CN109022463B - Bell seed ornithine decarboxylase ALODC gene and recombinant expression vector and application thereof - Google Patents

Abstract

The invention relates to a small bell seed ornithine decarboxylase ALODC gene, a recombinant expression vector and application thereof, wherein the gene is transferred into small bell seeds by cloning the small bell seed ornithine decarboxylase ALODC gene and a method mediated by agrobacterium tumefaciens to obtain a transgenic plant, and after AlODC is overexpressed, the content of putrescine and tropane alkaloids in the small bell plants can be obviously improved, so that an ideal method is provided for efficiently producing hyoscyamine and scopolamine by utilizing a transgenic technology.

Description

Bell seed ornithine decarboxylase ALODC gene and recombinant expression vector and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a small bell ornithine decarboxylase ALODC gene, a vector containing the gene and application of the gene.

Background

Tropane Alkaloids (TAs) are a class of secondary metabolites derived from a small number of solanaceae plants, and have been used as traditional drugs for nearly 3000 years because of their remarkable pharmacological activity. The hyoscyamine (or atropine racemate) and scopolamine (scopolamine) which are commonly used in modern clinic also comprise anisodamine (anisodamine) with low drug effect, which are anticholinergic drugs acting on a parasympathetic nervous system, are mainly used for analgesia, anesthesia, anti-motion sickness drugs, treatment of Parkinson's disease, microcirculation improvement, drug rehabilitation and withdrawal, treatment of pesticide poisoning and the like, and have huge market demands, wherein the scopolamine has stronger drug effect, weaker side effect and higher price. However, most plants have extremely low content of TAs, especially scopolamine, and if the content of scopolamine in the plants is only 0.01-0.08 percent of the dry weight, the market demand cannot be met. Therefore, the improvement of the content of TAs in resource plants by metabolic engineering techniques is a long-sought goal of the related industries. The first report of TAs metabolic engineering starts in 1992, namely, after the first TAs functional gene HnH6H is cloned for one year, HnH6H gene is transferred into plants to be over-expressed, and surprisingly, nearly all scopolamine is found in stems and leaves of transgenic plants of the current generation and the next generation and is completely transformed, which indicates that H6H is a very effective genetically-modified target gene, and the plants of Rongjingrong and the like simultaneously over-express two key enzyme genes PMT and H6H at the upstream and the downstream of a TAs synthetic pathway, so that the dry weight content of the final product scopolamine reaches 9mg/g, thereby proving the superiority of the strategy. However, there has been no effective means for greatly increasing the total content of tropane alkaloids, especially the content of scopolamine, in plants.

Bell seed, also known as Tibet eggplant, Tangchona, Himalayas anisodamine, Solanaceae anisodamine, native to the national Tibet (southeast), Yunnan (northwest), Nipol, Plumbum preparatium, and tin are also distributed, grow in the high altitude area of 3200 one and 4000 meters, and the plant height is 50-150 cm. Is a perennial root herb, and the tropine alkaloid is extracted from the root commonly used in medicine. The small bell plant is luxuriant and large, the whole plant is densely covered with villi and stellate hair, the root is thick and strong, and the whole plant has high fleshy degree. Compared with the traditional medicine source plant belladonna in China, the biomass is large, the alkaloid content is high, and the method is extremely suitable for large-area planting in high altitude areas such as Tibet in China. The content of hyoscyamine and scopolamine in wild small bell seeds is still very low, and metabolic engineering is the most effective method for effectively improving the hyoscyamine and scopolamine in the small bell seeds.

