WO2021043189A1 - 莨菪醛还原酶及其应用 - Google Patents

莨菪醛还原酶及其应用 Download PDF

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WO2021043189A1
WO2021043189A1 PCT/CN2020/113138 CN2020113138W WO2021043189A1 WO 2021043189 A1 WO2021043189 A1 WO 2021043189A1 CN 2020113138 W CN2020113138 W CN 2020113138W WO 2021043189 A1 WO2021043189 A1 WO 2021043189A1
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scopolaldehyde
reductase
transgenic
scopolamine
expression vector
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PCT/CN2020/113138
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English (en)
French (fr)
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廖志华
陈敏
杨春贤
邱飞
曾俊岚
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西南大学
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Priority to US17/788,105 priority Critical patent/US20230039694A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01021—Aldehyde reductase (1.1.1.21), i.e. aldose-reductase

Definitions

  • the invention relates to the field of biotechnology, in particular to scopolaldehyde reductase, and also relates to the application of scopolaldehyde reductase.
  • Tropine alkaloids are a class of anticholinergic drugs with great medical value. They are widely used in anesthesia, analgesia, cough, asthma, and anti-motion sickness. They are also used to control the stiffness of Parkinson's disease. And tremor. Commonly used clinically are hyoscyamine and scopolamine, both of which have a huge market demand. Among them, scopolamine has weaker side effects, stronger drug effects, and more expensive prices.
  • TAs are extracted from a handful of TAs resource plants in the Solanaceae family, including belladonna (Atropa belladonna), datura (Datura stramonium) and hyoscymus niger (Hyoscyamus niger).
  • Belladonna is the main commercial cultivation source of scopolamine and hyoscyamine , Is also TAs medicinal plants included in the Pharmacopoeia.
  • the mass fraction of scopolamine in wild belladonna plants is 0.02% ⁇ 0.17% (dry weight), and the content of scopolamine is extremely low, only 0.01% ⁇ 0.08% of dry weight. Therefore, cultivating high-yield belladonna tropane alkaloids has been a long-term goal pursued by the industry.
  • Scopolamine is both an important anticholinergic drug and the direct precursor of scopolamine.
  • the application of plant secondary metabolism engineering depends on the analysis of the biosynthetic pathway of secondary metabolites. Therefore, cloning the scopolamine biosynthesis gene is of great significance for improving and increasing the content of tropane alkaloids in belladonna.
  • one of the objects of the present invention is to provide a scopolaldehyde reductase;
  • the second object of the present invention is to provide an scopolaldehyde reductase gene;
  • the third object of the present invention is to provide a scopolaldehyde reductase gene Recombinant expression vector;
  • the fourth object of the present invention is to provide a transgenic cell line or transgenic recombinant bacteria containing the scopolaldehyde reductase gene;
  • the fifth object of the present invention is to provide the scopolaldehyde reductase catalyzing scopolaldehyde in vivo or in vitro Application of reduction to produce scopolamine;
  • the sixth objective of the present invention is to provide the scopolaldehyde reductase gene and the recombinant expression vector to reconstruct scopolamine in prokaryotes or eukaryotes that do not have a tropan
  • the present invention provides the following technical solutions:
  • Scopolaldehyde reductase a protein having one of the following amino acid residue sequences:
  • amino acid residue in SEQ ID NO. 4 has been substituted and/or deleted and/or added by one or several amino acid residues and has an amino acid sequence with the function of scopolaldehyde reductase.
  • Scopolaldehyde reductase gene said scopolaldehyde reductase gene has one of the following nucleotide sequences:
  • the recombinant expression vector is a recombinant expression vector expressing scopolaldehyde reductase by inserting the scopolaldehyde reductase gene into a prokaryotic or eukaryotic expression vector.
  • the recombinant expression vector is obtained by linking the scopolaldehyde reductase gene, such as the nucleotide sequence shown in SEQ ID NO. 3, into the pET28a vector via EcoRI and the SacI restriction site.
