CN109022463A

CN109022463A - The sub- ornithine decarboxylase ALODC gene of small bell and its recombinant expression carrier and application

Info

Publication number: CN109022463A
Application number: CN201811066743.3A
Authority: CN
Inventors: 兰小中; 廖志华; 陈敏; 赵腾飞
Original assignee: Southwest University; Xizang Agriculture and Animal Husbandry College
Current assignee: Southwest University; Xizang Agriculture and Animal Husbandry College
Priority date: 2018-09-13
Filing date: 2018-09-13
Publication date: 2018-12-18
Anticipated expiration: 2038-09-13
Also published as: CN109022463B

Abstract

The present invention relates to the sub- ornithine decarboxylase ALODC gene of small bell and its recombinant expression carrier and applications, by cloning the sub- ornithine decarboxylase ALODC gene of small bell, and the gene is transferred in small bell by Agrobacterium tumefaciens mediated method, obtain transgenic plant, after being overexpressed AlODC, the content of putrescine and tropane alkaloid in the sub- plant of small bell can be significantly improved, efficiently to produce hyoscyamine using transgenic technology and hyoscine provides a kind of ideal method.

Description

The sub- ornithine decarboxylase ALODC gene of small bell and its recombinant expression carrier and application

Technical field

The invention belongs to field of biotechnology, are related to the sub- ornithine decarboxylase ALODC gene of small bell, further relate to contain the base The carrier of cause and application.

Background technique

Tropane alkaloids (tropane alkaloids, TAs) are a kind of secondary generations from a small number of plants of Solanaceae It thanks to product, because having significant pharmacological activity, has been used nearly 3000 by people as conventional medicament.On modern clinic The most commonly used is hyoscyamine (hyoscyamine) (or its racemic modification atropine) and hyoscines (scopolamine), also wrap The slightly lower anisodamine of drug effect (anisodamine) is included, they are all the anticholinergic agents for acting on parasympathetic, main It is used to ease pain, anaesthetize, antimotion sickness drug, treatment parkinsonism, improving microcirculation, detoxification and addiction-removing, treatment pesticide poisoning etc., market Demand is very huge, and wherein hyoscine drug effect is stronger, and side effect is weaker, and price is also more expensive.However in most plants TAs content especially hyoscine content is extremely low, if hyoscine content is only the 0.01%~0.08% of dry weight in plant, no It is able to satisfy the market demand.So improving TAs content in resource plant using metabolic engineering technology is related industry long-sought Target.The report of first case TAs metabolic engineering originates in 1992, i.e. be cloned 1 year first TAs functional gene HnH6H Later, HnH6H gene is transferred to and is allowed to overexpress in plant, be surprised to find in the cauline leaf of the transgenosis present age and first filial generation plant It is almost hyoscine entirely, hyoscyamine is completely converted, and shows that H6H is effectively genetic modification target Ji Jiyin, king is thin The plant such as honor overexpress two key genes PMT and H6H of TAs route of synthesis upstream and downstream simultaneously, make final product east Liang Henbane alkali dry weight content has reached 9mg/g and has demonstrated the superiority of the strategy.But how to be increased substantially in plant in plant The content of the total content of tropane alkaloid, especially hyoscyamine never has effective means.

Small bell, also known as hiding eggplant, Tang Chong that thin, Himalaya Tangut Anisodus Radix, Solanaceae Tangut Anisodus Radix category are originated in from China Tibet (southeast), Yunnan (northwestward), Nepal, Bhutan, Sillim are also distributed, and are grown on 3200-4000 meters of high altitude localities, Plant height 50-150cm.It is a kind of perennial herb, pharmaceutically commonly uses root and extract Tropane alkaloids.The sub- plant of small bell is numerous Cyclopentadienyl is tall and big, and the densely covered villus of complete stool and stellate hair, root thickness are strong, and complete stool carnification degree is high.With China's tradition medicine source plant resource belladonna phase It than having the characteristics that biomass is big, alkaloid is high, and is extremely suitable for being planted in the high altitude localities large area such as China Tibet.And Hyoscyamine and hyoscine content are still very low in wild small bell, and metabolic engineering is to effectively improve the sub- hyoscyamine of small bell and east Liang Henbane alkali most efficient method.

