CN101914121A

CN101914121A - Method for preparing salidroside by utilizing RsTyrDC

Info

Publication number: CN101914121A
Application number: CN2010102619658A
Authority: CN
Inventors: 师光禄; 马兰青; 王有年; 张继星; 于寒松
Original assignee: Beijing University of Agriculture
Current assignee: Beijing University of Agriculture
Priority date: 2010-08-24
Filing date: 2010-08-24
Publication date: 2010-12-15
Anticipated expiration: 2030-08-24
Also published as: CN101914121B

Abstract

The invention discloses a method for preparing salidroside by utilizing RsTyrDC. The method for preparing the salidroside comprises the following steps: 1) introducing an encoding gene of RSTYRDC protein into a target plant and obtaining a transgenic plant; and 2) extracting the salidroside from the transgenic plant and obtaining the salidroside; and the amino acid sequence of the RSTYRDC protein is sequence 2 in a sequence table. The encoding gene of the RSTYRDC protein is introduced into the target plant through a recombinant plant expression vector; and the recombinant plant expression vector is obtained by inserting the encoding gene of the RSTYRDC protein among multiple cloning sites of the vector pBRin713. The invention provides a related technical method with novelty and practicality and an application thereof for utilizing a Sachalin rhodiola rhizome transgenic plant system for overexpression of a key enzyme for the biosynthesis route of the salidroside-tyrosine decarboxylase (RsTyrDC).

Description

Utilize RsTyrDC to prepare the method for rhodioloside

Technical field

The present invention relates to the method that a kind of RsTyrDC of utilization prepares rhodioloside.

Background technology

Radix Rhodiolae (Rhodiola sachalinensis A.Bor) has another name called storehouse page or leaf Root of Kirilow Rhodiola, it is Crassulaceae (Crassulaceae) rhodiola (Rhodiola rosea L.) plant, be perennial herb or undershrub, the normal tool meat root stock of crawling is one of rare medicinal plant in Changbai Mountain.The main active ingredient of Rhodida plant is rhodioloside (salidroside) and Jujubogenin tyrosol (tyrosol) etc., the modern pharmacology result of study show rhodioloside have tangible anti-hypoxia, cold resistance, antifatigue, radioprotective, antiviral, delay effects such as body aging, for industries such as space flight, deep-sea, desert, plateau, physical culture is a kind of environmental adaptation medicine that has development prospect, uses frequent personnel, strong brain worker, mid-aged population to have positive nourishing health function to high radiation practitioner, microcomputer simultaneously.Rhodioloside biosynthesizing final step reaction mechanism is clear, the intravital uridine diphosphoglucose based transferase of plant (UDP-glucosyltransferase, UDPGT is the synthetic rhodioloside of substrate catalysis with uridine diphosphoglucose (UDPG) and tyrosol UGTs).The biosynthesizing of tyrosol tyrosol then might derive from two kinds of different approach: the one, and tyrosol may derive from the decarboxylic reaction product of p-coumaric acid, and the p-coumaric acid derives from the initial phenylpropyl alcohol alkane pathways metabolism of phenylalanine; Another kind may be that tyrosol may derive from 4-hydroxybenzene acetaldehyde, and it comes from the tyrosine decarboxylation reaction.What is interesting is that phenylalanine and tyrosine derive from precursor compound sieve Ah acid again jointly.Because the tyrosol molecule belongs to typical simple phenolic compound, the intravital most of phenolic compound of plant derives from phenylpropyl alcohol alkanes pathways metabolism, and phenylpropyl alcohol alkanes pathways metabolism provides the carbon skeleton structure closely similar precursor compound source for the biosynthesizing of tyrosol.

Summary of the invention

An object of the present invention is to provide the method that a kind of RsTyrDC of utilization prepares rhodioloside.

Preparation provided by the invention comprises the steps: 1) the proteic encoding gene of RSTYRDC is imported the purpose plant, obtain transgenic plant; 2) from described transgenic plant, extract rhodioloside, obtain rhodioloside; The proteic aminoacid sequence of described RSTYRDC is the sequence 2 in the sequence table.

Sequence 2 is made up of 507 amino-acid residues.

The proteic encoding gene of described RSTYRDC is by the explant of the Agrobacterium tumefaciens mediated importing purpose plant of reorganization;

The proteic encoding gene of described RSTYRDC imports the explant of purpose plant by the recombinant plant expression vector.

Described reorganization agrobacterium tumefaciens is for importing the reorganization agrobacterium tumefaciens that the host Agrobacterium obtains with described recombinant plant expression vector;

Described recombinant plant expression vector is to obtain between the BgLII of the proteic encoding gene insertion of described RSTYRDC carrier pBRin713 and Spe I recognition site.

