third edition), J. Sambrook, Edited by D.W. Russell (US), translated by Huang Peitang et al., Science Press, Beijing, 2002). The specific experimental conditions can be determined by simple experiments if necessary.

PCR amplification experiments were carried out according to the reaction conditions or kit instructions provided by the plasmid or DNA template suppliers. It can be adjusted by simple experiment if necessary.

LB medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, pH 7.0.

HPLC-MS detection conditions of amoline and its precursors:

Agilent 1260 Infinity HPLC-MS system was used for target metabolomics detection. The chromatographic column is an Agilent C18 column (chromatographic column: 3.5um, 4.0×100mm); the mobile phase is 0.1% formic acid aqueous solution (A) and chromatographically pure acetonitrile (B), and the separation is carried out according to the following gradient: 0-2min, 5% B; 22min, 40%B; 25min, 60%B; 28min, 95%B; 35min, 5%B. The flow rate was 0.8mL/min, and the column equilibrated for 5min. The total analysis time is 40 min. The mass spectrometer was a single quadrupole mass spectrometer, and was performed according to the default parameters of the Agilent manufacturer.

Deletion of the GUS screening marker in the original vector pCambia2301 of embodiment 1

1.1 Utilize recombinase Redα/β or RecE/T-mediated homologous recombination to precisely modify DNA molecules to delete GUS screening markers and their promoters and terminators. The steps include: adding the original pCambia2301 vector (Invitrogen Corporation ) was digested at 37° C. with restriction endonucleases SnaBI and MaubI (Thermofisher Company) to obtain a linearized pCambia2301 vector.

1.2 Then, the above-mentioned linearized pCambia2301 vector and primers GUS-deletion-F and GUS-deletion-R were recombined in DH10β Escherichia coli (NEB Company) in vivo using Red/ET technology. Recombinant Escherichia coli DH10β was cultured overnight at 37°C in LB medium containing kanamycin sulfate.

The primer sequences (5'-3') used are:

GUS-deletion-F:

CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATC;

GUS-deletion-R:

FCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATC.

1.3 Use a plasmid extraction kit (Axygen Company) to extract the plasmid, and verify correctness by sequencing to obtain the intermediate vector pCambia2301-Gus-Del with the GUS screening marker deleted.

Embodiment 2 optimizes the construction of carrier

2.1 Synthesis of the insert fragment (Insert fragment): The insert fragment contains seven parts: 35S promoter, 5'UTR, P19, F2A peptide, eyGFPuv, 3'UTR and NosT, and the structure is 35S promoter-5'UTR-P19- 2A peptide-eyGFPuv-3'UTR-NosT, the nucleotide sequence of the fusion gene is SEQ ID NO:1.

The fusion sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd.

2.2 Construction of an optimized vector: Digest the insert fragment synthesized in step 2.1 and the intermediate vector pCambia2301-Gus-Del vector obtained in Example 1 with restriction enzymes ECoRI and SmaI (NEB Company) at 37°C for 12h to obtain Linearized pCambia2301-Gus-Del vector. Then, T4 DNA Ligase (NEB Company) was used to perform ligation according to the instructions of the kit to construct an optimized vector pCambia2301-Optimized, named pF08, which was verified to be correct by sequencing. The nucleotide sequence of the vector pF08 is SEQ ID NO:2.

Example 3 Amplification of Amoline Biosynthesis-Related Genes

Referring to the method disclosed in the patent document CN202010092362.3, the target genes TDC, SLS1, STR, NPF2.9, SGD and HYS related to amoline biosynthesis were amplified. Including the following steps.

3.1 Extraction of periwinkle total RNA and reverse transcription

The periwinkle leaves were ground into powder with liquid nitrogen, and 100 mg of the ground powder was placed in a centrifuge tube with 1.5 mL of RNA free, and then RNA was extracted according to the instructions of Quanjin’s RNA extraction kit. The extracted RNA is directly reverse-transcribed to produce cDNA.

Take 1 μg of RNA and reverse transcribe to produce cDNA using Quanshijin reverse transcription kit.

3.2 Target gene cloning

FastPfu Flybμffer10μl，dNTP(2.5mM)4μL，引物(10μM)各2μL，cDNA 1μL，

FastPfu Fly DNA聚合酶1μL，补加ddH₂O至终体积50μL扩增。PCR反应程序：98℃预变性30s，98℃变性10s，58℃退火30s，72℃延伸1min 30s，35个循环，72℃终延伸5min，16℃保存。利用Axygen公司的Agarose Gel Fragment Recovery Kit Ver.2.0纯化PCR产物。分别得到TDC、SLS、STR、NPF2.9、SGD和HYS基因片段。Use the periwinkle cDNA obtained by reverse transcription in step 3.1 as a template, and use the primers shown in Table 1 to amplify according to the following system [5×

10 μl of FastPfu Flyb μffer, 4 μL of dNTP (2.5 mM), 2 μL of each primer (10 μM), 1 μL of cDNA,

FastPfu Fly DNA polymerase 1 μL, add ddH ₂ O to a final volume of 50 μL for amplification. PCR reaction program: pre-denaturation at 98°C for 30s, denaturation at 98°C for 10s, annealing at 58°C for 30s, extension at 72°C for 1min and 30s, 35 cycles, final extension at 72°C for 5min, storage at 16°C. The PCR product was purified using Agarose Gel Fragment Recovery Kit Ver.2.0 from Axygen. The gene fragments of TDC, SLS, STR, NPF2.9, SGD and HYS were obtained respectively.

Table 1. Primers required for gene amplification

Wherein, F is a forward primer; R is a reverse primer.