Ornithine Decarboxylase (ODC) is a key enzyme gene for putrescine synthesis in most plants, and putrescine is an important precursor of tropane alkaloid and synthesis, and it is reported that overexpression of the Ornithine decarboxylase gene (ODC) of mice in Datura can slightly increase the content of Datura alkaloid. However, the bell overexpression of the endogenous ODC gene is not reported at present. Therefore, the study of endogenous ODC genes derived from plants itself is important and significant.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an Ornithine decarboxylase ALODC gene of Bell seed; the second object of the present invention is to provide a recombinant expression vector containing the bell ornithine decarboxylase ALODC gene; the third purpose of the invention is to provide the application of the bell seed ornithine decarboxylase ALODC gene in improving the content of the scopolamine and the scopolamine of the solanaceae plants; the fourth purpose of the invention is to provide the application of the recombinant expression vector in improving the content of the scopolamine and the scopolamine in the solanaceae plants; the fifth purpose of the invention is to provide a method for increasing alkaloid content of small bell; another object of the present invention is to provide a solanaceae plant overexpressing the bell ornithine decarboxylase ALODC gene.

In order to achieve the above purpose, the invention provides the following technical scheme:

1. the bell seed ornithine decarboxylase ALODC gene is characterized in that: the amino acid sequence of the ALODC gene code is shown in SEQ ID NO. 2.

Preferably, the nucleotide sequence of the ALODC gene is shown as SEQ ID NO. 1.

2. A recombinant expression vector containing the bell seed ornithine decarboxylase ALODC gene.

Preferably, the recombinant expression vector is obtained by connecting a sequence shown in SEQ ID NO.1 into BamHI and Sac I of a pBI121 vector.

3. The bell seed ornithine decarboxylase ALODC gene is applied to the improvement of the content of scopolamine and scopolamine in solanaceae plants.

4. The recombinant expression vector is applied to improving the content of the belladonna and scopolamine in the solanaceae plants.

5. A method for increasing the alkaloid content of bell seeds is characterized in that bell seed ornithine decarboxylase ALODC genes are overexpressed in bell seed plants, and the amino acid sequence coded by the ALODC genes is shown as SEQ ID No. 2.

Preferably, the method for over-expressing the bell seed ornithine decarboxylase ALODC gene in the bell seed plant is a transgenic technology, and specifically comprises the following steps: cloning the ornithine decarboxylase ALODC gene of the small bell, constructing a recombinant expression vector containing the ALODC gene, transforming the obtained expression vector into agrobacterium tumefaciens, then impregnating the small bell explant with the agrobacterium tumefaciens of the recombinant expression vector containing the AlODC gene, and obtaining the ornithine decarboxylase ALODC gene plant through regeneration induction, resistance screening and PCR detection.

6. A solanaceae plant containing the bell seed ornithine decarboxylase ALODC gene.

Preferably, the solanaceae plant is bell.

The invention has the beneficial effects that: the invention clones AlODC in the small bell for the first time, transfers the gene into the small bell by the method of mediating the agrobacterium tumefaciens to obtain a transgenic plant, can obviously improve the content of putrescine and tropane alkaloid in the small bell plant after overexpressing the AlODC, and particularly provides an ideal method for the high-efficiency production of hyoscyamine and scopolamine by utilizing the transgenic technology in the biological metabolism engineering. Meanwhile, the invention also establishes a method for stably measuring the content of the hyoscyamine and the scopolamine in the plant, and lays a foundation for utilizing the plant to produce the phytochemicals in a large scale.

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 results of the measurement of hyoscyamine and scopolamine in the plants (A: the content of hyoscyamine in leaves; B: the content of hyoscyamine in roots; C: the content of scopolamine in leaves; B: the content of scopolamine in roots).

Detailed Description

Detailed description of the inventiontechnical contents of the present invention will be further described with reference to the accompanying drawings and examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as molecular cloning in Sambrook et al: a Laboratory manual is described in New York, Cold Spring Harbor Laboratory Press, 1989 edition, or as recommended by the manufacturer. The agrobacterium tumefaciens EHA105 related to the invention is already in Huang Yali, Jiang Jing Liang, Tian Yun Long, Guo Ping and Zhu Chang Xiong; study of agrobacterium tumefaciens-mediated genetic transformation of trichoderma harzianum, journal of biological engineering of china, 2008, 28 (3): 38-43, respectively. Agrobacterium tumefaciens EHA105 is commercially available from publicly available sources, such as the CAMBIA corporation, Australia, under the strain designation Gambar 1.