  • the scopolaldehyde reductase gene SEQ ID NO.3 the nucleotide sequence shown in SEQ ID NO.3, is replaced with the GUS gene of pBI121 modified, wherein the modified pBI121 is the AbPMT promoter instead of the 35S promoter of pBI121.
  • a transgenic cell line or transgenic recombinant bacteria containing the scopolaldehyde reductase gene is provided.
  • the cell line is a plant cell line, which can be a belladonna cell line, a plant cell line with a tropane alkaloid biosynthetic pathway, or a plant cell line without a tropane alkaloid biosynthetic pathway Plant cell lines.
  • the recombinant bacteria is BL21, or other bacteria with or without tropane alkaloid biosynthesis pathway.
  • transgenic cell line or transgenic recombinant bacteria in increasing the content of scopolamine in organisms with tropane alkaloid biosynthesis pathway.
  • the present invention discloses scopolaldehyde reductase. After research, it is found that the amino acid sequence of scopolaldehyde reductase is shown in SEQ ID NO. 4, and the encoded nucleotides are shown in SEQ ID NO. 3. Prokaryotic expression of scopolaldehyde reductase can catalyze the reduction of scopolaldehyde to produce scopolamine.
  • the use of scopolaldehyde reductase to transform belladonna can increase the content of scopolamine in the belladonna cell line, which is to increase the tropane in the belladonna. The content of alkali is of great significance.
  • Figure 1 shows the result of HAR-catalyzed reduction of scopolaldehyde to scopolamine.
  • Figure 2 shows the use of fluorescence quantitative detection of HAR gene expression in transgenic belladonna hairy roots.
  • Figure 3 shows the scopolamine content of the transgenic hairy roots of the HAR overexpression vector and the control group.
  • RNA quality was identified by formaldehyde denaturing gel electrophoresis, and the RNA concentration was measured on a spectrophotometer.
  • HAR-F 5’-atggattcttctggtgtcctct-3’ (SEQ ID NO.1);
  • HAR-R 5'-ttggttgctgctcaaacctag-3' (SEQ ID NO. 2).
  • the HAR gene was amplified from the total cDNA by PCR and sequenced.
  • the result of the sequencing is SEQ ID NO. 3, the start codon is ATG, and the stop codon is TAA; the translated protein coding sequence is as SEQ ID NO.4 is shown.
  • the HAR gene was amplified by PCR, the restriction site EcoRI was introduced into the forward primer, and the restriction site SacI was introduced into the reverse primer. Using the above two restriction sites, the entire sequence of the HAR coding region was connected to the plasmid pET28a to obtain the HAR prokaryotic expression vector pET28a-HAR.
  • the primers are as follows:
  • XhoI-HAR-R 5'-cgcctcgagctaggtttgagcagcaaccaa-3' (SEQ ID NO. 6).
  • the constructed pET28a-HAR plasmid was transformed into the prokaryotic expression strain BL21, and the positive clones were screened by PCR to obtain the prokaryotic expression engineering strain BL21-pET28a-HAR.
  • Take 100 ⁇ L of BL21-pET28a-HAR bacterial solution and inoculate it into 30 mL LB liquid medium containing 100 mg/L kanamycin, and cultivate overnight at 37°C and 200 rpm. Then according to the ratio of 1:50 inoculation volume were respectively inoculated into 400mL LB liquid medium at 37°C, 200rpm for activation culture, and IPTG was added to the final concentration of 1mM when the culture reached OD 600 0.6.
  • the HAR-catalyzed scopolaldehyde reduction reaction system is as follows: phosphate buffer (pH 6.4), 0.2mM NADPH-4Na, 1mM scopolaldehyde, 30 ⁇ g HAR protein, incubated at 37.5°C for 1h, and the reaction product was identified by high-resolution mass spectrometry.
