CN113563440B

CN113563440B - Tropane alkaloid transporter AbTAUP1 and application thereof

Info

Publication number: CN113563440B
Application number: CN202110832216.4A
Authority: CN
Inventors: 廖志华; 陈敏; 杨春贤; 曾俊岚; 邱飞
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2021-07-22
Filing date: 2021-07-22
Publication date: 2023-08-08
Anticipated expiration: 2041-07-22
Also published as: CN113563440A

Abstract

The invention discloses tropane alkaloid transport protein AbTAUP1 and application thereof, wherein the amino acid sequence of the tropane alkaloid transport protein AbTAUP1 is shown as SEQ ID NO.8, or the sequence shown as SEQ ID NO.8 is substituted, mutated or deleted by one or more amino acid residues and encodes an amino acid sequence with the same function; the protein has the highest expression level in lateral roots, lower expression level in main roots, is positioned in cytoplasmic membranes, is supposed to have a tropane transfer function, verifies the tropane transfer function through yeast transfer experiments, and over-expresses AbTAUP1 in belladonna plants to improve the tropane yield in belladonna, so that the protein can be used for providing the tropane transfer capability in the plant synthesized by the tropane, improving the tropane yield and has important significance for improving the tropane alkaloid content in plant seeds.

Description

Tropane alkaloid transporter AbTAUP1 and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a tropane alkaloid transporter AbTAUP1 and application of the tropane alkaloid transporter.

Background

Tropane Alkaloids (TA) are a class of anticholinergic agents that act on parasympathetic nerves. The representative drugs of scopolamine, scopolamine and derivatives thereof are widely applied to the fields of anesthetics, treatment of parkinsonism, various visceral angina and asthma caused by smooth muscle spasm and the like, and have great market demands. The tropane alkaloids still depend entirely on the use of a few Solanaceae medicinal plants at presentComprises belladonna (Atropa belladonna), datura (Datura stramonium) and Hyoscyamus (Hyoscyamus niger). Belladonna is a commercial cultivated herb source plant recorded and regulated in Chinese pharmacopoeia for producing hyoscyamine and scopolamine. The mass fraction of scopolamine in wild belladonna plants is 0.02-0.17% (dry weight), and the scopolamine content is only 0.01-0.08% of dry weight. Therefore, the cultivation of tropane alkaloids high-yield medicinal plants has been a long-sought goal of the industry. Along with the rapid development of genetic engineering technology, metabolic engineering methods based on targeted modification of biosynthetic pathways have become an important means for increasing the content of hyoscyamine and scopolamine in drug-derived plants. In recent years, the establishment of a yeast engineering platform and the development of synthetic biology provide a possible strategy for solving the problem of tropane alkaloid resource shortage in the market. However, in the current tropane alkaloid total synthesis yeast strain, the maximum scopolamine yield is only 80 mu g.L ^-1 The scopolamine yield is only 30 mu g.L at the highest ^-1 Far below the commercial production requirements.

Tropane alkaloid medicine source plants such as belladonna, stramonium, scopolamine and the like are full plants rich in scopolamine, but researches show that enzyme genes of the biosynthesis pathway of scopolamine are specifically expressed in secondary roots of the plants. Thus, scopolamine, although specifically synthesized only in secondary roots of plants, is transported to aerial parts for storage. Secondary roots are the "source" of biosynthesis, while the aerial parts are the "pool" for storage. Enhancing the "source" to "sink" transport process facilitates pulling the biosynthetic metabolic stream. Thus, tropane alkaloid transporter has very important application value for tropane alkaloid plant metabolic engineering and microorganism synthesis biology. Unfortunately, no report has been made to date concerning the transport of tropane alkaloids. Thus, there is an urgent need for tropane alkaloid transporters to increase scopolamine yield in plants.