3.3 Fusion gene cloning

FastPfu Flybμffer10μl，dNTP(2.5mM)4μL，引物(10μM)各2μL，cDNA 1μL，

FastPfu Fly DNA聚合酶1μL，补加ddH₂O至终体积50μL扩增。PCR反应程序：98℃预变性30s，98℃变性10s，58℃退火30s，72℃延伸1min 30s，35个循环，72℃终延伸5min，16℃保存。利用Axygen公司的Agarose Gel Fragment Recovery Kit Ver.2.0纯化PCR产物。分别得到带有融合蛋白接头2A肽基因的TDC、SLS1、STR、NPF2.9、SGD和HYS基因片段。Use the plasmid containing the target gene obtained in step 3.2 as a template, and use the primers shown in Table 2 to amplify the fusion gene according to the following system [5×

10 μl of FastPfu Flyb μffer, 4 μL of dNTP (2.5 mM), 2 μL of each primer (10 μM), 1 μL of cDNA,

FastPfu Fly DNA polymerase 1 μL, add ddH ₂ O to a final volume of 50 μL for amplification. PCR reaction program: pre-denaturation at 98°C for 30s, denaturation at 98°C for 10s, annealing at 58°C for 30s, extension at 72°C for 1min and 30s, 35 cycles, final extension at 72°C for 5min, storage at 16°C. The PCR product was purified using Agarose Gel Fragment Recovery Kit Ver.2.0 from Axygen. The TDC, SLS1, STR, NPF2.9, SGD and HYS gene fragments with fusion protein linker 2A peptide genes were obtained respectively.

Table 2. Primers required for fusion gene amplification

Wherein, F is a forward primer; R is a reverse primer.

The construction of embodiment 4 Agrobacterium expression vector

4.1 Construction of the original vector

The original Agrobacterium binary expression vector pCambia2301 (Invitrogen Company) was digested with appropriate restriction enzymes BamHI and SalI at 37°C for 12 hours, and the digested product was purified using a gel recovery kit (Axygen Company), and homologous recombination was used to Perform homologous recombination on the purified fragment and restriction vector in step 3.3 according to the instructions to obtain the original vector expressing the fusion gene. Including pCambia2301-TDC-2A-SLS, pCambia2301-STR-2A-NPF2.9, pCambia2301-SGD-2A-HYS, etc.

4.2 Construction of optimized vector

Referring to the method in step 4.1, the optimized vector pF08 constructed in step 2.2 was digested with the appropriate restriction enzymes BamHI and SalI at 37°C for 12 h, and the digested product was purified using a gel recovery kit (Axygen Company). The recombinase performs homologous recombination on the purified fragment and restriction vector in step 3.3 according to the instructions to obtain an optimized vector for expressing the fusion gene. Including pF08-TDC-2A-SLS, pF08-STR-2A-NPF2.9, pF08-SGD-2A-HYS, etc.

Embodiment 5 constructs Agrobacterium

Take 1 μg of the expression vector obtained in Example 4 with correct sequencing and Agrobacterium GV3101 competent cells (Shanghai Weidi Biological Co., Ltd.) and incubate on ice for 30 minutes, quick freeze in liquid nitrogen for 5 minutes, heat shock at 37°C for 5 minutes, and place on ice for 5 minutes , add 1 mL of LB medium, and place on a shaker at 28°C for 4 hours to recover. All the bacteria were plated on kana-resistant and rifamycin-resistant LB plates, and placed in a 28°C incubator for 2-4 days to obtain the original expression vectors (including pCambia2301-TDC-2A-SLS , pCambia2301-STR-2A-NPF2.9, pCambia2301-SGD-2A-HYS, etc.) and optimized expression vectors (including pF08-TDC-2A-SLS, pF08-STR-2A-NPF2.9, pF08-SGD-2A- HYS, etc.) Agrobacterium engineering bacteria.

Example 6 Agrobacterium Infection

Pick a single colony from the LB medium plate resistant to kanamycin and rifampicin and culture it in LB (Kan+Rif) tube for 48 hours until OD600≥2.0. Incubate overnight (12h) at 28°C. After the cells were collected, they were incubated with MMA buffer (10 mM MES, 10 mM MgCl ₂ and 100 μM acetosyringone) for 3 h, and then the tobacco was injected with a needle-free syringe. The substrate (100 mg/mL tryptophan and 100 mg/mL loganin (Loganin)) was fed 4 days after the tobacco infection, and the tobacco leaves were harvested 1 day later for target metabolomics analysis (HPLC-MS analysis).

Example 7 Metabolomics Analysis of Tobacco Leaf

As shown in Figure 2, after infecting tobacco leaves with Agrobacterium containing optimized expression vectors (pF08-TDC-2A-SLS, pF08-STR-2A-NPF2.9, pF08-SGD-2A-HYS), GFP labeling Smooth expression, under 365nm ultraviolet irradiation, the infected area is a green (light-colored) part, which is visible to the naked eye, and is obviously different from the red (dark) part of the infected area without injection, indicating that the optimized Agrobacterium binary expression vector constructed by the present invention pF08 can monitor the expression of foreign proteins in real time and in situ.

Figure 3 shows that the content of amoline in the tobacco leaves infected by Agrobacterium containing the original vector pCambia2301 (TDC-2A-SLS, STR-2A-NPF2.9, SGD-2A-HYS) is about 12.1 μg/g Fresh weight; the content of amoline in tobacco leaves infected with Agrobacterium containing optimized vector pF08 (pF08-TDC-2A-SLS, pF08-STR-2A-NPF2.9, pF08-SGD-2A-HYS) was about It is 20 μ g/g fresh weight, obviously higher than original carrier, shows that the optimized Agrobacterium binary expression carrier that the present invention constructs can improve the expression level of heterologous gene (TDC, SLS, STR, NPF2.9, SGD and HYS), and then Increase the production of amoline.

It should also be noted that the enumeration and discussion of previously disclosed documents in this specification should not be regarded as acknowledging that the documents are prior art or common knowledge.

references

1. Chin, D.P., Shiratori, I., Shimizu, A., Kato, K., Mii, M., and Waga, I. (2018). Generation of brilliant green fluorescent petunia plants by using a new and potent fluorescent protein transgene .Sci Rep 8,1-10.

2. Estrada-Navarrete, G., Alvarado-Affantranger, X., Olivares, J.-E., Guillén, G., Diaz-Camino, C., Campos, F., Quinto, C., Gresshoff, P.M., and Sanchez, F. (2007). Fast, efficient and reproducible genetic transformation of Phaseolus spp. by Agrobacterium rhizogenes. Nature protocols 2, 1819.

3. Farhi, M., Marhevka, E., Ben-Ari, J., Algamas-Dimantov, A., Liang, Z., Zeevi, V., Edelbaum, O., Spitzer-Rimon, B., Abeliovich, H., and Schwartz, B. (2011). Generation of the potent anti-malarial drug artemisinin in tobacco. Nat Biotechnol 29, 1072.