The proteic encoding gene of described RSTYRDC is imported the purpose plant, the method that obtains transgenic plant comprises the steps: to infect the explant of described purpose plant with the reorganization agrobacterium tumefaciens, carries out common cultivation, antibacterial cultivation, inducing culture, differentiation culture and root culture more successively and obtains described transgenic plant.

The described time of infection that infects the explant of described purpose plant with the reorganization agrobacterium tumefaciens is 9 minutes.

Described explant is the leaf dish.Lay the sequin that comes by punch tool from the blade and be the leaf dish, can be used for the transgenosis transfection.

Describedly cultivate used substratum altogether and be prepared as follows: in the MS substratum, add 6-benzyl aminopurine, 1-naphthylacetic acid and 2, the 4-dichlorobenzene oxygen butyl acetate, obtain the described used substratum of cultivating altogether, described 6-benzyl aminopurine is 2.0mg/L in described concentration of cultivating altogether in the used substratum, described 1-naphthylacetic acid is 0.15mg/L in described concentration of cultivating altogether in the used substratum, described 2,4 dichlorophenoxyacetic acid butyl ester is 0.5mg/L in described concentration of cultivating altogether in the used substratum;

The used substratum of described antibacterial cultivation is prepared as follows: add 6-benzyl aminopurine, 1-naphthylacetic acid and cephamycin in the MS substratum, obtain the used substratum of described antibacterial cultivation, the concentration of described 6-benzyl aminopurine in the used substratum of described antibacterial cultivation is 1.5mg/L, the concentration of described 1-naphthylacetic acid in the used substratum of described antibacterial cultivation is 0.15mg/L, and the concentration of described cephamycin in the used substratum of described antibacterial cultivation is 500mg/L;

The used substratum of described differentiation culture is prepared as follows: add 6-benzyl aminopurine, 1-naphthylacetic acid and Plant hormones regulators,gibberellins in the MS substratum, obtain the used substratum of described differentiation culture, the concentration of described 6-benzyl aminopurine in the used substratum of described differentiation culture is 1.5mg/L, the concentration of described 1-naphthylacetic acid in the used substratum of described differentiation culture is 0.05mg/L, and the concentration of described Plant hormones regulators,gibberellins in the used substratum of described differentiation culture is 0.15mg/L;

The used substratum of described root culture is prepared as follows: add 1-naphthylacetic acid and Totomycin in the MS substratum, obtain the used substratum of described root culture, the concentration of described 1-naphthylacetic acid in the used substratum of described root culture is 0.1mg/L, and the concentration of described Totomycin in the used substratum of described root culture is 1.0mg/L;

The substratum of described inducing culture is prepared as follows: add 6-benzyl aminopurine, 1-naphthylacetic acid, Totomycin and cephamycin in the MS substratum, obtain the substratum of described inducing culture, the concentration of described 6-benzyl aminopurine in the substratum of described inducing culture is 1.5mg/L, the concentration of described 1-naphthylacetic acid in the substratum of described inducing culture is 0.15mg/L, the concentration of described Totomycin in the substratum of described inducing culture is 2.5mg/L, and the concentration of described cephamycin in the substratum of described inducing culture is 500mg/L;

Every 1000ml is described to be cultivated used substratum altogether and prepare as follows: every 1000ml is described to be cultivated used substratum altogether and prepares as follows: add 2.0ml6-benzyladenine mother liquor, 0.15ml1-naphthylacetic acid mother liquor, 0.5ml 2 in the 700mlMS liquid nutrient medium, behind the 4-dichlorobenzene oxygen butyl acetate mother liquor, distilled water is settled to 1000ml, obtains the described substratum of cultivating usefulness altogether;

The used substratum of the described antibacterial cultivation of every 1000ml is prepared as follows: after adding 1.5ml 6-benzyl aminopurine mother liquor, 0.15ml1-naphthylacetic acid mother liquor, 5.0ml cephamycin mother liquor in the 700mlMS liquid nutrient medium, distilled water is settled to 1000ml, obtains the used substratum of described antibacterial cultivation;

The used substratum of the described differentiation culture of every 1000ml is prepared as follows: after adding 1.5ml6-benzyladenine mother liquor, 0.05ml1-naphthylacetic acid mother liquor, 0.15ml Plant hormones regulators,gibberellins mother liquor in the 700mlMS liquid nutrient medium, distilled water is settled to 1000ml, obtains the used substratum of described differentiation culture;

The used substratum of the described root culture of every 1000ml is prepared as follows: after adding 0.1ml1-naphthylacetic acid mother liquor, 1.0ml Totomycin mother liquor in the 700mlMS liquid nutrient medium, distilled water is settled to 1000ml, obtains the used substratum of described root culture;

