Tropine alkaloid transporter AbTAUP1 and application thereof
阅读说明：本技术 托品烷生物碱转运蛋白AbTAUP1及其应用 (Tropine alkaloid transporter AbTAUP1 and application thereof ) 是由 廖志华 陈敏 杨春贤 曾俊岚 邱飞 于 2021-07-22 设计创作，主要内容包括：本发明公开了托品烷生物碱转运蛋白AbTAUP1及其应用,托品烷生物碱转运蛋白AbTAUP1的氨基酸序列如SEQ ID NO.8所示,或SEQ ID NO.8所示序列经一个或几个氨基酸残基的取代、突变或缺失且编码具有相同功能的氨基酸序列；该蛋白在侧根中的表达量最高,在主根中表达量较低,且定位于细胞质膜,推测具有莨菪碱转运功能,通过酵母转运实验验证了其莨菪碱转运功能,并在颠茄植株中过表达AbTAUP1提高颠茄中的莨菪碱产量,因此可用于提供莨菪碱合成植物中莨菪碱转运能力,提高莨菪碱产量,对提高托品烷生物碱在植物种的含量具有重要意义。(The invention discloses tropine alkaloid transport protein AbTAUP1 and application thereof, wherein the amino acid sequence of tropine 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 and lower expression level in main roots, is positioned at a cytoplasmic membrane, is supposed to have a hyoscyamine transport function, is verified to have the hyoscyamine transport function through a yeast transport experiment, and is overexpressed AbTAUP1 in a belladonna plant to improve the hyoscyamine yield in the belladonna, so that the protein can be used for providing the hyoscyamine transport capability in a hyoscyamine synthetic plant and improving the hyoscyamine yield, and has important significance for improving the content of tropane alkaloids in plant species.)
1. Tropine alkaloid transporter AbTAUP1, characterized in that: the amino acid sequence of the tropine alkaloid transporter 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.
2. The tropane alkaloid transporter AbTAUP1 according to claim 1, wherein: the nucleotide sequence for coding the tropine alkaloid transporter AbTAUP1 is shown in SEQ ID NO.7, or the nucleotide sequence shown in SEQ ID NO.7 is substituted, mutated or deleted by one or more nucleotides and codes the nucleotide sequence with the same function.
3. A recombinant expression vector comprising a gene encoding the tropine alkaloid transporter AbTAUP1 according to claim 1 or 2.
4. The recombinant expression vector of claim 3, wherein: the recombinant expression vector is obtained by connecting a sequence shown in SEQ ID NO.7 into a pBI121 vector through BamHI and SacI.
5. A host comprising a gene encoding the tropine alkaloid transporter AbTAUP1 according to claim 1 or 2.
6. The host of claim 5, wherein: the host is belladonna.
7. Use of the tropane alkaloid transporter AbTAUP1 according to claim 1 or 2 for increasing the content of scopolamine in a scopolamine synthesis host.
8. Use of the tropane alkaloid transporter AbTAUP1 according to claim 1 or 2 for increasing the transport capacity of scopolamine in a scopolamine synthesis host.
9. Use according to claim 7, characterized in that: the hyoscyamine synthesis host is belladonna, stramonium, hyoscyami, radix Anisodi Acutanguli or yeast.
10. A method for increasing the content of hyoscyamine in a plant for synthesizing hyoscyamine is characterized in that: constructing a recombinant expression vector containing a coding tropine alkaloid transporter AbTAUP1 gene, and expressing the recombinant expression vector in a hyoscyamine synthetic plant to obtain a variety with high hyoscyamine content; the nucleotide sequence of the coding tropine alkaloid transporter AbTAUP1 gene is shown in SEQ ID NO.7 or the sequence shown in SEQ ID NO.7 is substituted, mutated or deleted by one or more nucleotides and codes the nucleotide sequence with the same function.
Technical Field
The invention relates to the technical field of biology, in particular to tropane alkaloid transporter AbTAUP1 and application of the tropane alkaloid transporter.