  • the high resolution mass spectrometer is Bruker impact II Q-TOF
  • the chromatographic column is Symmetry C-18 reversed phase silica gel column (3.5 ⁇ m, 100x2.1mm) from Waters
  • mobile phase A is 0.1% formic acid aqueous solution
  • B is acetonitrile
  • Table 1 the elution procedure is shown in Table 1:
  • the column temperature was set to 40°C, the flow rate was 0.15 mL/min, the injection volume was 1 ⁇ L, the mass detector used an electrospray ion source (ESI), and the ion mode was positive ion mode.
  • the test results showed that the product of the HAR-catalyzed reduction of scopolamine was scopolamine, with a mass-to-charge ratio m/z of 290.1744 and a retention time of 4.00 min ( Figure 1).
  • a high expression vector PMT promoter::HAR specific to periperitoneum was constructed, and the original plasmid was pBI121.
  • the AbPMT promoter uses belladonna cDNA as a template, and the sequences shown in SEQ ID NO. 7 and SEQ ID NO. 8 are The primers are used for PCR amplification, and the restriction sites BamHI and SacI are used to replace the GUS gene on the original plasmid with the HAR gene.
  • the HAR gene uses belladonna cDNA as a template, and the sequence shown in SEQ ID NO.9 and SEQ ID NO.10 Perform PCR amplification with primers to obtain HAR plant overexpression vector PMT promoter::HAR.
  • the primer sequences used are as follows:
  • HindIII-PMT promoter-F 5'-cgc aagctt ctgagttcggatctaggtca-3' (SEQ ID NO. 7);
  • BamHI-PMT promoter-R 5'-cgc ggatcc ttcttcacttttggccttgct-3' (SEQ ID NO. 8);
  • BamHI-HAR-F 5'-cgc ggatcc atggattcttctggtgtcctct-3' (SEQ ID NO.9);
  • SacI-HAR-R 5'-cgc gagctc ctaggtttgagcagcaaccaa-3' (SEQ ID NO. 10).
  • the belladonna explants co-cultured for 2 days were transferred to the screening medium (MS+Kan 100mg/L+Cef 500mg/L) and cultured in the dark at 25°C, subcultured once a week, after 1-2 times of subculture Kan-resistant hairy roots can be obtained after generation.
  • the well-growing hairy roots were cut and transferred to a medium (MS+Cef 200mg/L) to be cultured until completely sterile, so as to obtain Kan-resistant belladonna hairy roots.
  • the forward primer design and the reverse primer are respectively designed to detect the target gene.
  • the results show that specific DNA fragments can be amplified by using the designed PCR specific primers. When using non-transformed belladonna hairy root genomic DNA as a template, no fragments were amplified.
  • the obtained transgenic belladonna hairy roots were detected by fluorescence quantitative detection. As shown in Figure 2, the expression of AbHAR gene in the transgenic hairy roots was extremely significantly increased.
  • the extract was filtered to remove plant residues, and the filtrate was dried at 40°C. Dissolve the dried material in the previous step with 5mL chloroform and 2mL 0.5M sulfuric acid, fully emuls
  • HPLC instrument configuration Shimadzu LC-20AD binary pump system, equipped with DUG-20A online degasser, CTO-20A column thermostat, SPD-M20A full-wavelength diode array detector, SIL-20A autosampler, chromatographic column It is Shimadzu INERTSUSTAIN C18 column (5 ⁇ m, 4.6 ⁇ 250mm), using Shimadzu guard column (5 ⁇ m, 4.0 ⁇ 10mm).
  • Chromatographic conditions for alkaloid analysis 11% acetonitrile and 89% water (20mM ammonium acetate and 0.1% formic acid, pH4.0) were used as the mobile phase, the column oven was 40°C, the total flow rate was 1mL/min, and the detection wavelength was 226nm.
  • the transgenic hairy roots of the AbHAR overexpression vector in the present invention significantly increased the hyoscyamine content.
  • the content of scopolamine in ordinary hairy roots is 2.32mg/g DW
  • the content of scopolamine in the hairy roots of the AbHAR overexpression vector belladonna reached an average of 4.24-5.98mg/g DW during the same period, and its content is non-transgenic belladonna hairs
  • the content of shaped roots is 1.83-2.58 times.