The seeds of bell (Anisodus luridus Link et Otto) were collected in Sichuan province and stored in the Tibet college of agriculture and pasture.

Example 1 cloning of Ornithine decarboxylase ALODC Gene of Bell seed

(1) Extraction of total RNA of bell-gene genome

Total RNA of bell genome was extracted using an RNA kit from TIANGEN. Taking 50-100mg of plant fibrous roots, quickly grinding into powder in liquid nitrogen, adding 550 mu L of lysis solution RL (added with mercaptoethanol), and violently shaking and uniformly mixing by vortex. Centrifuging at 9800rpm for 5min, sucking 450 μ L, transferring to a filtration column CS, placing CS in a collection tube, centrifuging at 12000rpm for 5min, carefully sucking 400 μ L of supernatant in the collection tube into a RNase-free centrifuge tube, slowly adding anhydrous ethanol with the volume 0.5 times of that of the supernatant, mixing uniformly, transferring to an adsorption column CR3, centrifuging at 12000rpm for 1min, discarding waste liquid, and placing CR3 back into the collection tube. 350 μ L of deproteinizing solution RW1 was added to CR3, centrifuged at 12000rpm for 1min, the waste liquid was discarded, and CR3 was returned to the collection tube. 80. mu.L of DNase1 working solution (10. mu.L of DNase1 stock solution + 70. mu.L of RDD solution, gently mixed and mixed) was added to the center of CR3, and the mixture was allowed to stand at room temperature (18-25 ℃) for 15 minutes. 350 μ L of deproteinizing solution RW1 was added to CR3, centrifuged at 12000rpm for 1min, the waste liquid was discarded, and CR3 was returned to the collection tube. Adding 500 mu L of rinsing liquid RW (added with ethanol) into CR3, standing for 2 minutes at room temperature (18-25 ℃), centrifuging for 1min at 12000rpm, pouring off waste liquid in the collection tube, and putting CR3 back into the collection tube for one time. Centrifuge at 12000rpm for 5 min. Placing CR3 at room temperature for 5min, placing CR3 in new RNase-free, adding 70 μ LRNase-free ddH2O dropwise into the middle part of the adsorption membrane, standing at room temperature for 2min, and centrifuging at 12000rpm for 2min to obtain RNA solution. Putting 10 mu L of the gel into an RNase-free tubule for gel running and concentration measurement; the remaining 60. mu.L was stored in RNase-free centrifuge tubes at-80 ℃.

(2) Cloning of the ALODC Gene of Bell seed

Reverse transcription is carried out on the obtained genome total RNA by reverse transcriptase to obtain first strand cDNA, and an upstream primer and a downstream primer of a complete coding frame are designed and synthesized according to a DNA sequence shown in SEQ ID NO.1, wherein the upstream primer and the downstream primer are specifically as follows:

an upstream primer: F-AlODC-OE 5' -gcggatccatggccggccaaacagtcatcg-3' (SEQ ID NO.3) (BamHI cleavage site underlined);

a downstream primer: R-AlODC-OE 5' -gcgagctctcagcttggataagcataagc-3' (SEQ ID NO.4) (Sac I cleavage site underlined);

the synthesized first strand cDNA is used as a template, SEQ ID NO.3 and SEQ ID NO.4 are used as a primer pair, and sequencing is carried out after PCR amplification (Shanghai Sangni Biotech Co., Ltd.), wherein the nucleotide is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2.