Background
Tropane Alkaloids (TA) are a group of anticholinergic agents that act on the parasympathetic nerves. The representative medicaments of the compound are hyoscyamine, scopolamine and derivatives thereof, which are widely used in the aspects of anesthetics, treating Parkinson, various organ cramps and asthma caused by smooth muscle spasm and the like, and have huge market demands. Tropane alkaloids are still completely dependent on extraction from a few solanaceae medicinal plants, including belladonna (Atropa belladonna), Datura stramonium (Datura stramnonium) and hyoscyami (Hyoscyamus niger). Belladonna is a commercial cultivated medicinal source plant for producing hyoscyamine and scopolamine recorded and specified in Chinese pharmacopoeia. The weight fraction of the hyoscyamine in the wild belladonna plant is 0.02-0.17% (dry weight), and the content of the scopolamine is only 0.01-0.08% of the dry weight. Therefore, the cultivation of medicinal plants with high tropane alkaloid yield is always a long-sought goal in the industry. With the rapid development of genetic engineering technology, metabolic engineering methods based on targeted modification of biosynthetic pathways have become 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 shortage of tropane alkaloid resources in the market. However, in the prior tropine alkaloid total synthesis yeast strains, the maximum yield of the hyoscyamine is only 80 mu g-1The highest yield of scopolamine is only 30 mu g-1Far below commercial production requirements.
The whole plant of tropane alkaloid medicinal source plants such as belladonna, stramonium, hyoscyamine and the like is rich in hyoscyamine, but researches show that hyoscyamine biosynthesis pathway enzyme genes are specifically expressed in secondary roots of plants. Thus, hyoscyamine, although specifically synthesized only in the secondary roots of plants, is transported to the aerial parts for storage. Secondary roots are the "source" of biosynthesis, while aerial parts are the "pool" for storage. Enhancing the transport process of "sources" to "sinks" facilitates the pulling of biosynthetic metabolic flows. Therefore, the tropine alkaloid transporter has very important application value for both tropine alkaloid plant metabolic engineering and microbial synthetic biology. Unfortunately, no report on tropane alkaloid transporters has been found so far. Therefore, there is an urgent need for tropane alkaloid transporters to increase the yield of scopolamine in plants.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a tropane alkaloid transporter AbTAUP 1; the second purpose of the invention is to provide a recombinant expression vector containing a gene coding the tropine alkaloid transporter AbTAUP 1; the third object of the present invention is to provide a host containing a gene encoding the tropane alkaloid transporter AbTAUP 1; the fourth purpose of the invention is to provide the application of the tropine alkaloid transporter AbTAUP1 in improving the content of plant hyoscyamine synthesized by hyoscyamine; the fifth purpose of the invention is to provide the application of the tropine alkaloid transporter AbTAUP1 in improving the transport capacity of hyoscyamine in a plant for synthesizing hyoscyamine; the present invention also provides a method for increasing the content of hyoscyamine in a plant for synthesizing hyoscyamine.
In order to achieve the aim, a tropine alkaloid transporter AbTAUP1 is discovered for the first time, the tropine alkaloid transporter AbTAUP1 is verified through a yeast transport experiment, the tropine transport function is verified, and the over-expression AbTAUP1 in a belladonna plant improves the yield of hyoscyamine in belladonna, and the specific scheme is as follows:
1. tropine alkaloid transport protein AbTAUP1, wherein the amino acid sequence of the tropine 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.
Preferably, the nucleotide sequence coding the tropine alkaloid transporter AbTAUP1 is shown in SEQ ID NO.7, or the sequence shown in SEQ ID NO.7 is substituted, mutated or deleted by one or more nucleotides and codes the nucleotide sequence with the same function.
2. Contains a recombinant expression vector which codes the tropine alkaloid transporter AbTAUP1 gene.
Preferably, the recombinant expression vector is obtained by connecting a sequence shown in SEQ ID NO.7 into a pBI121 vector through BamHI and SacI.
3. A host containing a gene encoding the tropine alkaloid transporter AbTAUP 1.
Preferably, the host is belladonna.
4. The tropine alkaloid transporter AbTAUP1 is used for improving the content of hyoscyamine in a hyoscyamine synthesis host.
Preferably, the hyoscyamine synthesis host is belladonna, stramonium, hyoscyami, acutangular anisodus root or yeast.