Disclosure of Invention

Accordingly, it is an object of the present invention to provide a tropane alkaloid transporter abtau 1; the second object of the present invention is to provide a recombinant expression vector comprising a gene encoding said tropane alkaloid transporter abtau up1; it is a third object of the present invention to provide a host comprising a gene encoding said tropane alkaloid transporter abtau up1; the fourth object of the invention is to provide the application of the tropane alkaloid transporter AbTAUP1 in improving the hyoscyamine content of a plant synthesized by hyoscyamine; the fifth object of the present invention is to provide the use of said tropane alkaloid transporter abtau 1 for increasing the scopolamine transport capacity in a plant for synthesizing scopolamine; the sixth object of the present invention is to provide a method for increasing the content of hyoscyamine in hyoscyamine synthesized plants.

In order to achieve the aim, a tropane alkaloid transporter AbTAUP1 is discovered for the first time, the scopolamine transporting function is verified through a yeast transporting experiment, and the AbTAUP1 is overexpressed in a belladonna plant to improve the scopolamine yield in the belladonna, and the specific scheme is as follows:

1. the tropane alkaloid transporter AbTAUP1 has an amino acid sequence shown in SEQ ID NO.8, or a sequence shown in SEQ ID NO.8 is substituted, mutated or deleted by one or more amino acid residues and encodes an amino acid sequence with the same function.

Preferably, the nucleotide sequence for encoding the tropane alkaloid transporter AbTAUP1 is shown as SEQ ID NO.7, or the nucleotide sequence with the same function is encoded by substitution, mutation or deletion of one or a plurality of nucleotides of the sequence shown as SEQ ID NO. 7.

2. A recombinant expression vector containing the gene encoding the tropane alkaloid transporter abtau up1.

Preferably, the recombinant expression vector is obtained by ligating the sequence shown in SEQ ID No.7 into pBI121 vector through BamHI and SacI.

3. A host comprising a gene encoding said tropane alkaloid transporter abtau up1.

Preferably, the host is belladonna.

4. The tropane alkaloid transporter AbTAUP1 is applied to improving the hyoscyamine content of a hyoscyamine synthesis host.

Preferably, the scopolamine synthesis host is belladonna, stramonium, scopolamine, anisodamine or yeast.

5. The tropane alkaloid transporter AbTAUP1 is applied to improving the scopolamine transporting capacity in a scopolamine synthesis host.

6. Constructing a recombinant expression vector containing an AbTAUP1 gene for encoding tropane alkaloid transport protein, and then expressing the recombinant expression vector in a plant synthesized by hyoscyamine to obtain a variety with high hyoscyamine content; the nucleotide sequence of the encoding tropane alkaloid transporter AbTAUP1 gene is shown as SEQ ID NO.7 or the nucleotide sequence shown as SEQ ID NO.7 is substituted, mutated or deleted by one or a plurality of nucleotides and encodes the nucleotide sequence with the same function.

The invention has the beneficial effects that: the invention discovers that the tropane alkaloid transport protein AbTAUP1 has the highest expression level in lateral roots, has lower expression level in main roots and is positioned on cytoplasmic membranes, is supposed to have a tropane alkaloid transport function, the tropane alkaloid transport function is verified through yeast transport experiments, and the tropane alkaloid transport function is improved by over-expressing AbTAUP1 in belladonna plants, so that the tropane alkaloid transport protein can be used for providing the tropane alkaloid transport capacity in belladonna plants, improving the tropane alkaloid yield and having important significance for improving the tropane alkaloid content in plant species.

Drawings

In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:

FIG. 1 shows the expression profile analysis of the AbTAUP1 gene in various tissues of belladonna;

FIG. 2 is a subcellular localization result;

FIG. 3 is a graph showing the detection of scopolamine transport in cells by yeast;

FIG. 4 shows the results of detecting the expression level of AbTAUP1 gene in wild type belladonna and AbTAUP1 over-expressed belladonna roots;

figure 5 shows the scopolamine content of wild type belladonna and AbTAUP1 overexpressing belladonna roots.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.