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Abstract

提供了一种具有如SEQ ID NO.4所示氨基酸序列的莨菪醛还原酶。还提供了该莨菪醛还原酶的基因，重组表达载体，转基因细胞系和重组菌，以及在催化莨菪醛还原生成莨菪碱中的应用和重建莨菪碱合成途径或提高莨菪碱含量中的应用。

Description

莨菪醛还原酶及其应用 技术领域
本发明涉及生物技术领域，具体涉及莨菪醛还原酶，还涉及莨菪醛还原酶的应用。
背景技术
托品烷类生物碱(tropane alkaloids，Tas)是一类具有巨大医疗价值的抗胆碱药物，广泛用于麻醉、镇痛、止咳、平喘和抗晕动病，也用于控制帕金森病的僵直和震颤。临床上常用的是莨菪碱(hyoscyamine)和东莨菪碱(scopolamine)，两者的市场需求均十分巨大，其中以东莨菪碱毒副作用更弱，药效更强，价格也更昂贵。目前TAs都是从少数茄科TAs资源植物中提取，包括颠茄(Atropa belladonna)、曼陀罗(Datura stramonium)和莨菪(Hyoscyamus niger)，颠茄是东莨菪碱和莨菪碱最主要的商业栽培药源，也是药典收录的TAs药源植物。野生颠茄植株中莨菪碱质量分数为0.02％～0.17％(干重)，东莨菪碱含量极低，仅为干重的0.01％～0.08％。因此，培育托品烷生物碱高产颠茄一直是该行业长期追求的目标。
大部分植物次生代谢产物其在天然植物中的含量极低，而使用化学合成的方法，工艺流程复杂、成本太高，并且还有许多植物次生代谢产物的生物合成途径不清晰，无法实现化学全合成。因此，研究人员开始探索其他的提高植物次生代谢产物含量的方法。例如，在植物中过量表达次生代谢生物合成途径关键酶基因，打破代谢产物合成的限速步骤，从而促进最终有用代谢产物在植物体内的积累，获得更有经济价值的材料。在颠茄中，过量表达H6H基因后，颠茄中的莨菪碱大量转化为更有价值的东莨菪碱，极大地提高了颠茄的经济价值。莨菪碱既是重要的抗胆碱药物也是东莨菪碱的直接前体。植物次生代谢工程的应用依赖于次生代谢产物生物合成途径的解析。因此，克隆莨菪碱生物合成基因，对于改良并提高颠茄中托品烷生物碱的含量具有重要的意义。
发明内容
有鉴于此，本发明的目的之一在于提供一种莨菪醛还原酶；本发明的目的之二在于提供莨菪醛还原酶基因；本发明的目的之三在于提供含有所述莨菪醛还原酶基因的重组表达载体；本发明的目的之四在于提供含有所述莨菪醛还原酶基因的转基因细胞系或转基因重组菌；本发明的目的之五在于提供所述莨菪醛还原酶在体内或体外催化莨菪醛还原生成莨菪碱的应用；本发明的目的之六在于提供所述莨菪醛还原酶基因、所述的重组表达载体在不具有托品烷生物碱生物合成途径的原核生物或真核生物中重建莨菪碱合成途径中的应用；本发明之七在于提供所述莨菪醛还原酶、所述莨菪醛还原酶基因、所述的重组表达载体、所述的转基因细胞系或转基因重组菌在具有托品烷生物碱生物合成途径的生物中提高莨菪碱含量中的应 用；本发明的目的之八在于提供一种提高托品烷生物碱合成植物中莨菪碱含量的方法。
为达到上述目的，本发明提供如下技术方案：
1、莨菪醛还原酶，具有下述氨基酸残基序列之一的蛋白质：
1)如SEQ ID NO.4所示的氨基酸残基序列；
2)将SEQ ID NO.4中的氨基酸残基经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有莨菪醛还原酶功能的氨基酸酸序列。
2、莨菪醛还原酶基因，所述莨菪醛还原酶基因具有下述核苷酸序列之一：
1)如SEQ ID NO.3所示的核苷酸序列；
2)编码如SEQ ID NO.3所示核苷酸序列的多核苷酸；