Ornithine decarboxylase (ODC) is the key gene that putrescine synthesizes in most plants body, and putrescine is tropane The important prerequisite substance of alkaloid and synthesis, forefathers are reported in the ornithine decarboxylase gene that mouse is overexpressed in datura It (ODC) being capable of the micro content for improving datura alkaloid.And there has been no relevant at present for the endogenous ODC gene of small bell overexpression Report.Therefore just seem important and significant from the research of the endogenous ODC gene of plant itself.

Summary of the invention

In view of this, one of the objects of the present invention is to provide the sub- ornithine decarboxylase ALODC genes of small bell；Of the invention The second purpose is to provide the recombinant expression carrier containing the sub- ornithine decarboxylase ALODC gene of the small bell；Mesh of the invention Three be to provide the sub- ornithine decarboxylase ALODC gene of the small bell and improving plant of Solanaceae hyoscyamine and hyoscine Application in content；The fourth object of the present invention is that providing the recombinant expression carrier is improving plant of Solanaceae hyoscyamine and east Application in the content of hyoscyamine；The fifth object of the present invention is to provide a kind of method for improving the sub- alkaloid of small bell； The sixth object of the present invention is to provide the plant of Solanaceae for being overexpressed the sub- ornithine decarboxylase ALODC gene of small bell.

For achieving the above object, the invention provides the following technical scheme:

1. the sub- ornithine decarboxylase ALODC gene of small bell, it is characterised in that: the amino acid sequence of the ALODC gene coding Column are as shown in SEQ ID NO.2.

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

2, the recombinant expression carrier containing the sub- ornithine decarboxylase ALODC gene of the small bell.

Preferably, recombinant expression carrier sequence as shown in SEQ ID NO.1 be connected into pBI121 carrier BamHI and Sac I and obtain.

3, the sub- ornithine decarboxylase ALODC gene of the small bell is in the content for improving plant of Solanaceae hyoscyamine and hyoscine In application.

4, application of the recombinant expression carrier in the content for improving plant of Solanaceae hyoscyamine and hyoscine.

5, a kind of method for improving the sub- alkaloid of small bell, by being overexpressed the sub- ornithine of small bell in the sub- plant of small bell Decarboxylase ALODC gene, the amino acid sequence of the ALODC gene coding is as shown in SEQ ID NO.2.

Preferably, the method that the sub- ornithine decarboxylase ALODC gene of small bell is overexpressed in the sub- plant of small bell is transgenosis Technology, specifically: clone's sub- ornithine decarboxylase ALODC gene of small bell, then building contains the recombination table of the ALODC gene Agrobacterium tumefaciems is converted up to carrier, then by the expression vector of acquisition, then with the root of the recombinant expression carrier containing AlODC gene The sub- explant of cancer During Agrobacterium small bell obtains through regeneration induction, resistance screening, PCR detection and turns ornithine decarboxylase ALODC base Because of plant.

6, the plant of Solanaceae containing the sub- ornithine decarboxylase ALODC gene of the small bell.

Preferably, the plant of Solanaceae is small bell.

The beneficial effects of the present invention are: the present invention has cloned AlODC in small bell for the first time, and is situated between by Agrobacterium tumefaciems The gene is transferred in small bell by the method led, and obtains transgenic plant, after being overexpressed AlODC, can significantly improve small bell Hyoscyamine and hyoscine in the content of putrescine and tropane alkaloid, especially plant in plant, for biological metabolic engineering, benefit Hyoscyamine is efficiently produced with transgenic technology and hyoscine provides a kind of ideal method.The present invention has also set up stabilization simultaneously The method for measuring hyoscyamine and hyoscine content in plant is laid a good foundation for plant element is mass produced using plant.