Example 1 tissue expression profiling screening for potential tropane alkaloid transporters

(1) Transcriptome analysis screening for potential tropane alkaloid transporter

The family of purine base transporters (Purine nucleobase transmembrane transporter, PUNUT) is widely available in plants and fungi and is involved in the transport of plant metabolites such as cytokinins, caffeine and nicotine. The corresponding hidden Markov model (Hidden Markov Model, HMM) in the Pfam database has accession number PF16913.5, 19 candidate genes are identified in a belladonna transcriptome by using the PF16913.5 model, and the candidate genes and tropane alkaloid synthesis pathway enzyme genes are subjected to tissue expression cluster analysis to obtain a PUNUT family transporter gene which is highly expressed in roots, which has higher correlation with the tropane alkaloid synthesis pathway enzyme gene expression, and is presumed to be possibly involved in tropane alkaloid transport, and named as belladonna tropane alkaloid transporter 1 (Tropane Alkaloid Uptake Permease 1, abTAUP1).

(2) qPCR verifies its expression pattern

And respectively taking small parts of fresh wild belladonna main root, fibrous root, stem and leaf materials, putting the materials into liquid nitrogen for quick freezing, and extracting total RNA according to the specification of a TIANGEN kit. The concentration of RNA was determined and cDNA was synthesized according to the instructions of the first strand synthesis kit of the FastKing cDNA of the root, and cDNA libraries of the main root, side root, stem and leaf of belladonna were established. Fluorescent quantitative PCR was performed using AbPGK as an internal reference gene and AbTAUP1 as a target gene. According to 2 ^-△△Ct The method calculates the relative expression of each gene, and the specific primer sequences are as follows:

qAbTAUP1-F：5’-gagtcacatgctatgcttca-3’(SEQ ID NO.1)；

qAbTAUP1-R：5’-tgagtcatctcagcttctgt-3’(SEQ ID NO.2)；

qAbPGK-F：5’-tcgctcttggagaaggttgac-3’(SEQ ID NO.3)；

qAbPGK-R：5’-cttgtcggcaatcactacatcag-3’(SEQ ID NO.4)；

the expression of the AbTAUP1 gene in various tissues of belladonna was analyzed according to qPCR results, and the results are shown in FIG. 1. The results show that AbTAUP1 has higher expression level in roots and lower expression level in stems and leaves, which is consistent with the analysis result of transcriptome expression, and further shows that the transporter AbTAUP1 is very likely to be involved in the transportation of tropane alkaloid.

(3) Gene cloning

Total RNA of the side roots of the sterile seedlings of belladonna is extracted according to the instruction of a TIANGEN kit, and cDNA is synthesized according to the instruction of a first strand synthesis kit of the FastKing cDNA of the root of the Chinese belladonna. Analysis is carried out according to the AbTAUP1 gene core sequence obtained by belladonna transcriptome screening, the 5' end of the gene is found to be complete, only a 3' end RACE specific primer is required to be designed, and the sequence at the 3' end is complemented by the RACE technology to obtain the full-length electronic sequence of AbTAUP1. The specific primer of the gene is designed according to the full-length sequence of electronic splicing, and the specific primer of the gene coding region is as follows:

AbTAUP1-F：5’-atggaatctcaaattgctag-3’(SEQ ID NO.5)；

AbTAUP1-R：5’-ttatattggaggggattgag-3’(SEQ ID NO.6)；

using belladonna cDNA as a template, carrying out PCR amplification by using a gene coding region specific primer, detecting and confirming the size of a PCR product by agarose electrophoresis, recovering a target strip, and sequencing to finally successfully obtain the cDNA sequence of AbTAUP1, wherein the nucleotide sequence is shown as SEQ ID NO.7, and the coded amino acid sequence is shown as SEQ ID NO. 8.

Example 2 subcellular localization analysis of AbTAUP1

(1) Construction of subcellular localization vectors

The transmembrane domain of the abtau up1 protein is predicted to find that the protein contains ten transmembrane domains and almost the whole protein penetrates the membrane structure, so that the abtau up1 protein is likely to exert the transport capacity for the substrate on the cell membrane. To verify that the abtau up1 protein is a transmembrane protein, a subcellular localization vector was constructed and the abtau up1 was subjected to subcellular localization analysis. The purchased PM-ck/CD3-1001 plasmid (purchased linked https:// abcc. Osu. Edu/stocks/764634) can express the plasma membrane localization protein PM-CFP fused with the cyan fluorescent protein CFP as a plasma membrane marker signal. After transformation of Agrobacterium GV3101, the engineering strain GV3101-pCD3-1001 was obtained.