5. The tropine alkaloid transporter AbTAUP1 is used for improving the tropine transport capacity in a tropine synthesis host.
6. A method for increasing the content of hyoscyamine in plant for synthesizing hyoscyamine comprises constructing a recombinant expression vector containing encoding tropine alkaloid transporter AbTAUP1 gene, and expressing the recombinant expression vector in plant for synthesizing hyoscyamine to obtain a high-content product; the nucleotide sequence of the coding tropine alkaloid transporter AbTAUP1 gene is shown in SEQ ID NO.7 or the sequence shown in SEQ ID NO.7 is substituted, mutated or deleted by one or more nucleotides and codes the nucleotide sequence with the same function.
The invention has the beneficial effects that: the invention discovers a tropane alkaloid transporter AbTAUP1 for the first time, the tropine alkaloid transporter AbTAUP1 has the highest expression level in lateral roots and lower expression level in main roots and is positioned at a cytoplasmic membrane, the tropine transporter AbTAUP1 is presumed to have a tropine transport function, the tropine transporter AbTAUP1 is verified through a yeast transport experiment, the tropine transport function is verified, the over-expression AbTAUP1 in a belladonna plant improves the hyoscyamine yield in belladonna, and therefore, the tropine alkaloid transporter AbTAUP1 can be used for providing the hyoscyamine transport capacity in a tropane synthesis plant, improving the hyoscyamine yield and has important significance for improving the content of tropane alkaloids in plant species.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 shows the expression profile analysis of AbTAUP1 gene in belladonna tissues;
FIG. 2 shows the result of subcellular localization;
FIG. 3 shows the transport of hyoscyamine in yeast test cells;
FIG. 4 shows the result of detecting AbTAUP1 gene expression level in wild belladonna and AbTAUP1 overexpression belladonna root;
FIG. 5 shows the hyoscyamine content in wild type belladonna and AbTAUP1 overexpression belladonna root.
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1 screening of potential tropane alkaloid transporters by tissue expression Pattern analysis
(1) Transcriptome assay for screening potential tropane alkaloid transporters
The Purine nucleotide transporter (punit) family is widely present in plants and fungi and is involved in the transport of plant metabolites such as cytokinins, caffeine and nicotine. The corresponding Hidden Markov Model (HMM) in the Pfam database has the accession number of PF16913.5, 19 candidate genes are identified in a belladonna transcriptome by using a PF16913.5 Model, the candidate genes and a Tropane Alkaloid synthesis pathway enzyme gene are subjected to tissue expression cluster analysis to obtain a PUNUT family transporter gene highly expressed in roots, the expression correlation with the Tropane Alkaloid synthesis pathway enzyme gene is high, the candidate gene is presumed to possibly participate in Tropane Alkaloid transport, and the gene is named as belladonna Tropane Alkaloid transporter 1(Tropane Alkaloid Uptake Permease 1, AbTAUP 1).
(2) qPCR verification of its expression patterns
Small portions of fresh wild belladonna main root, fibrous root, stem and leaf material are respectively put into liquid nitrogen for quick freezing, and total RNA is extracted according to the instruction of a TIANGEN kit. The concentration of RNA was determined and cDNA was synthesized according to the instructions of the Tiangen FastKing cDNA first strand synthesis kit to create a cDNA library of the major root, lateral root, stem and leaf of belladonna. Fluorescent quantitative PCR was performed using AbPGK as an internal reference gene and AbTAUP1 as a target gene. According to 2-△△CtThe method calculates the relative expression quantity 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 AbTAUP1 gene in various belladonna tissues was analyzed based on the 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 results of transcriptome expression analysis, and further indicate that the transport protein AbTAUP1 is likely to be involved in the transport of tropane alkaloids.