4. Frank Sainsbury, Eva C. Thuenemann and George P. Lomonossoff, pEAQ: versatile expression vectors for easy and quick transient expression of heterologous proteins in plants, Plant Biotechnology Journal (2009) 7, 682–693.

5. Fuentes, P., Zhou, F., Erban, A., Karcher, D., Kopka, J., and Bock, R. (2016). A new synthetic biology approach allows transfer of an entire metabolic pathway from a medicinal plant to a biomass crop. eLife 5.

6. Harper, B.K., Mabon, S.A., Leffel, S.M., Halfhill, M.D., Richards, H.A., Moyer, K.A., and Stewart, C.N. (1999). Green fluorescent protein as a marker for expression of a second gene in transgenic plants. Nat Biotechnol 17, 1125-1129.

7. Li, J., Mutanda, I., Wang, K., Yang, L., Wang, J., and Wang, Y. (2019). Chloroplastic metabolic engineering coupled with isoprenoid pool enhancement for committed taxanes biosynthesis in Nicotiana benthamiana .Nat Commun 10, 4850.

8. Malhotra, K., Subramaniyan, M., Rawat, K., Kalamuddin, M., Qureshi, M.I., Malhotra, P., Mohmmed, A., Cornish, K., Daniell, H., and Kumar, S .(2016).Compartmentalized Metabolic Engineering for Artemisinin Biosynthesis and Effective Malaria Treatment by Oral Delivery of Plant Cells.Mol Plant 9,1464-1477.

9. Miettinen, K., Dong, L., Navrot, N., Schneider, T., Burlat, V., Pollier, J., Woittiez, L., Der Krol, S.V., Lugan, R., and Ilc, T. (2014). The seco-iridoid pathway from Catharanthus roseus. Nat Commun 5, 3606-3606.

10. Paddon, C.J., Westfall, P.J., Pitera, D.J., Benjamin, K., Fisher, K., McPhee, D., Leavell, M., Tai, A., Main, A., and Eng, D. ( 2013). High-level semi-synthetic production of the potent antimalarial artemisinin. Nature 496,528.

11. Reed, J., Stephenson, M.J., Miettinen, K., Brouwer, B., Leveau, A., Brett, P., Goss, R.J., Goossens, A., O’Connell, M.A., and Osbourn, A. .(2017).A translationalsynthetic biology platform for rapid access to gram-scale quantities of noveldrug-like molecules.Metab Eng 42,185-193.

12. Sparkes, I.A., Runions, J., Kearns, A., and Hawes, C. (2006). Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nature protocols 1, 2019.

13. Wu Shiwen, Yang Mengquan, and Xiao Youli (2018). Research progress in synthetic biology of monoterpene indole alkaloids. Chin J Org Chem 38, 2243-2258.

sequence listing

<110> Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences

<120> A binary expression vector for Agrobacterium

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aaggaaggtg gctcctacaa atgccatcat tgcgataaag gaaaggccat cgttgaagat 120

gcctctgccg acagtggtcc caaagatgga cccccaccca cgaggagcat cgtggaaaaa 180

gaagacgttc caaccacgtc ttcaaagcaa gtggattgat gtgatatctc cactgacgta 240

agggatgacg cacaatccca ctatccttcg caagaccctt cctctatata aggaagttca 300

tttcatttgg agaggtatta aaatcttaat aggttttgat aaaagcgaac gtggggaaac 360

ccgaaccaaa ccttcttcta aactctctct catctctctt aaagcaaact tctctcttgt 420

ctttcttgcg tgagcgatct tcaacgttgt cagatcgtgc ttcggcacca gtacaacgtt 480

ttctttcact gaagcgaaat caaagatctc tttgtggaca cgtagtgcgg cgccattaaa 540

taacgtgtac ttgtcctatt cttgtcggtg tggtcttggg aaaagaaagc ttgctggagg 600

ctgctgttca gccccataca ttacttgtta cgattctgct gactttcggc gggtgcaata 660

tctctacttc tgcttgacga ggtattgttg cctgtacttc tttcttcttc ttcttgctga 720

ttggttctat aagaaatcta gtattttctt tgaaacagag ttttcccgtg gttttcgaac 780

ttggagaaag attgttaagc ttctgtatat tctgcccaaa ttcgcgatgg aacgagctat 840

acaaggaaac gacgctaggg aacaagctaa cagtgaacgt tgggatggag gatcaggagg 900

aagcacttct cccttcaaac ttcctgacga aagtccgagt tggactgagt ggcggctaca 960

taacgatgag acgaactcga atcaagataa tccccttggt ttcaaggaaa gctggggttt 1020

cgggaaagtt gtatttaaga gatatctcag atacgacagg acggaggcat cactgcacag 1080

agtccttgga tcttggacgg gagattcggt taactatgca gcatctcgat ttttcggttt 1140

cgaccagatc ggatgtacct atagtattcg gtttcgagga gttagtatca ccgtttctgg 1200

agggtctcga actcttcagc atctctgtga gatggcaatt cggtctaagc aagaactgct 1260

acagcttgcc ccaatcgaag tggaaagtaa tgtatcaaga ggatgccctg aaggtactga 1320

gaccttcgaa aaagaaagcg agggatctgg agttaagcaa actttgaatt ttgatttgtt 1380

gaagttggct ggagatgttg aatctaatcc tggacctgag ctcatgacaa ccttcaaaat 1440

cgagtcccgc atccacggca acctcaacgg ggagaagttc gagttggttg gaggtggagt 1500

aggtgaggag ggtcgcctcg agattgagat gaagactaaa gataaaccac tggcattctc 1560

tcccttcctg ctgaccactt gcatgggtta cgggttctac cacttcgcca gcttcccaaa 1620

ggggattaag aacatctatc ttcatgctgc aacgaacgga ggttacacca acaccaggaa 1680

ggagatctat gaagacggcg gcatcttgga ggtcaacttc cgttacactt acgagttcaa 1740

caagatcatc ggtgacgtcg agtgcattgg acatggattc ccaagtcaga gtccgatctt 1800

caaggacacg atcgtgaaga gttgtcccac ggtggacctg atgttgccaa tgtccgggaa 1860

catcatcgcc agctcctacg cttacgcctt ccaactgaag gacggctctt tctacacggc 1920

agaagtcaag aacaacatag acttcaagaa tccaatccac gagtcccttct cgaagtcagg 1980

gcccatgttc accccacagac gtgtcgagga gactctcacc aaggagaacc ttgccatagt 2040

ggagtaccag caggttttca acagcgcccc aagagacatg tagactagtt taactctggt 2100

ttcattaaat tttctttagt ttgaatttac tgttattcgg tgtgcatttc tatgtttggt 2160

gagcggtttt ctgtgctcag agtgtgttta ttttatgtaa tttaatttct ttgtgagcac 2220

ctgtttagca ggtcgtccct tcagcaagga cacaaaaaga ttttaatttt attgatcgtt 2280

caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta 2340

tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt 2400

tatttatgag atgggttttt atgattatag tcccgcaatt atacatttaa tacgcgatag 2460

aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac 2520

tagatc 2526

<210> 2

<211> 12724

<212>DNA

<213> artificial sequence ()