The restriction sites of the coding region of the AbTAUP1 gene were analyzed and combined with the multiple cloning sites of pGD3G-YFP (https:// doi.org/10.1046/j.1365-313X.2002.01360. X) vector. Finally, xhoI and HindIII are selected as enzyme cutting sites to design upstream and downstream specific primers. The primer sequences were as follows:

pGD3G-AbTAUP1-F:5’-cgc ctcgagatggaatctcaaattgctag-3’(SEQ ID NO.9)；

pGD3G-AbTAUP1-R:5’-cgc aagctttattggaggggattgagtca-3’(SEQ ID NO.10)。

after PCR amplification, the vector and the PCR product are subjected to double enzyme digestion and then are connected, and the recombinant plasmid pGD3G-AbTAUP1-YFP is obtained after sequencing is successful. The AbTAUP1 expressed by the plasmid and yellow fluorescent protein YFP form fusion proteins. After transferring the recombinant plasmid and pGD3G-YFP into agrobacterium GV3101, engineering strains GV3101-pGD3G-AbTAUP1-YFP and GV3101-YFP are obtained.

(2) Subcellular localization analysis

The activated GV3101-YFP, GV3101-pGD3G-AbTAUP1-YFP and GV3101-pCD3-1001 engineering bacteria were cultured overnight at 28℃and 200rpm, respectively. The positive bacteria were centrifuged at 4500rpm for 6min and the medium was discarded. With MES at a final concentration of 10mM, mgCl at a final concentration of 10mM ₂ And 40mM AS (acetosyringone) mixed solution to gently suspend the bacterial cells, and regulating the bacterial liquid OD ₆₀₀ =0.6, left at room temperature for 3h. Different bacterial solutions were mixed and two experiments were set up. The mixed bacterial liquid is injected into the Nicotiana benthamiana leaves with the size of 6-8 weeks by a syringe without adding a needle, the leaves are subjected to weak light culture for 48 hours, then the leaves are cracked by cellulase and pectase to obtain protoplasts, and a laser confocal microscope Olympus FV1000 is used for photographing, and the result is shown in figure 2. The results showed that the free YFP yellow fluorescent signal did not coincide exactly with the PM-CFP cyan fluorescent signal, since the YFP fluorescent protein alone was not specifically spatially localized in the tobacco cells. The yellow fluorescence of AbTAUP1-YFP almost completely coincides with the blue fluorescence of plasma membrane localization signal PM-CFP, and the blue fluorescence is only expressed on plasma membrane, which indicates that AbTAUP1 is localizedCytoplasmic membranes.

Example 3 use of AbTAUP1 in Yeast

(1) Construction of Yeast transport Carrier and engineering Strain

Whether the AbTAUP1 gene has the ability to transport tropane alkaloids or not was investigated by constructing yeast transport vectors. The upstream and downstream primers with PstI and XhoI restriction endonuclease cleavage sites were designed based on the multiple cloning site of the yeast expression vector pDR 196. The specific primers are as follows:

pDR-AbTAUP1-F：5’-ggg ctgcagatggaatctcaaattgctag-3’(SEQ ID NO.11)；

pDR-AbTAUP1-F：5’-ccc ctcgagttatattggaggggattgag-3’(SEQ ID NO.12)。

after PCR amplification, the pDR196 plasmid and the PCR product are subjected to double digestion and recovery and then are connected, and finally the recombinant plasmid pDR196-AbTAUP1 is successfully obtained.

The yeast cells are transformed by a chemical method, the pDR196-AbTAUP1 recombinant plasmid is transferred into a Saccharomyces cerevisiae mutant strain ADR8, and the mutant strain lacks 8 yeast endogenous membrane transport proteins and is widely applied to research of the functions of transport proteins. Meanwhile, the ADR8 mutant yeast strain transformed with pDR196 empty load is used as a control, and the transgenic yeast is used for subsequent feeding experiments.