The substratum of the described inducing culture of every 1000ml is prepared as follows: after adding 0.15ml 6-benzyl aminopurine mother liquor, 0.15ml1-naphthylacetic acid mother liquor, 2.5ml Totomycin mother liquor, 5.0ml cephamycin mother liquor in the 700mlMS liquid nutrient medium, distilled water is settled to 1000ml, obtains the substratum of described inducing culture;

Described 6-benzyl aminopurine mother liquor disposes as follows: add the water constant volume to 50ml after taking by weighing the NaOH aqueous solution dissolving of 50mg6-benzyladenine with 10ml1M, obtain described 6-benzyl aminopurine mother liquor;

Described 1-naphthylacetic acid mother liquor disposes as follows: add water constant volume 50ml after taking by weighing the NaOH aqueous solution dissolving of 50mg1-naphthylacetic acid with 10ml1M, obtain described 1-naphthylacetic acid mother liquor;

Described 2,4 dichlorophenoxyacetic acid butyl ester mother liquor disposes as follows: take by weighing 20mg2, the 4-dichlorobenzene oxygen butyl acetate adds water constant volume 20ml after with the 15ml anhydrous alcohol solution, obtains described 2,4 dichlorophenoxyacetic acid butyl ester mother liquor;

Described cephamycin mother liquor disposes as follows: take by weighing the 1000mg cephamycin soluble in water after, add the water constant volume to 10ml, obtain described cephamycin mother liquor;

Described Totomycin mother liquor disposes as follows: take by weighing the 1000mg Totomycin soluble in water after, add the water constant volume to 10ml, obtain described Totomycin mother liquor;

Described Plant hormones regulators,gibberellins mother liquor disposes as follows: take by weighing 10mg Plant hormones regulators,gibberellins soluble in water after, add the water constant volume to 10ml, obtain described Plant hormones regulators,gibberellins mother liquor;

The described time of cultivating altogether is 3 days, and described temperature of cultivating altogether is 25 ℃, and described the cultivation altogether is continuous illumination;

The time of described antibacterial cultivation is 5 days, and the temperature of described antibacterial cultivation is 25 ℃, and described antibacterial cultivation is continuous illumination;

The temperature of described inducing culture is 25 ℃, and described inducing culture is continuous illumination.

The time of described differentiation culture was 8 weeks, and the temperature of described differentiation culture is 25 ℃, and described differentiation culture is continuous illumination;

The time of described root culture was 6 weeks, and the temperature of described root culture is 25 ℃, and described root culture is continuous illumination.

The proteic encoding gene of described RsTyrDC is following 1) or 2) or 3):

1) sequence in the sequence table 1 from the dna molecular shown in 5 ' terminal the 69th to the 1592nd Nucleotide;

2) sequence in the sequence table 1 from the dna molecular shown in 5 ' terminal the 63rd to the 1592nd Nucleotide;

3) dna molecular shown in the sequence in the sequence table 1.

Above-mentioned sequence 1 is made up of 1715 Nucleotide, its OFR be sequence 1 from the dna molecular shown in 5 ' terminal the 69th to the 1592nd Nucleotide.

Described purpose plant is dicotyledons or monocotyledons, and described dicotyledons is preferably the Crassulaceae plant, and described Crassulaceae plant optimization is a Rhodida plant, and described Rhodida plant especially is preferably Radix Rhodiolae.

Another object of the present invention provides a kind of transgenic plant method that salidroside content improves of cultivating.

The transgenic plant method that cultivation salidroside content provided by the invention improves obtains transgenic plant for the proteic encoding gene of RsTyrDC is imported the purpose plant, and the salidroside content of described transgenic plant is higher than described purpose plant;

The proteic aminoacid sequence of described RsTyrDC is the sequence 2 in the sequence table;

The salidroside content that it is the blade of described transgenic plant that the salidroside content of described transgenic plant is higher than described purpose plant optimization is higher than the content in the described purpose plant leaf.

Of the present invention experimental results show that, the RsTyrDC gene is placed 35S promoter+Ω enhanser downstream of high efficiency plant expression vector pBRin713, transform Radix Rhodiolae by agrobacterium-mediated transformation, by the enhancement expression and the gene transformation medium optimization of potent promotor, the efficiently expressing of RsTyrDC cause in the transgenosis Radix Rhodiolae plant salidroside content contrast improve doubly 2.7 times.Content of the present invention provides related art method and the application with novelty and practicality for utilizing Radix Rhodiolae transfer-gen plant system to cross expression rhodioloside biosynthetic pathway key enzyme-tyrosine deearboxylase (RsTyrDC).