Detailed description of the invention

In order to keep the purpose of the present invention, technical scheme and beneficial effects clearer, the present invention provides following attached drawing and carries out Illustrate:

Fig. 1 is hyoscyamine and hyoscine measurement result (A: henbane alkali content in leaf in plant；B: hyoscyamine contains in root Amount；C: hyoscine content in leaf；B: hyoscine content in root).

Specific embodiment

Specific embodiment is with reference to the accompanying drawings and examples described further technology contents of the invention.Below Embodiment will be helpful to those skilled in the art and further understand the present invention, but the invention is not limited in any way.It is following Test method without specific conditions in embodiment, usually according to normal condition, such as the molecular clonings such as Sambrook: experiment Room handbook is shown in condition described in the versions in 1989 of NewYork:Cold Spring Harbor Laboratory Press, or According to the normal condition proposed by manufacturer.Agrobacterium tumefaciems EHA105 of the present invention is in " Huang Yali, Jiang Xiliang, Tian Yun Dragon, Guo Ping, Zhu Changxiong；The research of Agrobacterium tumefaciens mediated trichoderma harzianum genetic transformation, Chinese biological engineering magazine, 2008,28 (3): 38-43 " it discloses in document.Agrobacterium tumefaciems EHA105 can be obtained by disclosing commercially available commercial channel, such as may be used To be bought from Australian CAMBIA company, strain number Gambar1.

Sub (the Anisodus luridus Link et Otto) seed collection of experimental material small bell is protected in Tibet, China It is stored in Tibet Agricultural and Animal Husbandry College.

The clone of the sub- ornithine decarboxylase ALODC gene of embodiment 1, small bell

(1) extraction of small bell subgenom total serum IgE

The extraction of small bell subgenom total serum IgE uses the RNA kit of TIANGEN company.Take 50-100mg plant fibrous root 550 μ L lysate RL (mercaptoethanol has been added) are added in the rapid grind into powder in liquid nitrogen, and the acutely concussion that is vortexed mixes. 9800rpm is centrifuged 5min, draws 450 μ L and is transferred on Filter column CS, and CS is placed in collecting pipe, and 12000rpm is centrifuged 5 minutes, small The heart is drawn in the centrifuge tube of the 400 μ L to RNase-free of supernatant in collecting pipe, and the anhydrous second of 0.5 times of supernatant volume is slowly added to Alcohol is mixed, is transferred in adsorption column CR3, and 12000rpm is centrifuged 1min, abandons waste liquid, CR3 is put back in collecting pipe.It is added into CR3 350 μ L protein liquid removal RW1,12000rpm are centrifuged 1min, abandon waste liquid, CR3 is put back in collecting pipe.80 μ L are added to the center CR3 DNase1 working solution (+70 μ L RDD solution of 10 μ L DNase1 storing liquid, soft mix), (18~25 DEG C) of room temperature stand 15 Minute.350 μ L protein liquid removal RW1,12000rpm are added into CR3 and are centrifuged 1min, abandons waste liquid, CR3 is put back in collecting pipe.To 500 μ L rinsing liquid RW (ethyl alcohol is added) is added in CR3, (18~25 DEG C) of room temperature stand 2 minutes, and 12000rpm is centrifuged 1min, Fall the waste liquid in collecting pipe, CR3 is put back in collecting pipe, is repeated once.12000rpm is centrifuged 5min.CR3 is placed in room temperature to put 5min is set, CR3 is put into new RNase-free, 70 μ LRNase-free dd are vacantly added dropwise to the intermediate position of adsorbed film H2O, is stored at room temperature 2min, and 12000rpm is centrifuged 2min, obtains RNA solution.It takes 10 μ L to be put in RNase-free tubule, is used for It runs glue and surveys concentration；Remaining 60 μ L is put in -80 DEG C of preservations in RNase-free centrifuge tube.