(2) Scopolamine feeding engineering yeast

Inoculating transgenic engineering yeast into 50mL uracil-deficient liquid culture medium at 30deg.C and shaking culture at 200rpm to OD ₆₀₀ =1, 3 biological replicates were set per group. Centrifuging the thallus, discarding the supernatant, adding 50mL 1/2 uracil-deficient medium again, and simultaneously adding 0.5mM hyoscyamine, scopolamine and anisodamine into the medium respectively, and shake culturing at 30deg.C and 220 rpm. Sampling for 3 hours, centrifugally collecting thalli, adding precooled ddH ₂ O was washed three times. 100. Mu.L of the extract (containing 50% ethanol, 49.5% methanol and 0.05% acetic acid in volume ratio) was added, then 100. Mu.L of glass beads were added, and the mixture was broken by shaking with a vortex shaker for 15min, and the supernatant was centrifuged and analyzed by HPLC.

(3) HPLC determination of hyoscyamine content in Yeast cells

The scopolamine content in the yeast cells was determined by using Shimadzu high performance liquid chromatograph. The specific instrument parameters are as follows:

chromatograph: shimadzu Shimadzu high performance liquid chromatograph (system controller: CBM-20Alite, pump: LC-20 AD);

column incubator: CTO-20A, detector: SPD-M20A, autosampler: SIL-20A);

chromatographic column: PHenomenex Gemini 5 u C18 110A liquid chromatography column (250X 4.6mm,5 m. Acron);

mobile phase: acetonitrile in ammonium acetate buffer (containing 20mM ammonium acetate, 0.1% formic acid, ph=4.0) =11:89;

flow rate: 1mL/min;

detection wavelength: 226nm;

column temperature: 40 ℃;

sample injection amount: 20. Mu.L;

the detection results are shown in FIG. 3. HPLC result analysis shows that the control group pDR196 yeast strain has no transporting capacity to scopolamine, anisodamine and scopolamine; the transformed yeast strain of pDR196-AbTAUP1 has obvious transfer capacity to scopolamine only and has no transfer capacity to scopolamine and anisodamine. The results indicate that abtau 1 is a hyoscyamine-specific transporter localized to the plasma membrane.

Example 4 use of AbTAUP1 in plant Metabolic engineering

(1) Construction of overexpression vector

In order to evaluate whether over-expression of abtau up1 in plants affects the transport and content changes of tropane alkaloids in belladonna, the present study used belladonna plants to develop plant metabolic engineering by constructing over-expression vectors. The plant expression vector pBI121 is selected as an over-expression vector of a belladonna plant, and the enzyme digestion site contained in the sequence is analyzed according to the AbTAUP1 coding region sequence obtained above and combined with the enzyme digestion site carried by the cloning site of the over-expression vector pBI 121. The upstream introduced cleavage site was designed as BamHI and the downstream introduced cleavage site was designed as SacI. The specific primer sequences are as follows:

pBI121-AbTAUP1-F:5’-cgc ggatccatggaatctcaaattgctag-3’(SEQ ID NO.13)；

pBI121-AbTAUP1-R:5’-cgc gagctcttatattggaggggattgag-3’(SEQ ID NO.14)；

and (3) carrying out PCR amplification by using the primers, and carrying out double digestion recovery on the pBI121 plasmid and a PCR product and then connecting to finally obtain the recombinant plasmid pBI121-AbTAUP1.

(2) Genetic transformation of belladonna plants

The plant over-expression vector pBI121-AbTAUP1 constructed in the above is transformed into agrobacterium EHA105 by adopting a freeze thawing method, and the engineering strain is successfully obtained for subsequent genetic transformation through PCR positive identification.