3)与1)或2)限定的核苷酸序列具有80％以上的同源性且编码具有所述莨菪醛还原酶功能的核苷酸序列；
4)与1)或2)所述的序列杂交的核苷酸序列。
3、含有所述莨菪醛还原酶基因的重组表达载体。
优选的，所述重组表达载体为将所述的莨菪醛还原酶基因插入原核或真核表达载体得到表达莨菪醛还原酶的重组表达载体。
更优选的，所述重组表达载体为将莨菪醛还原酶基因，如SEQ ID NO.3所示的核苷酸序列，经EcoRI和连入SacI酶切位点连入pET28a载体而得。或将莨菪醛还原酶基因SEQ ID NO.3，如SEQ ID NO.3所示的核苷酸序列，替换改造pBI121的GUS基因，其中改造pBI121为AbPMT启动子替换pBI121的35S启动子。
3、含有所述莨菪醛还原酶基因的转基因细胞系或转基因重组菌。
优选的，所述细胞系为植物细胞系，可以为颠茄细胞系，也可以为具有托品烷生物碱生物合成途径的植物细胞系，也可以为不具有托品烷生物碱生物合成途径的植物细胞系。所述重组菌为BL21，或其他具有或不具有托品烷生物碱生物合成途径的细菌。
5、所述莨菪醛还原酶在体内或体外催化莨菪醛还原生成莨菪碱中的应用。
6、所述莨菪醛还原酶基因在不具有托品烷生物碱生物合成途径的原核生物或真核生物中重建莨菪碱合成途径中的应用。
所述的重组表达载体在不具有托品烷生物碱生物合成途径的原核生物或真核生物中重建莨菪碱合成途径中的应用。
7、所述莨菪醛还原酶在具有托品烷生物碱生物合成途径的生物中提高莨菪碱含量中的应用。
所述莨菪醛还原酶基因在具有托品烷生物碱生物合成途径的生物中提高莨菪碱含量中的应用。
所述的重组表达载体在具有托品烷生物碱生物合成途径的生物中提高莨菪碱含量中的应用。
所述的转基因细胞系或转基因重组菌在具有托品烷生物碱生物合成途径的生物中提高莨菪碱含量中的应用。
8、一种提高托品烷生物碱合成植物中莨菪碱含量的方法，将通过在具有托品烷生物碱生物合成途径的植物中过量表达所述的莨菪醛还原酶基因。
本发明的有益效果在于：本发明公开了莨菪醛还原酶，经研究发现莨菪醛还原酶的氨基酸序列如SEQ ID NO.4所示，其编码的核苷酸如SEQ ID NO.3所示，将莨菪醛还原酶进行原核表达后能够催化莨菪醛还原生成莨菪碱，将莨菪醛还原酶用于转化颠茄后能够提高颠茄细胞系中莨菪碱的含量，为提高颠茄中托品烷生物碱的含量具有重要的意义。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1为HAR催化莨菪醛还原为莨菪碱结果图。
图2为转基因颠茄毛状根采用荧光定量检测HAR基因表达量。
图3为HAR超表达载体的转基因毛状根和对照组莨菪碱含量。
具体实施方式
下面对本发明的实施例作详细说明：本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等分子克隆：实验室手册(New York：Cold Spring Harbor Laboratory Press，1989)中所述的条件，或按照制造厂商所建议的条件。
实施例1、莨菪醛还原酶(hyoscyamine aldehyde reductase synthase，HAR)基因的克隆
(1)颠茄须根总RNA的提取
取适量颠茄须根组织，置于液氮中研碎，加入盛有裂解液的1.5mL Eppendorf(EP)离心管中，充分振荡后，按照TIANGEN试剂盒的说明书抽提总RNA。用甲醛变性胶电泳鉴定总RNA质量，在分光光度计上测定RNA浓度。
(2)HAR基因的克隆
以所提取的总RNA为模板，按照天根FastKing cDNA第一链合成试剂盒说明书合成 cDNA；设计HAR基因特异性引物，具体引物如下：