<400> 2

ggatcctcta gagtcgacct gcaggcatgc cctgctttaa tgagatatgc gagacgccta 60

tgatcgcatg atatttgctt tcaattctgt tgtgcacgtt gtaaaaaacc tgagcatgtg 120

tagctcagat ccttaccgcc ggtttcggtt cattctaatg aatatatcac ccgttactat 180

cgtattttta tgaataatat tctccgttca atttactgat tgtccaagct tggcactggc 240

cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt accccaactta atcgccttgc 300

agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc 360

cgataaatta tcgcgcgcgg tgtcatctat gttactagat cgggaattaa actatcagtg 420

tttgacagga tatattggcg ggtaaaccta agagaaaaga gcgtttatta gaataacgga 480

tattaaaag ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac 540

agggttcccc tcgggatcaa agtactttga tccaacccct ccgctgctat agtgcagtcg 600

gcttctgacg ttcagtgcag ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta 660

cgcgacaggc tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca 720

taaagtagaa tacttgcgac tagaaccgga gacattacgc catgaacaag agcgccgccg 780

ctggcctgct gggctatgcc cgcgtcagca ccgacgacca ggacttgacc aaccaacggg 840

ccgaactgca cgcggccggc tgcaccaagc tgttttccga gaagatcacc ggcaccaggc 900

gcgaccgccc ggagctggcc aggatgcttg accacctacg ccctggcgac gttgtgacag 960

tgaccaggct agaccgcctg gcccgcagca cccgcgacct actggacatt gccgagcgca 1020

tccaggaggc cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac accaccacgc 1080

cggccggccg catggtgttg accgtgttcg ccggcattgc cgagttcgag cgttccctaa 1140

tcatcgaccg cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc 1200

cccgccctac cctcaccccg gcacagatcg cgcacgcccg cgagctgatc gaccaggaag 1260

gccgcaccgt gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg 1320

cacttgagcg cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt gccttccgtg 1380

aggacgcatt gaccgaggcc gacgccctgg cggccgccga gaatgaacgc caagaggaac 1440

aagcatgaaa ccgcaccagg acggccagga cgaaccgttt ttcattaccg aagagatcga 1500

ggcggagatg atcgcggccg ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg 1560

gctgcatgaa atcctggccg gtttgtctga tgccaagctg gcggcctggc cggccagctt 1620

ggccgctgaa gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag 1680

cttgcgtcat gcggtcgctg cgtatatgat gcgatgagta aataaacaaa tacgcaaggg 1740

gaacgcatga aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc 1800

gcaacccatc tagcccgcgc cctgcaactc gccggggccg atgttctgtt agtcgattcc 1860

gatccccagg gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt 1920

gtcggcatcg accgcccgac gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc 1980

gtagtgatcg acggagcgcc ccaggcggcg gacttggctg tgtccgcgat caaggcagcc 2040

gacttcgtgc tgattccggt gcagccaagc ccttacgaca tatgggccac cgccgacctg 2100

gtggagctgg ttaagcagcg cattgaggtc acggatggaa ggctacaagc ggcctttgtc 2160

gtgtcgcggg cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg 2220

tacgagctgc ccattcttga gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc 2280

gccgccggca caaccgttct tgaatcagaa cccgagggcg acgctgcccg cgaggtccag 2340

gcgctggccg ctgaaattaa atcaaaactc atttgagtta atgaggtaaa gagaaaatga 2400

gcaaaagcac aaacacgcta agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa 2460