(3) Cloning of genes
Total RNA from the aseptic seedling lateral root of belladonna was extracted according to the instructions of the TIANGEN kit, and cDNA was synthesized according to the instructions of the first strand synthesis kit of the Tiangen FastKing cDNA. The 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 3 ' end RACE specific primers are needed to be designed, and the full-length electronic sequence of the AbTAUP1 is obtained by supplementing the sequence of the 3 ' end through RACE technology. Designing a specific primer of the gene according to the full-length sequence of electronic splicing, wherein 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, using a gene coding region specific primer to carry out PCR amplification, using agarose electrophoresis to detect and confirm the size of a PCR product, recovering a target band and then carrying out sequencing to finally successfully obtain a 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
Prediction of transmembrane domain of AbTAUP1 protein shows that the protein contains ten transmembrane domains, almost the whole protein penetrates through the membrane structure, so that AbTAUP1 protein is likely to exert the transport capacity to the substrate on the cell membrane. To verify that the AbTAUP1 protein is a transmembrane protein, a subcellular localization vector was constructed and a subcellular localization assay was performed on AbTAUP 1. The PM-ck/CD3-1001 plasmid (purchased as https:// abrc. osu. edu/stocks/764634 ligated) was purchased to express the plasma membrane localization protein PM-CFP fused to the cyan fluorescent protein CFP as a plasma membrane marker signal. After agrobacterium GV3101 is transformed, engineering strain GV3101-pCD3-1001 is obtained.
The cloning site of pGD3G-YFP (https:// doi.org/10.1046/j.1365-313X.2002.01360.x) vector was determined by analyzing the restriction sites of the coding region of AbTAUP1 gene. And finally, selecting XhoI and HindIII as enzyme cutting sites to design upstream and downstream specific primers. The primer sequences are 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 after sequencing is successful, the recombinant plasmid pGD3G-AbTAUP1-YFP is obtained. AbTAUP1 expressed by the plasmid and yellow fluorescent protein YFP form a fusion protein. After the recombinant plasmid and pGD3G-YFP are transferred into agrobacterium GV3101, engineering strains GV3101-pGD3G-AbTAUP1-YFP and GV3101-YFP are obtained.
(2) Subcellular localization analysis
Respectively activating GV3101-YFP, GV3101-pGD3G-AbTAUP1-YFP and GV3101-pCD3-1001 engineering bacteria at 28 ℃ and 200rpmThe culture was performed overnight. 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 10mM2And 40mM AS (acetosyringone) mixed solution, gently resuspending the cells, and adjusting OD of the cell suspension600Leave for 3h at room temperature, 0.6. Two sets of experiments were set up with different bacterial solutions mixed. Injecting the mixed bacterial liquid into tobacco leaves of Benedict 6-8 weeks old by using an injector without a needle, culturing for 48 hours under low light, cracking the tobacco leaves by using cellulase and pectinase to obtain protoplasts, and taking a picture by using a laser confocal microscope Olympus FV1000, wherein the result is shown in figure 2. The results show that the free YFP yellow fluorescence signal does not completely coincide with the PM-CFP cyan fluorescence signal, since YFP fluorescent protein alone has no specific spatial localization in tobacco cells. AbTAUP1-YFP yellow fluorescence almost completely coincided with the plasma membrane localization signal PM-CFP cyan fluorescence, expressed only on the plasma membrane, indicating that AbTAUP1 localizes to the plasma membrane of the cell.
Example 3 application of AbTAUP1 in Yeast
(1) Construction of Yeast transport vectors and engineered strains
Whether the AbTAUP1 gene has the capacity of transferring tropane alkaloid is researched by constructing a yeast transfer vector. Based on the multiple cloning site of the yeast expression vector pDR196, upstream and downstream primers were designed with PstI and XhoI restriction endonuclease cleavage sites. 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 recovered by double enzyme digestion 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 transporters and is widely applied to researching the functions of the transporters. Meanwhile, the ADR8 mutant yeast strain with the transformed pDR196 unloaded is used as a control, and the transgenic yeast is used for subsequent feeding experiments.
(2) Hyoscyamine feeding engineering yeast
Inoculating the transgenic engineering yeast into 50mL uracil-deficient liquid culture medium according to the inoculation amount of 0.05%, performing shake culture at 30 ℃ and 200rpm until OD is reached600Each set of 3 biological replicates as 1. The cells were centrifuged, the supernatant was discarded, 50mL of 1/2 uracil-deficient medium was added again, and 0.5mM of hyoscyamine, scopolamine, and anisodamine were added to the medium, respectively, and shaking-cultured at 30 ℃ and 220 rpm. Sampling for 3 hours, centrifugally collecting thalli, and adding precooled ddH2O washes three times. Adding 100 μ L of extractive solution (containing 50% ethanol, 49.5% methanol and 0.05% acetic acid, volume ratio), adding 100 μ L of glass beads, vortex-breaking for 15min, centrifuging, and collecting supernatant for HPLC analysis.