HAR-F：5’-atggattcttctggtgtcctct-3’(SEQ ID NO.1)；
HAR-R：5’-ttggttgctgctcaaacctag-3’(SEQ ID NO.2)。
通过PCR从总cDNA中扩增HAR基因，并测序，测序结果获得HAR基因的核苷酸序列如SEQ ID NO.3，起始密码子为ATG，终止密码子为TAA；翻译出蛋白编码序列如SEQ ID NO.4所示。
实施例2、原核表达验证HAR基因的功能
(1)HAR的原核表达与蛋白纯化
HAR基因进行PCR扩增，在正向引物中引入酶切位点EcoRI，在反向引物中引入酶切位点SacI。利用上述两个酶切位点将HAR编码区完整序列连接质粒pET28a，获得HAR原核表达载体pET28a-HAR。引物如下所示：
EcoRI-HAR-F：5’-cgcgaattcatggattcttctggtgtcctct-3’(SEQ ID NO.5)；
XhoI-HAR-R：5’-cgcctcgagctaggtttgagcagcaaccaa-3’(SEQ ID NO.6)。
将构建好的pET28a-HAR质粒转化原核表达菌株BL21，用PCR筛选阳性克隆子，获得原核表达工程菌BL21-pET28a-HAR。取BL21-pET28a-HAR菌液100μL接种到含100mg/L卡那霉素的30mL LB液体培养基中，37℃，200rpm过夜培养。再按1：50比例接种量分别接种到400mL LB液体培养基中37℃，200rpm进行活化培养，培养至OD 600＝0.6左右时加入IPTG至终浓度1mM。在37℃，200rpm条件下继续培养6h。将收获的菌液8000rpm离心，去掉上清。再把菌体沉淀用磷酸盐缓冲液重悬，并在超声破碎后用GE公司的Ni-NTA填料纯化获得HAR蛋白。
(2)HAR酶活验证
HAR催化的莨菪醛还原反应体系如下：磷酸盐缓冲液(pH 6.4)，0.2mM NADPH-4Na，1mM莨菪醛，HAR蛋白30μg，37.5℃孵育1h，反应产物使用高分辨质谱进行鉴定。
高分辨质谱仪为Bruker impact II Q-TOF，色谱柱为Waters公司的Symmetry C-18反相硅胶柱(3.5μm，100x2.1mm)，流动相A为0.1％甲酸水溶液，B为乙腈，采用梯度方式洗脱，洗脱程序见表1：
表1、洗脱程序
时间(min) 流动相A(％) 流动相B(％)
0 95 5
2 75 25
5.5 0 100
6.5 0 100
6.6 95 5
9 95 5
柱温设定为40℃，流速0.15mL/min，进样量1μL，质谱检测器使用电喷雾离子源(ESI)，离子模式为正离子模式。检测结果表明，HAR催化的莨菪醛还原反应产物为莨菪碱，质荷比m/z为290.1744，保留时间为4.00min(图1)。
实施例3、过表达HAR提高颠茄莨菪碱含量
(1)HAR植物过表达载体的构建
为研究HAR基因对颠茄中托品烷类生物碱的影响，构建了中柱鞘特异性高表达载体PMT promoter::HAR，原始质粒为pBI121。首先，利用酶切位点HindIII和XbaI将原始质粒上pBI121的35S启动子替换为AbPMT启动子；AbPMT启动子以颠茄cDNA为模板，SEQ ID NO.7和SEQ ID NO.8所示序列为引物进行PCR扩增，再利用酶切位点BamHI和SacI将原始质粒上的GUS基因替换为HAR基因，HAR基因以颠茄cDNA为模板，SEQ ID NO.9和SEQ ID NO.10所示序列为引物进行PCR扩增，获得HAR植物过表达载体PMT promoter::HAR。使用的引物序列如下：