cgttggccag cctggcagac acgccagcca tgaagcgggt caactttcag ttgccggcgg 2520

aggatcacac caagctgaag atgtacgcgg tacgccaagg caagaccatt accgagctgc 2580

tatctgaata catcgcgcag ctaccagagt aaatgagcaa atgaataaat gagtagatga 2640

attttagcgg ctaaaggagg cggcatggaa aatcaagaac aaccaggcac cgacgccgtg 2700

gaatgcccca tgtgtgggagg aacgggcggt tggccaggcg taagcggctg ggttgtctgc 2760

cggccctgca atggcactgg aacccccaag cccgaggaat cggcgtgagc ggtcgcaaac 2820

catccggccc ggtacaaatc ggcgcggcgc tgggtgatga cctggtggag aagttgaagg 2880

ccgcgcaggc cgcccagcgg caacgcatcg aggcagaagc acgccccggt gaatcgtggc 2940

aagcggccgc tgatcgaatc cgcaaagaat cccggcaacc gccggcagcc ggtgcgccgt 3000

cgattaggaa gccgcccaag ggcgacgagc aaccagattt tttcgttccg atgctctatg 3060

acgtgggcac ccgcgatagt cgcagcatca tggacgtggc cgttttccgt ctgtcgaagc 3120

gtgaccgacg agctggcgag gtgatccgct acgagcttcc agacgggcac gtagaggttt 3180

ccgcagggcc ggccggcatg gccagtgtgt gggattacga cctggtactg atggcggttt 3240

cccatctaac cgaatccatg aaccgatacc gggaagggaa gggagacaag cccggccgcg 3300

tgttccgtcc acacgttgcg gacgtactca agttctgccg gcgagccgat ggcggaaagc 3360

agaaagacga cctggtagaa acctgcattc ggttaaacac cacgcacgtt gccatgcagc 3420

gtacgaagaa ggccaagaac ggccgcctgg tgacggtatc cgagggtgaa gccttgatta 3480

gccgctacaa gatcgtaaag agcgaaaccg ggcggccgga gtacatcgag atcgagctag 3540

ctgattggat gtaccgcgag atcacagaag gcaagaaccc ggacgtgctg acggttcacc 3600

ccgattactt tttgatcgat cccggcatcg gccgttttct ctaccgcctg gcacgccgcg 3660

ccgcaggcaa ggcagaagcc agatggttgt tcaagacgat ctacgaacgc agtggcagcg 3720

ccggagagtt caagaagttc tgtttcaccg tgcgcaagct gatcgggtca aatgacctgc 3780

cggagtacga tttgaaggag gaggcggggc aggctggccc gatcctagtc atgcgctacc 3840

gcaacctgat cgagggcgaa gcatccgccg gttcctaatg tacggagcag atgctagggc 3900

aaattgccct agcaggggaa aaaggtcgaa aaggtctctt tcctgtggat agcacgtaca 3960

ttgggaaccc aaagccgtac attgggaacc ggaacccgta cattgggaac ccaaagccgt 4020

acattgggaa ccggtcacac atgtaagtga ctgatataaa agagaaaaaa ggcgattttt 4080

ccgcctaaaa ctctttaaaa ctttattaaaa ctcttaaaac ccgcctggcc tgtgcataac 4140

tgtctggcca gcgcacagcc gaagagctgc aaaaagcgcc tacccttcgg tcgctgcgct 4200

ccctacgccc cgccgcttcg cgtcggccta tcgcggccgc tggccgctca aaaatggctg 4260

gcctacggcc aggcaatcta ccagggcgcg gacaagccgc gccgtcgcca ctcgaccgcc 4320

ggcgcccaca tcaaggcacc ctgcctcgcg cgtttcggtg atgacggtga aaacctctga 4380

cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa 4440

gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca 4500

cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga 4560

gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 4620

ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 4680

cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 4740

gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 4800

tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 4860

agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 4920

tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 4980

cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 5040

ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 5100

ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 5160

ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 5220

ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc 5280

cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 5340

gcggtggtttttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 5400

atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 5460

ttttggtcat gcattctagg tactaaaaca attcatccag taaaatataa tattttattt 5520

tctcccaatc aggcttgatc cccagtaagt caaaaaatag ctcgacatac tgttcttccc 5580

cgatatcctc cctgatcgac cggacgcaga aggcaatgtc ataccacttg tccgccctgc 5640

cgcttctccc aagatcaata aagccactta ctttgccatc tttcacaaag atgttgctgt 5700

ctcccaggtc gccgtgggaa aagacaagtt cctcttcggg cttttccgtc tttaaaaaat 5760

catacagctc gcgcggatct ttaaatggag tgtcttcttc ccagttttcg caatccacat 5820

cggccagatc gttattcagt aagtaatcca attcggctaa gcggctgtct aagctattcg 5880

tatagggaca atccgatatg tcgatggagt gaaagagcct gatgcactcc gcatacagct 5940

cgataatctt ttcagggctt tgttcatctt catactcttc cgagcaaagg acgccatcgg 6000

cctcactcat gagcagattg ctccagccat catgccgttc aaagtgcagg acctttggaa 6060

caggcagctt tccttccagc catagcatca tgtccttttc ccgttccaca tcataggtgg 6120

tccctttata ccggctgtcc gtcattttta aatataggtt ttcattttct cccaccagct 6180

tatatacctt agcaggagac attccttccg tatcttttac gcagcggtat ttttcgatca 6240

gttttttcaa ttccggtgat attctcattt tagccattatta ttattcctt cctcttttct 6300

acagtattta aagatacccc aagaagctaa ttataacaag acgaactcca attcactgtt 6360

ccttgcattc taaaacctta aataccagaa aacagctttt tcaaagttgt tttcaaagtt 6420

ggcgtataac atagtatcga cggagccgat tttgaaaccg cggtgatcac aggcagcaac 6480

gctctgtcat cgttacaatc aacatgctac cctccgcgag atcatccgtg tttcaaaccc 6540

ggcagcttag ttgccgttct tccgaatagc atcggtaaca tgagcaaagt ctgccgcctt 6600

acaacggctc tcccgctgac gccgtcccgg actgatgggc tgcctgtatc gagtggtgat 6660

tttgtgccga gctgccggtc ggggagctgt tggctggctg gtggcaggat atattgtggt 6720

gtaaacaaat tgacgcttag acaacttaat aacacattgc ggacgttttt aatgtactga 6780

attaacgccg aattaattcg ggggatctgg attttagtac tggattttgg ttttaggaat 6840

tagaaatttt attgatagaa gtattttaca aatacaaata catactaagg gtttcttata 6900

tgctcaacac atgagcgaaa ccctatagga accctaattc ccttatctgg gaactactca 6960

cacatttatta tggagaaact cgagcttgtc gatcgactct agctagagga tcgatccgaa 7020

ccccagagtc ccgctcagaa gaactcgtca agaaggcgat agaaggcgat gcgctgcgaa 7080

tcgggagcgg cgataccgta aagcacgagg aagcggtcag cccattcgcc gccaagctct 7140

tcagcaatat cacgggtagc caacgctatg tcctgatagc ggtccgccac accccagccgg 7200

ccacagtcga tgaatccaga aaagcggcca ttttccacca tgatattcgg caagcaggca 7260

tcgccatgtg tcacgacgag atcctcgccg tcgggcatgc gcgccttgag cctggcgaac 7320

agttcggctg gcgcgagccc ctgatgctct tcgtccagat catcctgatc gacaagaccg 7380

gcttccatcc gagtacgtgc tcgctcgatg cgatgtttcg cttggtggtc gaatgggcag 7440

gtagccggat caagcgtatg cagccgccgc attgcatcag ccatgatgga tactttctcg 7500

gcaggagcaa ggtgagatga caggagatcc tgccccggca cttcgcccaa tagcagccag 7560

tcccttcccg cttcagtgac aacgtcgagc acagctgcgc aaggaacgcc cgtcgtggcc 7620

agccacgata gccgcgctgc ctcgtcctgg agttcattca gggcaccgga caggtcggtc 7680

ttgacaaaaa gaaccgggcg cccctgcgct gacagccgga acacggcggc atcagagcag 7740

ccgattgtct gttgtgccca gtcatagccg aatagcctct ccacccaagc ggccggagaa 7800

cctgcgtgca atccatcttg ttcaatcccc atggtcgatc gacagatctg cgaaagctcg 7860

agagagatag atttgtagag agagactggt gatttcagcg tgtcctctcc aaatgaaatg 7920

aacttcctta tatagaggaa ggtcttgcga aggatagtgg gattgtgcgt catcccttac 7980

gtcagtggag atatcacatc aatccacttg ctttgaagac gtggttggaa cgtcttcttt 8040

ttccacgatg ctcctcgtgg gtgggggtcc atctttggga ccactgtcgg cagaggcatc 8100

ttgaacgata gcctttcctt tatcgcaatg atggcatttg taggtgccac cttccttttc 8160

tactgtcctt ttgatgaagt gacagatagc tgggcaatgg aatccgagga ggtttcccga 8220

tattaccctt tgttgaaaag tctcaatagc cctttggtct tctgagactg tatctttgat 8280

attcttggag tagacgagag tgtcgtgctc caccatgtta tcacatcaat ccacttgctt 8340

tgaagacgtg gttggaacgt cttctttttc cacgatgctc ctcgtgggtgggggtccatc 8400

tttgggacca ctgtcggcag aggcatcttg aacgatagcc tttcctttat cgcaatgatg 8460

gcatttgtag gtgccacctt ccttttctac tgtccttttg atgaagtgac agatagctgg 8520

gcaatggaat ccgaggaggt ttcccgatat taccctttgt tgaaaagtct caatagccct 8580

ttggtcttct gagactgtat ctttgatatt cttggagtag acgagagtgt cgtgctccac 8640

catgttggca agctgctcta gccaatacgc aaaccgcctc tccccgcgcg ttggccgatt 8700

cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca 8760

attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct 8820

cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgacatg 8880

attacgaatt cggaaacctc ctcggattcc attgcccagc tatctgtcac tttattgaga 8940

agatagtgga aaaggaaggt ggctcctaca aatgccatca ttgcgataaa ggaaaggcca 9000

tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg accccccaccc acgaggagca 9060

tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca agtggattga tgtgatatct 9120