(3) HPLC determination of the content of hyoscyamine in Yeast cells
The content of hyoscyamine in yeast cells was determined by Shimadzu high performance liquid chromatography. The specific instrument parameters are as follows:
chromatograph: shimadzu high performance liquid chromatograph (system controller: CBM-20Alite, pump: LC-20 AD);
column oven: CTO-20A, detector: SPD-M20A, autosampler: SIL-20A);
a chromatographic column: PHenomenex Gemini 5 u C18110A liquid chromatography column (250 × 4.6mm, 5 microns);
mobile phase: acetonitrile ammonium acetate buffer (containing 20mM amine acetate, 0.1% formic acid, pH 4.0) 11: 89;
flow rate: 1 mL/min;
detection wavelength: 226 nm;
column temperature: 40 ℃;
sample introduction amount: 20 mu L of the solution;
the results of the detection are shown in FIG. 3. HPLC result analysis shows that the control group pDR196 yeast strain has no transport capacity for hyoscyamine, anisodamine and scopolamine; the transformed yeast strain pDR196-AbTAUP1 has obvious transport capacity to hyoscyamine and no transport capacity to scopolamine and anisodamine. This result indicates that AbTAUP1 is a hyoscyamine-specific transporter localized to the plasma membrane.
Example 4 application of AbTAUP1 in plant metabolic engineering
(1) Overexpression vector construction
To assess whether overexpression of AbTAUP1 in plants affected the transport and content changes of tropane alkaloids in belladonna, the present study utilized belladonna plants to perform plant metabolic engineering by constructing overexpression vectors. The plant expression vector pBI121 is selected as an over-expression vector of a belladonna plant, and enzyme cutting sites contained in the sequence are analyzed according to the sequence of the coding region of AbTAUP1 obtained above and are combined with the enzyme cutting sites carried by the cloning site of the over-expression vector pBI 121. The restriction enzyme site introduced at the upstream is designed to be BamHI, and the restriction enzyme site introduced at the downstream is designed to be 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)；
after PCR amplification is carried out by using the primers, pBI121 plasmid and PCR product are subjected to double digestion recovery and then are connected, and finally the over-expression recombinant plasmid pBI121-AbTAUP1 is successfully obtained.
(2) Genetic transformation of belladonna plants
The constructed plant over-expression vector pBI121-AbTAUP1 is transformed into agrobacterium EHA105 by a freeze-thaw method, and an engineering strain is successfully obtained for subsequent genetic transformation through PCR positive identification.
Belladonna seed is soaked in gibberellin solution overnight, washed with tap water overnight for 1 day, and then sterilized. Sterilizing with 75% ethanol for 1min and 50% NaClO solution for 20min, respectively, and shaking sufficiently to ensure sufficient sterilization. And finally, washing the glass substrate with sterile water for 5-6 times. Placing the sterilized belladonna seeds on sterile absorbent paper by using tweezers to suck excessive water, inoculating the belladonna seeds on a solid culture medium of MS +200mg/LCef at 25 ℃, culturing for about 15 days under 16h/8h (light/dark) light conditions, and transferring the germinated belladonna wild type seedlings into a glass bottle of the MS solid culture medium for later use.