Example 2 construction of plant expression vector containing AlODC Gene and construction of engineering bacterium

Firstly, the ALODC gene cloned in the embodiment 1 is connected with a PJET vector (Takara Corp.) to construct an intermediate vector PJET-AlODDC; and then carrying out enzyme digestion on the intermediate vector PJET-AlODC by using (BamH I/Sac I), simultaneously using the same enzyme digestion expression vector pBI121 to recover an AlODC gene segment and a pBI121 vector large segment, carrying out connection transformation, selecting a single clone, extracting a plasmid to carry out PCR detection and enzyme digestion verification, and obtaining the plant expression vector containing the AlODC gene, which is called AlODC-pBI121 for short.

The recombinant expression vector AlODC-pBI121 is transformed into agrobacterium tumefaciens (EHA105), positive clones are screened, and PCR verification is carried out.

Example 3 Agrobacterium tumefaciens-mediated ALODC Gene-transformed plants and transgenic Screen

(1) Pre-culturing explants; soaking seeds of bell seeds in 1g/L GA3 solution overnight, slightly washing the seeds with tap water, disinfecting the seeds with ethanol with the volume fraction of 70% for 1min, then sterilizing the seeds with 50% by mass sodium hypochlorite solution for 10min, washing the seeds with sterile water for multiple times, and then inoculating the seeds on MS +200mg/L Cef solid culture medium without adding hormone. When the seeds are cultured for about 15 days under the light condition of 25C and 16h/8h (light/dark), two cotyledons germinate from the seeds, and the hypocotyl length is about 1cm, so that the seeds can be used for genetic transformation.

(2) Agrobacterium activation and explant co-culture: selecting an Agrobacterium EHA105-pBI121-AlODC monoclonal from the streaked plate, inoculating into 15mL YEP (Rif + Str + Kan) liquid culture medium, and performing shake culture at 28 ℃ and 200r/min for 24-48 h; centrifuging at 5000rpm for 6min to collect thallus, suspending thallus in transforming co-culture medium (1/2MS + AS100 μmol/L), adjusting OD600 to 0.5 for transformation, shearing plant cotyledon and hypocotyl, soaking in activated Agrobacterium liquid for 5min, sucking to remove bacteria liquid, spreading the explant on the transforming co-culture medium with filter paper, and dark culturing at 25 deg.C for 4 d. Meanwhile, the control was leaf explants dropped on 1/2MS liquid medium suspension of Agrobacterium tumefaciens without the target gene.

(3) Screening of resistant regenerated plants: after the co-culture is finished, the explants are transferred to a degerming and regeneration culture medium (MS +1.0mg/L ZT +0.5mg/L IAA) to be cultured under the illumination condition of 16h/8h (light/dark) at the temperature of 25 ℃, and the explants are transferred to a primary culture medium after about 5 days. And after continuing culturing for 1 month, transferring the resistance callus stripped from the resistance buds or with bud points into a rooting culture medium without screening pressure for continuous culturing until a complete regeneration plant is formed, numbering and subculturing the regeneration plant on a basic culture medium to obtain a kan resistance regeneration plant for subsequent experiments.

Detecting the obtained kan resistance regeneration plant by PCR, respectively designing a forward primer and a reverse primer according to an expression cassette p35s-ALODC-nos sequence p35s and AlODC where a target gene is located by a detection primer, wherein the specific primers are as follows:

an upstream primer: f-p35s:5'-gatgcctctgccgacagtggtc-3' SEQ ID NO. 5;

a downstream primer: 5'-tcagcttggataagcataagc-3' (SEQ ID NO. 6);

the result shows that the designed PCR specific primer can amplify specific DNA segment, and when non-transformed plant genome DNA is used as template, no segment is amplified.

In this embodiment, the plant expression vector is transformed into agrobacterium tumefaciens to obtain an agrobacterium tumefaciens strain containing an AlODC gene plant expression vector for transforming a plant, and the constructed agrobacterium tumefaciens strain is used to transform the plant to obtain a transgenic plant detected by PCR. The acquisition of transgenic plant provides direct material for screening plant lines with higher hyoscyamine and scopolamine contents.