ccactgacgt aagggatgac gcacaatccc actatccttc gcaagaccct tcctctatat 9180

aaggaagttc atttcatttg gagaggtatt aaaatcttaa taggttttga taaaagcgaa 9240

cgtggggaaa cccgaaccaa accttcttct aaactctctc tcatctctct taaagcaaac 9300

ttctctcttg tctttcttgc gtgagcgatc ttcaacgttg tcagatcgtg cttcggcacc 9360

agtacaacgt tttctttcac tgaagcgaaa tcaaagatct ctttgtggac acgtagtgcg 9420

gcgccattaa ataacgtgta cttgtcctat tcttgtcggt gtggtcttgg gaaaagaaag 9480

cttgctggag gctgctgttc agccccatac attacktgtt acgattctgc tgactttcgg 9540

cgggtgcaat atctctactt ctgcttgacg aggtattgtt gcctgtactt ctttcttctt 9600

cttcttgctg attggttcta taagaaatct agtattttct ttgaaacaga gttttcccgt 9660

ggttttcgaa cttggagaaa gattgttaag cttctgtata ttctgcccaa attcgcgatg 9720

gaacgagcta tacaaggaaa cgacgctagg gaacaagcta acagtgaacg ttgggatgga 9780

ggatcaggag gaagcacttc tcccttcaaa cttcctgacg aaagtccgag ttggactgag 9840

tggcggctac ataacgatga gacgaactcg aatcaagata atccccttgg tttcaaggaa 9900

agctggggtt tcgggaaagt tgtatttaag agatatctca gatacgacag gacggaggca 9960

tcactgcaca gagtcccttgg atcttggacg ggagattcgg ttaactatgc agcatctcga 10020

tttttcggtt tcgaccagat cggatgtacc tatagtattc ggtttcgagg agttagtatc 10080

accgtttctg gagggtctcg aactcttcag catctctgtg agatggcaat tcggtctaag 10140

caagaactgc tacagcttgc cccaatcgaa gtggaaagta atgtatcaag aggatgccct 10200

gaaggtactg agaccttcga aaaagaaagc gagggatctg gagttaagca aactttgaat 10260

tttgatttgt tgaagttggc tggagatgtt gaatctaatc ctggacctga gctcatgaca 10320

accttcaaaa tcgagtcccg catccacggc aacctcaacg gggagaagtt cgagttggtt 10380

ggaggtggag taggtgagga gggtcgcctc gagattgaga tgaagactaa agataaacca 10440

ctggcattct ctcccttcct gctgaccact tgcatgggtt acgggttcta ccacttcgcc 10500

agcttcccaa aggggattaa gaacatctat cttcatgctg caacgaacgg aggttacacc 10560

aacaccagga aggagatcta tgaagacggc ggcatcttgg aggtcaactt ccgttacact 10620

tacgagttca acaagatcat cggtgacgtc gagtgcattg gacatggatt cccaagtcag 10680

agtccgatct tcaaggacac gatcgtgaag agttgtccca cggtggacct gatgttgcca 10740

atgtccggga acatcatcgc cagctcctac gcttacgcct tccaactgaa ggacggctct 10800

ttctacacgg cagaagtcaa gaacaacata gacttcaaga atccaatcca cgagtccttc 10860

tcgaagtcag ggcccatgtt cacccacaga cgtgtcgagg agactctcac caaggagaac 10920

cttgccatag tggagtacca gcaggttttc aacagcgccc caagagacat gtagactagt 10980

ttaactctgg tttcattaaa ttttctttag tttgaattta ctgttattcg gtgtgcattt 11040

ctatgtttgg tgagcggttt tctgtgctca gagtgtgttt attttatgta atttaatttc 11100

tttgtgagca cctgtttagc aggtcgtccc ttcagcaagg acacaaaaag attttaattt 11160

tattgatcgt tcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct 11220

tgcgatgatt atcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta 11280

atgcatgacg ttattattga gatgggtttt tatgattaga gtcccgcaat tatacattta 11340

atacgcgata gaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc 11400

atctatgtta ctagatcccc ggggatctcc tttgccccag agatcacaat ggacgacttc 11460

ctctatctct acgatctagt caggaagttc gacggagaag gtgacgatac catgttcacc 11520

actgataatg agaagattag ccttttcaat ttcagaaaga atgctaaccc acagatggtt 11580

agagaggctt acgcagcagg tctcatcaag acgatctacc cgagcaataa tctccaggag 11640

atcaaatacc ttcccaagaa ggttaaagat gcagtcaaaa gattcaggac taactgcatc 11700

aagaacacag agaaagatat atttctcaag atcagaagta ctattccagt atggacgatt 11760

caaggcttgc ttcacaaacc aaggcaagta atagagattg gagtctctaa aaaggtagtt 11820

cccactgaat caaaggccat ggagtcaaag attcaaatag aggacctaac agaactcgcc 11880

gtaaagactg gcgaacagtt catacagagt ctcttacgac tcaatgacaa gaagaaaatc 11940

ttcgtcaaca tggtggagca cgacacactt gtctactcca aaaatatcaa agatacagtc 12000

tcagaagacc aaagggcaat tgagactttt caacaaaggg taatatccgg aaacctcctc 12060

ggattccatt gcccagctat ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc 12120