(2) clone of the sub- ALODC gene of small bell

The genome total serum IgE obtained is obtained into the first chain cDNA by reverse transcriptase reverse transcription, according to SEQ ID NO.1 institute The DNA sequence dna shown designs and synthesizes the upstream primer and downstream primer of complete encoder block, specific as follows:

Upstream primer: F-AlODC-OE:5'-gcggatccatggccggccaaacagtcatcg-3'(SEQ ID NO.3) (being BamHI restriction enzyme site at underscore)；

Downstream primer: R-AlODC-OE:5'-gcgagctctcagcttggataagcataagc-3'(SEQ ID NO.4) (being Sac I restriction enzyme site at underscore)；

Using the first chain cDNA of synthesis as template, using SEQ ID NO.3 and SEQ ID NO.4 as primer pair, through PCR amplification After (Shanghai Sani Biotechnology Co., Ltd) is sequenced, nucleotide is as shown in SEQ ID NO.1, and amino acid sequence is such as Shown in SEQ ID NO.2.

Embodiment 2, the plant expression vector for constructing the gene containing AlODC and engineering bacteria building

It connect, constructs with PJET carrier (Dalian Takara company) firstly, embodiment 1 is cloned obtained ALODC gene Intermediate vector PJET-AlODC；Again by intermediate vector PJET-AlODC (BamH I/Sac I) digestion, while with identical enzyme Expression vector pBI121 is cut, AlODC genetic fragment and pBI121 carrier large fragment, connection conversion are recycled, picking monoclonal extracts Plasmid does PCR detection and digestion verification, obtains the plant expression vector of the gene containing AlODC, referred to as AlODC-pBI121.

Recombinant expression carrier AlODC-pBI121 is converted into Agrobacterium tumefaciems (EHA105), screening positive clone, and carry out PCR verifying.

Embodiment 3, Agrobacterium tumefaciens mediated ALODC genetic transformation plant and transgenosis screening

(1) preculture of explant；The GA3 solution soaked overnight of small bell seed 1g/L, after slightly being rinsed with tap water The ethanol disinfection 1min for being 70% with volume fraction, the liquor natrii hypochloritis degerming 10min for being then 50% with mass fraction are used After aseptic water washing is multiple, it is inoculated on the MS+200mg/L Cef solid medium for not adding hormone.25C, 16h/8h (light/dark) about 15d or so is cultivated under illumination condition, seed sprouts two panels cotyledon out, and hypocotyl is about 1cm, can be used for losing Pass conversion.

(2) co-cultivation of Agrobacterium activation and explant: from one Agrobacterium EHA105- of picking on the plate of scribing line culture PBI121-AlODC monoclonal is inoculated in 15mL YEP (Rif+Str+Kan) fluid nutrient medium, 28 DEG C, 200r/min oscillation Cultivate 24-48h；5000rpm is centrifuged 6min and collects thallus, trains culture medium (1/2MS+AS100 μm of ol/L) suspended bacteria altogether with conversion Body adjusts OD600 to can be used to convert to 0.5, and clip plant cotyledon and hypocotyl are impregnated in activated Agrobacterium bacterium solution 5min blots bacterium solution, and explant is layered on the conversion added with filter paper and is trained on culture medium altogether, and 25 DEG C, dark culture 4d.Simultaneously with drop Being added in without the blade explant of the 1/2MS fluid nutrient medium suspension of the Agrobacterium tumefaciems of target gene is control.

(3) altogether after training, explant the screening of resistance regeneration plant: is transferred to degerming and regeneration culture medium (MS+ 1.0mg/l ZT+0.5mg/L IAA), it 25 DEG C, is cultivated under 16h/8h (light/dark) illumination condition, 5d or so switching One subculture.Continue after cultivating January, removes resistant buds or the kanamycin-resistant callus tissue with bud point is transferred to the culture of rootage without screening pressure Continue to cultivate in base, until complete regeneration plant is formed, numbers and subculture regeneration plant is cultivated on minimal medium Kan resistance regeneration plant is obtained, subsequent experimental is used for.