Will activateThe good engineering bacteria are used for sterilizing MS liquid culture medium added with AS, gently suspending the bacteria, and adjusting OD600To about 0.3 to 0.5. And (3) cutting the belladonna leaves in a super clean workbench, adding the belladonna leaves into the resuspended agrobacterium liquid, and infecting for about 5-8 min. It was spread on MS solid co-culture medium (MS + AS +6-BA1.0mg/L + NAA1.0mg/L) and cultured in the dark at 28 ℃ for 2 days. After 2 days, the culture medium is transferred to a differentiation solid culture medium of different antibiotics, the screening culture medium of an overexpression vector is (MS + NAA1.0mg/L +6-BA1.0mg/L + Cef 200mg/L + Kan 8mg/L), and the screening culture medium is replaced every other week until cluster buds grow out. The cluster buds are cut off and transferred to a rooting medium (1/2MS +0.05g/L NAA) to induce rooting. The regenerated plants were removed from the pots, cleaned of the culture medium at the roots, seeded in a culture medium (vermiculite: peatmoss: perlite: 6: 3: 1) and cultivated in a climatic chamber at 25 ℃ for 16h/8h (light/dark). Positive AbTAUP1 overexpression transgenic belladonna plants were screened by genomic PCR detection. Wild belladonna and AbTAUP1 overexpression belladonna root are taken to carry out real-time fluorescent quantitative PCR analysis on AbTAUP1 gene expression quantity, and the method is the same as the above method. As shown in FIG. 4, the expression level of AbTAUP1 in the over-expressed strain was 10-26 times that of the wild type control, indicating that the belladonna plant in which AbTAUP1 gene was over-expressed was successfully obtained.
(3) HPLC analysis of belladonna Hyoscyamine content
Collecting the root and leaf of positive belladonna seedling growing in culture room for 3 months in clean envelope, oven drying at 40 deg.C, grinding into powder, and extracting alkaloid. Weighing about 0.1g of dry powder, transferring the dry powder into a 50mL EP tube, adding 10mL of alkaloid extract, carrying out ultrasonic crushing for 1h, and standing overnight; performing ultrasonic extraction for 1h after overnight extraction, and standing for more than 1 h; filtering the above extractive solution with filter paper, washing the residue with 5mL chloroform, collecting the filtrate and washing solution in 50mL beaker, oven drying at 40 deg.C; the volatilized residue was washed with 5mL of chloroform and 2mL of 1M H2SO4Ultrasonically dissolving the mixed solution, fully and uniformly mixing, transferring the mixed solution into a10 mL EP tube, and centrifuging the mixed solution at 4000rpm for 10 min; discarding the lower organic waste liquid, adding 1.5mL ammonia water into an EP tube to alkalize the solution, then extracting alkaloid by using 3mL chloroform, and collecting a chloroform phase in a 50mL beaker; 2mL of chloroform was again usedExtracting once, and combining the chloroform phases twice; drying chloroform at 40 ℃, and dissolving alkaloid by using 1mL of methanol; filtering alkaloid with filter head, and detecting by HPLC. The specific HPLC instrument parameters were as described above and the results are shown in fig. 5. HPLC result analysis shows that the content of hyoscyamine in the wild type belladonna leaf is 1.54mg/g, and the content of hyoscyamine in the root is 1.34 mg/g. The hyoscyamine content in the leaves and roots of the belladonna plant over-expressed AbTAUP1 is remarkably improved, the hyoscyamine content in the leaves is 2.36-3.01mg/g, and is 153% -195% of that of the wild type; the content of hyoscyamine in root is 1.81-2.44mg/g, which is 135% -182% of wild type. The results indicate that overexpression of AbTAUP1 is beneficial to enhance the transport of hyoscyamine from "source" to "sink" in belladonna and pulls anabolic flux, finally effectively increasing accumulation of hyoscyamine.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university of southwest