tcctacaaat gccatcattg cgataaagga aaggccatcg ttgaagatgc ctctgccgac 12180

agtggtccca aagatggacc cccaccacg aggagcatcg tggaaaaaga agacgttcca 12240

accacgtctt caaagcaagt ggattgatgt gataacatgg tggagcacga cacacttgtc 12300

tactccaaaa atatcaaaga tacagtctca gaagaccaaa gggcaattga gacttttcaa 12360

caaagggtaa tatccggaaa cctcctcgga ttccattgcc cagctatctg tcactttatt 12420

gtgaagatag tggaaaagga aggtggctcc tacaaatgcc atcattgcga taaaggaaag 12480

gccatcgttg aagatgcctc tgccgacagt ggtcccaaag atggaccccc accacgagg 12540

agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat 12600

atctccactg acgtaaggga tgacgcacaa tcccactatc cttcgcaaga cccttcctct 12660

atataaggaa gttcatttca tttggagagg acacgctgaa atcaccagtc tctctctcaa 12720

gctt 12724

<210> 3

<211> 1503

<212>DNA

<213> Catharanthus roseus

<400> 3

atgggcagca ttgattcaac aaatgtagcc atgtccaatt ctccagttgg agaatttaag 60

ccacttgaag ctgaggaatt ccgaaaacaa gcccatcgta tggtagattt catagccgat 120

tattacaaaa atgtggaaac atatccggtc cttagcgaag tcgaacctgg atatctccga 180

aaacgtatcc ccgaaaccgc tccttacctc cccgaatcac tcgacgacat catgaaagat 240

attcagaagg atattatccc aggaatgaca aattggatga gccctaattt ttatgcattt 300

tttcctgcca ctgttagttc agctgccttt ttaggagaaa tgttgtctac tgccctaaat 360

tcagtaggct ttacttgggt ttcttcacca gccgccaccg aattagaaat gattgttatg 420

gattggttgg ctcagatcct taaactcccc aaatctttca tgttttcagg tacgggtggc 480

ggcgtcatcc aaaacaccac tagtgagtcc attctttgta caatcattgc cgcccgggaa 540

agggccctgg agaagctcgg tcccgatagt attggaaaac ttgtctgtta cggatcagat 600

caaacccata ccatgttccc aaaaacttgc aaattggcgg gaattttccc gaataatatt 660

aggttaatac ctacaaccgt cgaaacggat ttcggcatct cacctcaagt tctacgaaaa 720

atggtcgagg atgacgtggc ggccggatat gtaccgctgt tcttatgcgc taccctgggt 780

accacctcga ccacggctac cgatcctgtg gactcacttt ctgaaatcgc taacgagttt 840

ggtatttgga tccacgtgga tgcggcttat gcgggcagcg cctgtatatg tcccgagttc 900

agacattact tggatggaat cgagcgagtt gactcactga gtctgagtcc acacaaatgg 960

ctactcgctt acttagattg cacttgcttg tgggtcaagc aaccacattt gttactaagg 1020

gcactcacta cgaatcctga gtatttaaaa aataaacaga gtgattaga caaagttgtg 1080

gacttcaaaa attggcaaat cgcaacggga cgaaaatttc ggtcgcttaa actttggctc 1140

attttacgta gctatggagt tgttaattta cagagtcata ttcgttctga cgtcgcaatg 1200

gcgaaaatgt tcgaagaatg ggttagatca gactccagat tcgaaattgt ggtaccaaga 1260

aacttttctc ttgtttgttt tagattaaag cctgacgttt cgagtttaga tgtagaagaa 1320

gtgaataaga aacttttgga tatgcttaac tcgacgggac gagtttatat gactcatact 1380

attgtggggag gcatatacat gctaagactg gctgttggct catcgctaac tgaagaacat 1440

catgtacgcc gtgtttggga tttgattcaa aaattaaccg atgatttgct caaagaagct 1500

tga 1503

<210> 4

<211> 1575

<212>DNA

<213> Catharanthus roseus

<400> 4

atggagatgg atatggatac cattagaaag gcaattgctg ccactatttt tgcattggta 60

atggcttggg catggagagt gttggattgg gcatggtta ctcctaagag gatcgagaaa 120

cgtctaaggc agcaaggttt tagaggaaat ccttatagat tcttggttgg agatgttaag 180

gagagtggaa aaatgcatca agaagccttg tctaaaccca tggagttcaa caatgatatt 240

gttcctcgcc tcatgccaca tattaaccac actatcaata cttacggtag gaattccttt 300

acatggatgg gaaggattcc aagaattcat gttatggaac ctgaacttat taaggaagta 360

ttgacccact caagcaaata ccaaaagaac tttgatgttc acaatcccct tgttaagttc 420

cttctcaccg gagttggaag ctttgagggt gcaaaatggt caaaacacag aagaattatt 480

tcccctgcct tcactcttga gaaactaaag tcaatgctgc cagcttttgc catatgctac 540

catgacatgt tgaccaaatg ggagaaaata gctgaaaaac aaggatccca tgaagttgat 600

atctttccca cgtttgatgt tttaacaagt gatgtgattt caaaggttgc atttggtagc 660

acatatgaag aaggaggcaa aatcttcaga ctattgaaag aactcatgga tctcacaatt 720

gactgcatga gagatgtcta cattccagga tggagctact tgccaaccaa gaggaacaag 780

aggatgaaag aaattaacaa agagatcaca gatatgctaa ggttcatcat caacaagaga 840

atgaaggctt tgaaggctgg agagccaggt gaggatgact tgctgggagt attgttggaa 900

tcaaacattc aagaaattca aaagcaagga aacaagaagg atggtggaat gtcaatcaat 960

gatgtaattg aggagtgcaa attgttctac tttgctggtc aagaaactac tggagtttta 1020

ctgacatgga ccaccatctt attgagcaag caccctgagt ggcaagagcg agctagagaa 1080

gaagttctcc aagcctttgg caagaataaa cctgaatttg aacgcttaaa tcacctcaaa 1140

tatgtgtcta tgatcttgta cgaggttcta aggttgtacc caccagtgat tgatctaacc 1200