The kan resistance regeneration plant of acquisition is detected with PCR, detection primer is according to expression cassette p35s- where target gene ALODC-nos sequence p35s and AlODC separately design forward primer and reverse primer, and specific primer is as follows:

Upstream primer: F-p35s:5'-gatgcctctgccgacagtggtc-3'SEQ ID NO.5；

Downstream primer: R-AlODC:5'-tcagcttggataagcataagc-3'(SEQ ID NO.6)；

The result shows that specific DNA fragment can be amplified using designed PCR special primer, and with non transformed plants base Because group DNA be template when, do not amplify any segment.

The plant expression vector is converted Agrobacterium tumefaciems by the present embodiment, is obtained and is contained AlODC for transformed plant The Agrobacterium tumefaciens strain of gene plant expression vector is obtained using constructed Agrobacterium tumefaciens strain transformed plant through PCR The transgenic plant plant of detection.The acquisition of transgenic plant plant is that screening obtains higher hyoscyamine and hyoscine content Plant strains provide direct material.

Embodiment 4 utilizes hyoscyamine and hyoscine content in HPLC-ELSD measurement transgenic plant

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

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

Chromatographic column: YMC-Pack ODS-A (150 × 6.0mm, 5micron, 12nm)；

Mobile phase: acetonitrile and the isometric mixed liquor of methanol: ammonium acetate buffer (20mM ammonium acetate, 0.1% formic acid, pH= 4.0)=17:83；

Flow velocity: 1mL/min

Detection wavelength: 215nm

Column temperature: 40 DEG C

Sample volume: 20 μ L

The production of standard curve weighs hyoscyamine, anisodamine and hyoscine standard items (Sigma) respectively and is configured to 500 μg·mL^-1、250μg·mL^-1、100μg·mL^-1、50μg·mL^-1、25μg·mL^-1With 10 μ gmL^-1The gradient standard of concentration Solution measures according to above-mentioned testing conditions and draws the standard curve of three kinds of alkaloids, calculates respective equation of linear regression and uses After the quantitative calculating of sample reaches the florescence, by complete stool blade mixed collection, 40 DEG C of drying pulverize, cross 50 meshes, press The methods of Kamada [200] extracts alkaloid, specific as follows:

1) 0.2g plant drymeal addition 20mL methanol, ultrasonication 30min, if water temperature increases an intermediate interchangeable cold water, After be stored at room temperature 1h；

2) filter paper filters, and washs residue with 10mL methanol, 40 DEG C of filtrate volatilize；

3) residue volatilized 10mL chloroform+5mL 1N H₂SO₄Solution ultrasonic dissolution, mixes well；

4) upper layer H is collected₂SO₄Phase, 3mL ammonium hydroxide is added on ice bath to be made basified, and pH is in 10-12；

5) primary with the above-mentioned solution of 5mL chloroform extraction, collect chloroform phase；

6) primary with the extraction of 3mL chloroform again, chloroform phase is collected, chloroform phase twice is merged；

7) chloroform is volatilized for 40 DEG C, residue stores buffer solution with 2mL alkaloid.

Equation calculation goes out hyoscyamine and hyoscine content (mg) in sample, then divided by the plant leaf dry weight (g) of sample, To calculate the content of hyoscyamine and hyoscine in plant plant, as a result as shown in Figure 1.

The results show that turning AlODC gene significantly improves hyoscyamine and hyoscine content in plant.Turn the plant of ALODC gene The content of hyoscyamine and hyoscine can reach the 3.5mg/g and 5.5mg/g of dry weight in blade in strain, be non-transformed respectively 4.7 and 3.67 times of common plant (0.5mg/g dry weight).WT indicates non-transformed common plant in figure, and what VC was indicated is empty carrier Adjoining tree；L1,2,3,4,5 and L6 indicates different transgenic lines.The content of alkaloid has also obtained similar big in root The promotion of amplitude.