The belladonna seeds are soaked in gibberellin solution overnight, and then are washed by tap water for 1 day for disinfection. Sterilizing with 75% ethanol for 1min and 50% NaClO solution for 20min, respectively, and shaking thoroughly to ensure adequate sterilization. Finally, cleaning the product for 5 to 6 times by using sterile water. Placing sterilized belladonna seeds on sterile absorbent paper with forceps, absorbing excessive water, inoculating to solid culture medium of MS+200mg/LCef at 25deg.C for about 15 days under 16h/8h (light/dark) light condition, and transferring the budded belladonna wild type seedlings into glass bottle of MS solid culture medium.

The activated engineering bacteria are gently resuspended by using a sterilized MS liquid culture medium added with AS, and OD is regulated ₆₀₀ To about 0.3 to 0.5. Cutting off belladonna leaves in an ultra-clean workbench, adding the belladonna leaves into the re-suspended agrobacterium tumefaciens bacteria liquid, and infecting for about 5-8 min. It was plated on MS solid co-culture medium (MS+AS+6-BA 1.0mg/L+NAA1.0 mg/L) and dark-cultured at 28℃for 2 days. After 2 days, the plant is transferred to differentiated solid culture media of different antibiotics, and the screening culture media of the overexpression vector are (MS+NAA 1.0mg/L+6-BA1.0 mg/L+Cef200mg/L+Kan8mg/L), and the screening culture media are replaced once every other week until cluster buds grow. The cluster buds are cut off and transferred to rooting medium (1/2MS+0.05 g/L NAA) for rooting induction. The regenerated plants were removed from the pots, cleaned of root medium, planted in a cultivation substrate (vermiculite: turfy soil: perlite=6:3:1) and cultivated in a climatic chamber at 25 ℃ for 16h/8h (light/dark). Detection by genomic PCRAnd (5) detecting and screening positive AbTAUP1 over-expression transgenic belladonna plants. The wild belladonna and the AbTAUP1 over-expressed belladonna root are taken for real-time fluorescence quantitative PCR analysis of the AbTAUP1 gene expression quantity, and the method is the same as the previous method. As shown in the results of FIG. 4, the expression amount of AbTAUP1 in the over-expressed strain is 10-26 times that of the wild type control, which indicates that belladonna plants in which the AbTAUP1 gene was over-expressed have been successfully obtained.

(3) HPLC analysis of scopolamine content in belladonna

Collecting root and leaf of positive belladonna seedling grown in culture room for 3 months respectively in clean envelopes, oven drying at 40deg.C, grinding into powder, and extracting alkaloid. The specific method comprises the steps of weighing about 0.1g of dry powder, transferring the dry powder into a 50mL EP tube, adding 10mL of alkaloid extract, performing ultrasonic crushing for 1h, and standing overnight; after the night extraction, carrying out ultrasonic extraction for 1 hour, and standing for more than 1 hour; filtering the extracting solution by using filter paper, washing residues by using 5mL of chloroform, collecting filtrate and washing solution in a 50mL beaker, and drying in a 40 ℃ oven; the residue after the evaporation was purified by using 5mL of chloroform and 2mL of 1M H ₂ SO ₄ The mixed solution is dissolved by ultrasonic, is transferred into a10 mL EP tube after being fully and evenly mixed, and is centrifuged for 10min at 4000 rpm; discarding the lower organic waste liquid, adding 1.5mL of ammonia water into an EP tube to alkalize the solution, extracting alkaloid with 3mL of chloroform, and collecting chloroform phase in a 50mL beaker; extracting once with 2mL chloroform, and combining the chloroform phases; drying chloroform at 40deg.C, dissolving alkaloid with 1mL of methanol; the alkaloids were filtered with a filter head and tested by HPLC. HPLC specific instrument parameters were as described above and the results are shown in FIG. 5. HPLC result analysis shows that the content of scopolamine in wild type belladonna leaves is 1.54mg/g, and the content of scopolamine in roots is 1.34mg/g. The scopolamine content in the leaves and roots of the plant of the over-expressed AbTAUP1 belladonna is greatly improved, the scopolamine content in the leaves is 2.36-3.01mg/g, and is 153-195% of that of the wild plant; the scopolamine content in root is 1.81-2.44mg/g, which is 135% -182% of wild type. The results demonstrate that over-expression of abtau up1 is beneficial to enhancing the transport of scopolamine from "source" to "pool" in belladonna, and pulls anabolic flow, ultimately effectively enhancing accumulation of scopolamine.