<120> tropine alkaloid transporter AbTAUP1 and application thereof
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atggaatctc aaattgctag tcccactatg aaaaaattcc tcatcctgat aaactctatt 60
cttctcttca ctagcaattg cgctggccct ttaattattc gcctctattt catccgtggt 120
ggatcaagaa tatggctatc atgttctcta ataaccggtg gcttcccttt tactctcttc 180
ctcctcatca tagcctattt ccatcgtcga aaatccaatg gaccggacaa tagtactaag 240
atactgctca tgactcgtaa attatttata gcttgcttaa tttctggctt agtcactggc 300
atggttgatt atttctacgc ttttggttca gccaaattac ccgtctccac atcctctctc 360
cttacttcaa ctcaactagt ttttacagcg attttcgctt tcctaattgt caagcaaaaa 420
ttcacatcgt attcgattaa tgctgtggtt gtattaactc ttggagctgg aattttggcc 480
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
attccaaggg aggcaaaaca atttgagtta ggtgaaggca aatattacat gcttatagta 780
ttgagtgccc tcatatggca aattttcttt gttggagcca ttggagtcac atgctatgct 840
tcagcattac tctctggaat tttgatagct gcttcacttt cagttacaga agtgttggct 900
gttattttct ttcatgaaaa aattggaggt gaaaagggat tctcacttgc cctctctctt 960
tggggatttg tttcttattt ttatggtgag atcaaacaaa ccaataaaaa gaagaattta 1020
actacagaag ctgagatgac tcaatcccct ccaatataa 1059
<210> 9
<211> 352
<212> PRT
<213> belladonna (Atropa belladonna)
<400> 9
Met Glu Ser Gln Ile Ala Ser Pro Thr Met Lys Lys Phe Leu Ile Leu
1 5 10 15
Ile Asn Ser Ile Leu Leu Phe Thr Ser Asn Cys Ala Gly Pro Leu Ile
20 25 30
Ile Arg Leu Tyr Phe Ile Arg Gly Gly Ser Arg Ile Trp Leu Ser Cys
35 40 45
Ser Leu Ile Thr Gly Gly Phe Pro Phe Thr Leu Phe Leu Leu Ile Ile
50 55 60
Ala Tyr Phe His Arg Arg Lys Ser Asn Gly Pro Asp Asn Ser Thr Lys
65 70 75 80
Ile Leu Leu Met Thr Arg Lys Leu Phe Ile Ala Cys Leu Ile Ser Gly
85 90 95
Leu Val Thr Gly Met Val Asp Tyr Phe Tyr Ala Phe Gly Ser Ala Lys
100 105 110
Leu Pro Val Ser Thr Ser Ser Leu Leu Thr Ser Thr Gln Leu Val Phe
115 120 125
Thr Ala Ile Phe Ala Phe Leu Ile Val Lys Gln Lys Phe Thr Ser Tyr
130 135 140
Ser Ile Asn Ala Val Val Val Leu Thr Leu Gly Ala Gly Ile Leu Ala
145 150 155 160
Leu Gly Ala Ser Ser Asp Arg Pro Ala Gly Glu Ser Ser Lys Ala Tyr
165 170 175
Ile Val Gly Phe Ile Met Thr Leu Leu Ala Ala Leu Phe Tyr Gly Phe
180 185 190
Val Leu Ala Phe Asn Glu Val Ser Phe Arg Lys Thr Lys Lys Val Ile
195 200 205
Ala Phe Thr Leu Val Leu Glu Phe Gln Met Met Met Cys Phe Phe Ala
210 215 220
Thr Ala Phe Cys Val Thr Gly Met Leu Ile Asn Lys Asp Ile Gln Ala
225 230 235 240
Ile Pro Arg Glu Ala Lys Gln Phe Glu Leu Gly Glu Gly Lys Tyr Tyr
245 250 255
Met Leu Ile Val Leu Ser Ala Leu Ile Trp Gln Ile Phe Phe Val Gly
260 265 270
Ala Ile Gly Val Thr Cys Tyr Ala Ser Ala Leu Leu Ser Gly Ile Leu
275 280 285
Ile Ala Ala Ser Leu Ser Val Thr Glu Val Leu Ala Val Ile Phe Phe
290 295 300
His Glu Lys Ile Gly Gly Glu Lys Gly Phe Ser Leu Ala Leu Ser Leu
305 310 315 320
Trp Gly Phe Val Ser Tyr Phe Tyr Gly Glu Ile Lys Gln Thr Asn Lys
325 330 335
Lys Lys Asn Leu Thr Thr Glu Ala Glu Met Thr Gln Ser Pro Pro Ile
340 345 350
<210> 10
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cgcctcgaga tggaatctca aattgctag 29
<210> 11
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cgcaagcttt attggagggg attgagtca 29
<210> 12
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gggctgcaga tggaatctca aattgctag 29
<210> 13
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
cccctcgagt tatattggag gggattgag 29
<210> 14
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cgcggatcca tggaatctca aattgctag 29
<210> 15
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cgcgagctct tatattggag gggattgag 29