The present embodiment determines hyoscyamine and hyoscine content in transgenic plant using HPLC method, using conversion The metabolic engineering strategies of AlODC gene obtain the plant plant of hyoscyamine and hyoscine high yield, are large-scale production henbane Alkali and hyoscine provide a kind of ideal method.

Finally, it is stated that preferred embodiment above is only used to illustrate the technical scheme of the present invention and not to limit it, although logical It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be Various changes are made to it in form and in details, without departing from claims of the present invention limited range.

Sequence table

<110>Tibet Agricultural and Animal Husbandry College

Southwest University

<120>the sub- ornithine decarboxylase ALODC gene of small bell and its recombinant expression carrier and application

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1293

<212> DNA

<213>small bell is sub (Anisodus luridus Link et Otto)

<400> 1

atggccggcc aaacagtcat cgtttccggg ttgaacccgg cggccattct tcagtccaca 60

atcggcggag ctcctccatc cacggcggaa aacggccata ccagaaaagt tgttcctatg 120

tcaaaagatg ccctccaaga tttcatggtt tcaattataa cccaaaaatt acaagacgaa 180

aaacaacctt tttacgtgct agatttgggt gaagttgttt cccttatgga ccaatggaat 240

aacgctttac caaatatccg tccattttat gctgttaagt gtaacccgga accttctttc 300

ctgtccatgt tgtctgcttt gggctcaaac tttgattgtg ctagccgtgc cgaaattgag 360

tacgttttat ctctcggtat ttccccagac cggatcgttt tcgctaaccc gtgtaaaccc 420

gaatccgaca tcattttcgc agcgaaagtt ggagttaatc tcacaacgta tgactccgaa 480

gacgaggttt acaagatccg aagacatcac ccgaaatctg agcttttgct ccgtatcaag 540

cccatggacg acgctaacgc caggtgtcca atgggcccga aatacggagc attgcccgaa 600

gaagttgaac cgttgctccg gaccgcccat gcggcccggc ttactgtctc tggcgtctcc 660

tttcatattg gaagcggaga cgccgattcg aacgcctatt taggcgctat agctgcggct 720

aaggaagttt tccaaaccgc ggctaggttt ggtatgtcta aaatgacgat tttagacata 780

ggcggcggtt ttacatctgg tcaccaattc acaaccgccg caaccgcgat taaatcagct 840

ttatctcaac acttccatga tgaaccggag ttaacaatca tagctgaacc gggccgtttt 900

tttgcggaaa cggcgtttac tttagcgacg acaattatag ggaaaagagt gaggggagaa 960

ttaagagaat attggattaa cgacgggtta tacggttcaa tgaactgtgt actttacgac 1020

cacgcaacgg taactgcgac ggcgttagcg tgtatgtcga accgtggtaa cgttaactgt 1080

ggcgggtcga aaacgtttcc gtcaactgtg tttgggccca catgtgatgc acttgatact 1140

gtgttgaggg attaccagtt accggagctg caggttaatg attggttggt ttttcctaat 1200

atgggtgctt atactaaggc tgctgggtct aatttcaatg gatttaatac atctgccatt 1260

gttactcacc ttgcttatgc ttatccaagc tga 1293

<210> 2

<211> 430

<212> PRT

<213>small bell is sub (Anisodus luridus Link et Otto)