The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.

Sequence listing

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cttggtgcaa gtagtgatag gccagcaggg gagtcaagta aggcatatat tgtagggttt 540

attatgacac tgcttgctgc attattttat ggatttgttc tggcattcaa cgaagtaagt 600

tttaggaaaa caaagaaggt tattgccttt acattggttt tggagtttca gatgatgatg 660

tgtttctttg ctactgcttt ttgtgtcacg gggatgctta ttaacaagga tattcaggca 720

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tcagcattac tctctggaat tttgatagct gcttcacttt cagttacaga agtgttggct 900

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Thr Ala Ile Phe Ala Phe Leu Ile Val Lys Gln Lys Phe Thr Ser Tyr

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Ser Ile Asn Ala Val Val Val Leu Thr Leu Gly Ala Gly Ile Leu Ala

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Leu Gly Ala Ser Ser Asp Arg Pro Ala Gly Glu Ser Ser Lys Ala Tyr

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Ile Val Gly Phe Ile Met Thr Leu Leu Ala Ala Leu Phe Tyr Gly Phe

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Val Leu Ala Phe Asn Glu Val Ser Phe Arg Lys Thr Lys Lys Val Ile

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Ala Phe Thr Leu Val Leu Glu Phe Gln Met Met Met Cys Phe Phe Ala

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Met Leu Ile Val Leu Ser Ala Leu Ile Trp Gln Ile Phe Phe Val Gly

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Ala Ile Gly Val Thr Cys Tyr Ala Ser Ala Leu Leu Ser Gly Ile Leu

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Ile Ala Ala Ser Leu Ser Val Thr Glu Val Leu Ala Val Ile Phe Phe

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His Glu Lys Ile Gly Gly Glu Lys Gly Phe Ser Leu Ala Leu Ser Leu

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Trp Gly Phe Val Ser Tyr Phe Tyr Gly Glu Ile Lys Gln Thr Asn Lys

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gggctgcaga tggaatctca aattgctag 29

<210> 12

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

cccctcgagt tatattggag gggattgag 29

<210> 13

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

cgcggatcca tggaatctca aattgctag 29

<210> 14

<211> 29

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

cgcgagctct tatattggag gggattgag 29

Claims

1. Tropane alkaloid transporter abtau up1, characterized in that: the amino acid sequence of the tropane alkaloid transporter AbTAUP1 is shown as SEQ ID NO. 8.

2. A gene encoding the tropane alkaloid transporter abtau up1 of claim 1, characterized in that: the nucleotide sequence of the gene is shown as SEQ ID NO. 7.

3. A recombinant expression vector comprising a gene encoding the tropane alkaloid transporter abtau 1 of claim 1.

4. A recombinant expression vector according to claim 3, wherein: the recombinant expression vector is obtained by ligating the sequence shown in SEQ ID No.7 into a pBI121 vector through BamHI and SacI.

5. Use of the tropane alkaloid transporter abtau up1 of claim 1 or the gene of claim 2 for increasing the hyoscyamine content of a hyoscyamine synthesis host.

6. Use of the tropane alkaloid transporter abtau up1 of claim 1 or the gene of claim 2 for increasing the tropane-transporting capacity in a host for the synthesis of hyoscyamine.

7. The use according to claim 6, characterized in that: the scopolamine synthesis host is belladonna, datura, scopolamine or Anisodus acutangulus.

8. A method for increasing the content of hyoscyamine in hyoscyamine synthesized plants is characterized in that: constructing a recombinant expression vector containing an AbTAUP1 gene for encoding tropane alkaloid transport protein, and then expressing the recombinant expression vector in a hyoscyamine synthetic plant to obtain a variety with high hyoscyamine content; the nucleotide sequence of the encoding tropane alkaloid transporter AbTAUP1 gene is shown as SEQ ID NO. 7.