Example 4 determination of the content of scopolamine and scopolamine in transgenic plants by HPLC-ELSD

(1) HPLC condition and system applicability and preparation of standard solution

Chromatograph: shimadzu high performance liquid chromatograph (system controller: CBM-20Alite, pump: LC-20AD, column oven: CTO-20A, diode array detector: SPD-M20A, autosampler: SIL-20A);

a chromatographic column: YMC-Pack ODS-A (150X 6.0mm,5micron,12 nm);

mobile phase: an equal volume of acetonitrile and methanol in ammonium acetate buffer (20mM amine acetate, 0.1% formic acid, pH 4.0) 17: 83;

flow rate: 1mL/min

Detection wavelength: 215nm

Column temperature: 40 deg.C

Sample introduction amount: 20 μ L

Preparation of Standard Curve Scopolamine, anisodamine and scopolamine Standard (Sigma) were weighed to 500. mu.g.mL^-1、250μg·mL^-1、100μg·mL^-1、50μg·mL^-1、25μg·mL^-1And 10. mu.g.mL^-1Measuring and drawing standard curves of three alkaloids according to the above detection conditions, calculating respective linear regression equation for quantitative calculation of sample, mixing and collecting whole leaf after flowering, oven drying at 40 deg.C, grinding into powder, sieving with 50 mesh sieve, and sieving with Kamada [200 ] as required]The method for extracting the alkaloid comprises the following steps:

1) adding 0.2g plant dry powder into 20mL methanol, ultrasonically crushing for 30min, if the water temperature rises, replacing cold water once, and standing at room temperature for 1 h;

2) filtering with filter paper, washing residue with 10mL of methanol, and volatilizing filtrate at 40 ℃;

3) the volatilized residue was combined with 10mL of chloroform +5mL of 1N H₂SO₄Ultrasonically dissolving the solution, and fully and uniformly mixing;

4) collecting the upper layer H₂SO₄Adding 3mL of ammonia water on ice bath to alkalize the solution, wherein the pH value is 10-12;

5) extracting the solution once with 5mL of chloroform, and collecting a chloroform phase;

6) extracting with 3mL of chloroform once, collecting chloroform phases, and combining the two chloroform phases;

7) chloroform was evaporated at 40 ℃ and the residue was dissolved in 2mL of alkaloid stock buffer.

The equation calculates the content (mg) of the hyoscyamine and the scopolamine in the sample, and then divides the content by the dry weight (g) of the plant leaves of the sample, thereby calculating the content of the hyoscyamine and the scopolamine in the plant plants, and the result is shown in figure 1.

The result shows that the content of hyoscyamine and scopolamine in the plant is obviously improved by the AlODC gene. The content of hyoscyamine and scopolamine in the leaves of the transgenic ALODC plant can reach 3.5mg/g and 5.5mg/g of dry weight at most, and is 4.7 times and 3.67 times of that of non-transformed ordinary plants (0.5mg/g of dry weight) respectively. In the figure, WT represents a non-transformed normal plant, and VC represents an empty vector control plant; l1,2,3,4,5 and L6 represent different transgenic lines. The content of alkaloid in roots is also improved by similar large margin.

In the embodiment, the content of hyoscyamine and scopolamine in transgenic plants is determined by an HPLC method, and the high-yield plant of the hyoscyamine and the scopolamine are obtained by adopting a metabolic engineering strategy of transforming an AlODC gene, so that an ideal method is provided for large-scale production of the hyoscyamine and the scopolamine.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

<110> Tibet college of agriculture and animal husbandry

SOUTHWEST University

<120> small bell ornithine decarboxylase ALODC gene, recombinant expression vector and application thereof

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50 55 60

Tyr Val Leu Asp Leu Gly Glu Val Val Ser Leu Met Asp Gln Trp Asn

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Asn Ala Leu Pro Asn Ile Arg Pro Phe Tyr Ala Val Lys Cys Asn Pro