HindIII-PMT promoter-F：5’-cgc aagcttctgagttcggatctaggtca-3’(SEQ ID NO.7)；
BamHI-PMT promoter-R：5’-cgc ggatccttcttcacttttggccttgct-3’(SEQ ID NO.8)；
BamHI-HAR-F：5’-cgc ggatccatggattcttctggtgtcctct-3’(SEQ ID NO.9)；
SacI-HAR-R：5’-cgc gagctcctaggtttgagcagcaaccaa-3’(SEQ ID NO.10)。
(2)发根农杆菌工程菌的获得
将PMT promoter::bHAR载体采用冻融法转入发根农杆菌(如C58C1)，并进行PCR验证。结果表明，含HAR的植物双元过量表达载体已成功构建到发根农杆菌菌株中。
(3)转基因发根的获得
A.颠茄外植体准备
颠茄种子用75％乙醇浸泡1min，再用50％NaClO浸泡20min，无菌水冲洗3-4次，用无菌吸水纸吸干表面水分，接种于无激素的1/2MS固体培养基中，25℃，16h/8h(光亮/黑暗)光照培养，即可获得颠茄无菌苗。该条件下培养2周左右后，剪取无菌苗叶片和下胚轴外植体用于转化。
B.农杆菌与外植体的共培养
将所述外植体，加入活化好的所述含HAR植物双元超表达载体的发根农杆菌工程菌的重悬液(MS+AS 100μmol/L)中，菌液与外植体充分接触5分钟，转到共培养培养基(MS+AS  100μmol/L)上，28℃暗培养2d。
C.抗性毛状根的筛选
将所述的共培养2d的颠茄外植体转入到筛选培养基(MS+Kan 100mg/L+Cef 500mg/L)上于25℃暗培养，每周继代培养一次，经过1-2次继代后即可获得Kan抗性毛状根。将生长良好的毛状根剪下转入培养基(MS+Cef 200mg/L)上培养至完全无菌，从而获得Kan抗性颠茄毛状根。
D.毛状根的基因组PCR和表达量检测
根据目的基因所在表达盒上游的35S启动子区域和HAR分别设计正向引物设计和反向引物对目的基因进行检测。结果表明，利用所设计的PCR特异引物，能扩增出特异DNA片段。而以非转化颠茄毛状根基因组DNA为模板时，没有扩增出任何片段。
将获得的转基因颠茄毛状根采用荧光定量检测，结果如图2所示，转基因毛状根的AbHAR基因表达量极显著提高。
E.毛状根莨菪碱提取与测定
毛状根在MS培养液中振荡培养30天后收获，材料于冷冻干燥机中冻干至恒重。磨成粉末，准确称取0.1g干燥至恒重的植物材料。加入10mL生物碱提取液(氯仿：甲醇：氨水＝15:5:1)，超声提取30min，于室温静置1h。提取液过滤去除植物残渣，滤液置于40℃干燥。用5mL氯仿和2mL 0.5M的硫酸溶解上一步干燥物，充分乳化以使生物碱转移至水相，弃掉氯仿。将水相置于冰上，并用氨水(28％)调节水相至pH 10.0。加入2mL氯仿以萃取生物碱，重复萃取两次，合并所有的氯仿，无水硫酸钠干燥去除水分，过滤，滤液置于40℃干燥。1mL液相色谱级甲醇溶解生物碱，0.22μm滤膜过滤，HPLC测定生物碱含量，结果为三次重复的平均值，误差线表示标准差。统计分析用t-test检验。
HPLC仪器配置：岛津LC-20AD二元泵系统，配备DUG-20A在线脱气机，CTO-20A柱温箱，SPD-M20A全波长二极管阵列检测器，SIL-20A自动进样器，色谱柱为岛津INERTSUSTAIN C18色谱柱(5μm，4.6×250mm)，使用岛津保护柱(5μm，4.0×10mm)。
生物碱分析色谱条件：流动相使用11％乙腈和89％水(20mM乙酸铵和0.1％甲酸，pH4.0)，柱温箱为40℃，总流速1mL/min，检测波长为226nm。