aagattgtcc acgaggacac aaagttaggt ccgtacacaa ttcctgcagg aacacaagtg 1260

atgttgccaa cagtaatgct tcacagagag aagagcattt ggggagaaga tgcaacagaa 1320

ttcaacccaa tgagatttgc tgatggagtt gccaatgcaa ccaagaacaa tgtcacatat 1380

ttgccattca gttggggacc tagggtttgt cttggccaaa actttgcact tctgcaagca 1440

aaattaggat tggcaatgat tttacaacgc ttcacgtttg atgttgctcc atcctatgtt 1500

catgctcctt ttaccattct cacagttcaa ccccagtttg gttctcatgt catctacaag 1560

aagcttgaga gctag 1575

<210> 5

<211> 1668

<212>DNA

<213> Catharanthus roseus

<400> 5

atgggatcta aagatgatca gtcccttgtt gttgccattt ctccagctgc tgaaccaaat 60

ggaaatcatt ctgtccccat cccattcgcc taccccagta tccccattca acctagaaag 120

cacaacaagc ccatcgttca tcgtcgagat ttcccctcag atttcatctt gggtgccgga 180

ggatctgctt atcagtgtga gggtgcatat aatgaaggca accgcggtcc cagtatatgg 240

gatactttca caaaccgata tccagccaaa atagctgatg gatctaatgg caatcaagcc 300

atcaattctt acaatttgta caaggaagat atcaagatta tgaagcaaac aggcttggaa 360

tcatataggt tttcaatttc atggtcaaga gtattgccag gtggaaatct atccggtgga 420

gtgaataaag atggtgtcaa gttctatcat gactttatag atgagcttct agccaatggc 480

atcaaaccct ttgcaactct cttccactgg gatcttcccc aagctcttga agacgagtat 540

ggaggcttct tgagtgatcg aattgtggaa gattttacgg agtatgcaga attttgcttt 600

tgggaattcg gtgacaaagt aaaattttgg acgactttca atgaaccaca tacttatgtt 660

gcaagtggat atgccactgg tgaatttgca ccaggaagag gtggtgcaga tggcaagggg 720

aaccctggca aagaacccta tatagcgaca cataatttac ttctttctca caaagctgct 780

gtggaagtat ataggaaaaa ttttcagaaa tgtcaaggag gtgaaattgg aattgtactt 840

aattcaatgt ggatggagcc tctcaatgaa accaaagaag atattgatgc tcgggaaagg 900

ggtcttgatt tcatgctcgg atggttcata gagccattaa caacgggtga atacccaaaa 960

tccatgagag ctcttgtagg aagccgtctt ccagaatttt caacagaaga ttccgaaaaa 1020

ttaacaggat gctatgattt tatcggaatg aattattata caactactta tgtttctaat 1080

gcagacaaaa ttcccgatac tccgggttac gaaacagatg ctcgaattaa taagaatatt 1140

tttgtcaaaa aagttgatgg gaaggaagtg cgcattggtg aaccgtgcta tgggggatgg 1200

cagcatgttg ttccatctgg actctacaat ctcttggttt acactaagga gaaataccat 1260

gttccagtga tttatgtctc agaatgtggt gtggttgagg aaaatagaac caacatatta 1320

cttacagaag gtaaaaccaa catattactt acagaagctc gtcacgataa actcagggtt 1380

gattttctac aaagtcatct cgctagcgtg cgagatgcta ttgatgatgg tgtgaatgta 1440

aaaggattct ttgtttggtc attcttcgac aacttcgaat ggaatttggg atatatatgc 1500

cgttatggaa ttatccatgt tgattataaa acttttcaaa gatatccaaa ggattctgcc 1560

atatggtaca agaatttcat tagtgaagga tttgttacga atacagctaa aaagagattc 1620

cgagaagaag ataaactagt tgagttagtc aagaagcaaa aatactaa 1668

Claims (3)

1. Agrobacterium binary expression carrier, it comprises: deleted the carrier pCambia2301 backbone of GUS selection marker gene, 35S promoter gene, P19 protein coding gene and GFP selection marker gene, wherein 35S promoter gene, P19 protein coding gene It is fused with the GFP screening marker gene by self-cleaving polypeptide 2A to form a fusion gene. The nucleotide sequence of the fusion gene is SEQ ID NO: 1; respectively encoding tryptophan decarboxylase TDC and split loganin synthesis The genes of enzyme SLS, isosidine synthase STR, isosidine beta-D glucosidase SGD, nitrate transporter NPF2.9, isoyohimbine alkaloid synthase HYS, among which The nucleotide sequence of the gene encoding tryptophan decarboxylase TDC is SEQ ID NO: 3; the nucleotide sequence of the gene encoding split loganin synthase SLS is SEQ ID NO: 4; The nucleotide sequence of gene is GenBank accession number: X61932; The nucleotide sequence of isosidine beta-D type glucosidase SGD coding gene is SEQID NO:5; The nucleotide of nitrate transport protein NPF2.9 coding gene The sequence is GenBank accession number: KX372303; the nucleotide sequence of the gene encoding heteroyohimbine alkaloid synthase HYS is GenBank accession number: KU865325. 2. An Agrobacterium transformed with the Agrobacterium binary expression vector according to claim 1. 3. the application of agrobacterium as claimed in claim 2 in the production of amoline, is characterized in that, infects tobacco with agrobacterium as claimed in claim 2, produces amoline with tobacco as biosynthesis system.

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