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Glu Pro Ser Phe Leu Ser Met Leu Ser Ala Leu Gly Ser Asn Phe Asp

100 105 110

Cys Ala Ser Arg Ala Glu Ile Glu Tyr Val Leu Ser Leu Gly Ile Ser

115 120 125

Pro Asp Arg Ile Val Phe Ala Asn Pro Cys Lys Pro Glu Ser Asp Ile

130 135 140

Ile Phe Ala Ala Lys Val Gly Val Asn Leu Thr Thr Tyr Asp Ser Glu

145 150 155 160

Asp Glu Val Tyr Lys Ile Arg Arg His His Pro Lys Ser Glu Leu Leu

165 170 175

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180 185 190

Pro Lys Tyr Gly Ala Leu Pro Glu Glu Val Glu Pro Leu Leu Arg Thr

195 200 205

Ala His Ala Ala Arg Leu Thr Val Ser Gly Val Ser Phe His Ile Gly

210 215 220

Ser Gly Asp Ala Asp Ser Asn Ala Tyr Leu Gly Ala Ile Ala Ala Ala

225 230 235 240

Lys Glu Val Phe Gln Thr Ala Ala Arg Phe Gly Met Ser Lys Met Thr

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Ala Ala Thr Ala Ile Lys Ser Ala Leu Ser Gln His Phe His Asp Glu

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Ala Phe Thr Leu Ala Thr Thr Ile Ile Gly Lys Arg Val Arg Gly Glu

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Val Leu Tyr Asp His Ala Thr Val Thr Ala Thr Ala Leu Ala Cys Met

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Claims (7)

1. Bell ornithine decarboxylaseALODCA gene characterized by: the above-mentionedALODCThe amino acid sequence of the gene code is shown in SEQ ID NO. 2.

2. The ornithine decarboxylase of bell as claimed in claim 1ALODCA gene characterized by: the above-mentionedALODCThe nucleotide sequence of the gene is shown in SEQ ID NO. 1.

3. The ornithine decarboxylase of bell as claimed in claim 2ALODCA recombinant expression vector for a gene, characterized in that: the recombinant expression vector is connected into a pBI121 vector by a sequence shown in SEQ ID NO.1BamH I andSaci between the enzyme cutting sites.

4. The ornithine decarboxylase of bell as claimed in claim 1 or 2ALODCThe application of the gene in improving the content of hyoscyamine and scopolamine in solanaceae plants; the Solanaceae plant is small bell.

5. The use of the recombinant expression vector of claim 3 to increase the content of scopolamine and scopolamine in the solanaceae family; the recombinant expression vector is connected into a pBI121 vector by a sequence shown in SEQ ID NO.1BamH I andSaci, and the solanaceae plant is bell seed.

6. A method for increasing alkaloid content of small bell is characterized by comprising the following steps: by overexpression of Bell ornithine decarboxylase in Bell plantsALODCA gene ofALODCThe amino acid sequence of the gene code is shown in SEQ ID NO. 2; the alkaloids are hyoscyamine and scopolamine.

7. The method for increasing alkaloid content of bell seeds of claim 6, wherein the method comprises the following steps: overexpression of bell ornithine decarboxylase in bell plantsALODCThe gene method is a transgenic technology, and specifically comprises the following steps: ornithine decarboxylase of clone small bellALODCGenes, then constructs containing the sameALODCRecombinant expression vector of gene, transforming Agrobacterium tumefaciens with the obtained expression vector, and culturing the transformed Agrobacterium tumefaciens containing the geneAlODCThe bell seed explant is soaked with agrobacterium tumefaciens of a gene recombinant expression vector, and the ornithine decarboxylase is obtained through regeneration induction, resistance screening and PCR detectionALODCAnd (4) gene plants.

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