结果如图3所示，在本发明中转AbHAR超表达载体的转基因毛状根显著提高了莨菪碱含量。在普通毛状根中莨菪碱含量为2.32mg/g DW时，同时期AbHAR过表达载体颠茄毛状根中莨菪碱的含量平均达到4.24-5.98mg/g DW，其含量是非转基因颠茄毛状根含量的1.83-2.58倍。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限 于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。

Claims (17)

  1. 莨菪醛还原酶，其特征在于：具有下述氨基酸残基序列之一的蛋白质：
    1)如SEQ ID NO.4所示的氨基酸残基序列；
    2)将SEQ ID NO.4中的氨基酸残基经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有莨菪醛还原酶功能的氨基酸酸序列。
  2. 莨菪醛还原酶基因，其特征在于：所述莨菪醛还原酶基因具有下述核苷酸序列之一：
    1)如SEQ ID NO.3所示的核苷酸序列；
    2)编码如SEQ ID NO.3所示核苷酸序列的多核苷酸；
    3)与1)或2)限定的核苷酸序列具有80％以上的同源性且编码具有权利要求1所述莨菪醛还原酶功能的核苷酸序列；
    4)与1)或2)所述的序列杂交的核苷酸序列。
  3. 含有权利要求2所述莨菪醛还原酶基因的重组表达载体。
  4. 根据权利要求3所述的重组表达载体，其特征在于：所述重组表达载体为将权利要求2所述的莨菪醛还原酶基因插入原核或真核表达载体得到表达莨菪醛还原酶的重组表达载体。
  5. 含有权利要求2所述莨菪醛还原酶基因的转基因细胞系或转基因重组菌。
  6. 根据权利要求5所述的转基因细胞系，其特征在于：所述细胞系为植物细胞系。
  7. 权利要求1所述莨菪醛还原酶在体内或体外催化莨菪醛还原生成莨菪碱中的应用。
  8. 权利要求2所述莨菪醛还原酶基因在不具有托品烷生物碱生物合成途径的原核生物重建莨菪碱合成途径中的应用。
  9. 权利要求2所述莨菪醛还原酶基因在不具有托品烷生物碱生物合成途径的真核生物中重建莨菪碱合成途径中的应用。
  10. 权利要求3所述的重组表达载体在不具有托品烷生物碱生物合成途径的原核生物或真核生物中重建莨菪碱合成途径中的应用。
  11. 权利要求4所述的重组表达载体在不具有托品烷生物碱生物合成途径的原核生物或真核生物中重建莨菪碱合成途径中的应用。
  12. 权利要求1所述莨菪醛还原酶的转基因细胞系或转基因重组菌在具有托品烷生物碱生物合成途径的生物中提高莨菪碱含量中的应用。
  13. 权利要求2所述莨菪醛还原酶基因的转基因细胞系或转基因重组菌在具有托品烷生物碱生物合成途径的生物中提高莨菪碱含量中的应用。
  14. 权利要求3所述的重组表达载体的转基因细胞系或转基因重组菌在具有托品烷生物 碱生物合成途径的生物中提高莨菪碱含量中的应用。
  15. 权利要求4所述的重组表达载体的转基因细胞系或转基因重组菌在具有托品烷生物碱生物合成途径的生物中提高莨菪碱含量中的应用。
  16. 权利要求5所述的转基因细胞系或转基因重组菌在具有托品烷生物碱生物合成途径的生物中提高莨菪碱含量中的应用。
  17. 一种提高托品烷生物碱合成植物中莨菪碱含量的方法，其特征在于：将通过在具有托品烷生物碱生物合成途径的植物中过量表达权利要求2或3所述的莨菪醛还原酶基因。
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