<400> 2

Met Ala Gly Gln Thr Val Ile Val Ser Gly Leu Asn Pro Ala Ala Ile

1 5 10 15

Leu Gln Ser Thr Ile Gly Gly Ala Pro Pro Ser Thr Ala Glu Asn Gly

20 25 30

His Thr Arg Lys Val Val Pro Met Ser Lys Asp Ala Leu Gln Asp Phe

35 40 45

Met Val Ser Ile Ile Thr Gln Lys Leu Gln Asp Glu Lys Gln Pro Phe

50 55 60

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

65 70 75 80

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

85 90 95

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

Leu Arg Ile Lys Pro Met Asp Asp Ala Asn Ala Arg Cys Pro Met Gly

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

245 250 255

Ile Leu Asp Ile Gly Gly Gly Phe Thr Ser Gly His Gln Phe Thr Thr

260 265 270

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

275 280 285

Pro Glu Leu Thr Ile Ile Ala Glu Pro Gly Arg Phe Phe Ala Glu Thr

290 295 300

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

305 310 315 320

Leu Arg Glu Tyr Trp Ile Asn Asp Gly Leu Tyr Gly Ser Met Asn Cys

325 330 335

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

340 345 350

Ser Asn Arg Gly Asn Val Asn Cys Gly Gly Ser Lys Thr Phe Pro Ser

355 360 365

Thr Val Phe Gly Pro Thr Cys Asp Ala Leu Asp Thr Val Leu Arg Asp

370 375 380

Tyr Gln Leu Pro Glu Leu Gln Val Asn Asp Trp Leu Val Phe Pro Asn

385 390 395 400

Met Gly Ala Tyr Thr Lys Ala Ala Gly Ser Asn Phe Asn Gly Phe Asn

405 410 415

Thr Ser Ala Ile Val Thr His Leu Ala Tyr Ala Tyr Pro Ser

420 425 430

<210> 3

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

gcggatccat ggccggccaa acagtcatcg 30

<210> 4

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

gcgagctctc agcttggata agcataagc 29

<210> 5

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

gatgcctctg ccgacagtgg tc 22

<210> 6

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

tcagcttgga taagcataag c 21

Claims

1. the sub- ornithine decarboxylase ALODC gene of small bell, it is characterised in that: the amino acid sequence of the ALODC gene coding is such as Shown in SEQ ID NO.2.

2. the sub- ornithine decarboxylase ALODC gene of small bell according to claim 1, it is characterised in that: the ALODC gene Nucleotide sequence is as shown in SEQ ID NO.1.

3. the recombinant expression carrier containing the sub- ornithine decarboxylase ALODC gene of small bell described in claim 2.

4. recombinant expression carrier according to claim 3, it is characterised in that: the recombinant expression carrier is by SEQ ID Sequence shown in NO.1 is connected into BamH I and the Sac I of pBI121 carrier and obtains.

5. the sub- ornithine decarboxylase ALODC gene of small bell as claimed in claim 1 or 2 is improving plant of Solanaceae hyoscyamine and Anisodus luridus Application in the content of alkali.

6. the recombinant expression carrier of claim 3 or 4 answering in the content for improving plant of Solanaceae hyoscyamine and hyoscine With.

7. a kind of method for improving the sub- alkaloid of small bell, it is characterised in that: by being overexpressed small bell in the sub- plant of small bell Sub- ornithine decarboxylase ALODC gene, the amino acid sequence of the ALODC gene coding is as shown in SEQ ID NO.2.

8. a kind of method for improving the sub- alkaloid of small bell according to claim 7, it is characterised in that: in the sub- plant of small bell The middle method for being overexpressed the sub- ornithine decarboxylase ALODC gene of small bell is transgenic technology, specifically: clone's sub- ornithine of small bell Decarboxylase ALODC gene, then building contains the recombinant expression carrier of the ALODC gene, then the expression vector of acquisition is turned Change Agrobacterium tumefaciems, then disseminates the sub- explant of small bell, warp with the Agrobacterium tumefaciems of the recombinant expression carrier containing AlODC gene Regeneration induction, resistance screening, PCR detection obtain and turn ornithine decarboxylase ALODC gene plant.

9. containing the plant of Solanaceae of the sub- ornithine decarboxylase ALODC gene of small bell as claimed in claim 1 or 2.

10. application according to claim 9, it is characterised in that: the plant of Solanaceae is small bell.