Description of drawings

Fig. 1 is the electrophoresis result of total RNA

Fig. 2 is the RT-PCR amplification

Fig. 3 is gene 5 ' RACE result

Fig. 4 is the pcr amplification of full-length cDNA

Fig. 5 is the proteic structure function domain analysis of RsTyrDC

Fig. 6 is the structure synoptic diagram of pBSRsTyrDC expression vector

Fig. 7 is that the BgLII and the Spe I double digestion of pBSRsTyrDC expression vector identified

Fig. 8 is that the PCR of pBSRsTyrDC expression vector identifies

Fig. 9 is the Radix Rhodiolae genetic transformation process (agrobacterium-mediated transformation) of RsTyrDC

Figure 10 is the PCR detected result of transgenosis Radix Rhodiolae plant

Figure 11 is the real-time fluorescence PCR detected result of transgenosis Radix Rhodiolae plant

Figure 12 is the real-time fluorescence RT-PCR detected result of part transgenosis Radix Rhodiolae

Figure 13 is that transgenosis Radix Rhodiolae plant salidroside content is measured

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

Embodiment 1, RsTyrDC gene isolation

(rapid-amplification of cDNA ends) successfully obtains 1 new tyrosine deearboxylase gene cDNA complete sequence, called after RsTyrDC from the Radix Rhodiolae separate tissue with the RACE method.

Detailed process is as follows:

1, the extraction of total RNA

The Trizol method is extracted the total RNA of Radix Rhodiolae callus, and agarose gel electrophoresis the results are shown in Figure 1.

2, the separation of Radix Rhodiolae tyrosine carboxylic acid gene (RsTyrDC) 3 ' end

Total RNA is a template with the Radix Rhodiolae callus, with Q _T(5 '-CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTT-3 ') is the reverse transcription primer, carries out the synthetic of cDNA first chain according to Promega reverse transcription test kit specification sheets.CDNA first chain with reverse transcription is a template afterwards, according to conserved regions design synthetic Block T (5 '-GAGTTCCAGCACTACCTCGAYGGNRTNGAR-3 ') degenerated primer, with the nido anchor primer Q of design ₀(5 '-CCAGTGAGCAGAGTGACG-3 ') make up and carry out the sleeve type PCR amplification, obtain a size and be about 770bp specific band (Fig. 2: M, DL-2000Marker; 1, the pcr amplification result.), expection size and design of primers position apart from other relevant TyrDC gene 3 ' end distance from conforming to.

Enzyme cut with PCR identify that correct plasmid serves Hai Shenggong company and carry out sequencing, measurement result is carried out BLAST P analyze the back discovery in the GenBank storehouse of NCBI, this result and registered plant tyrosine deearboxylase gene 3 ' end have certain homology (44%-52%), illustrate that this sequence really is Radix Rhodiolae tyrosine deearboxylase gene 3 ' end, and be a new gene.

3, the clone of RsTyrDC gene 5 ' terminal sequence

Separate 5 ' terminal sequence with reference to Invitrogen 5 ' RACE test kit specification sheets.Synthetic 35 ' the RACE experiment of cDNA sequence 5 ' the tip designs nested primers that obtain according to 3 ' RACE (Tyr5 '-1:5 '-CGCTAGAGATTGGATTAGG-3 '; Tyr5 '-2:5 '-CACATGAAGCAGCAATCGAG-3 '; Tyr5 '-3:5 '-CGTTCATGCTGATCGAATCG-3), after the sleeve type PCR amplification, specific band (Fig. 3: M, DL2000marker occur at the 950bp place; 1, the pcr amplification result.), this band through reclaim, the T carrier connects, transform, identify, order-checking, find that this sequence and the cDNA sequence 5 ' end of 3 ' RACE acquisition have one section tumor-necrosis factor glycoproteins after analyzing sequencing result, simultaneously higher with other plant TyrDC homology, illustrate that this sequence is the 5 ' terminal sequence of this cDNA.

4, the clone of RsTyrDC full length gene cDNA

Utilize DNAman software that isolating cDNA3 ' terminal sequence and 5 ' terminal sequence sequencing result are carried out the electronics merging, obtain the sequence (sequence 1 of sequence table) of total length 1715bp.At this sequence ORF two ends designs total length primer (TyrQC5 ' in the table 1-1 and TyrQC3 '-1), be template with cDNA first chain of reverse transcription, it is (shown in Figure 4: M, DL2000marker for the PCR product of 1524bp to amplify size; 1, the RsTyrDC full length gene cDNA fragment of amplification), with the order-checking after recovery, T carrier connect, transform, identify of this PCR product, this PCR product has the 63-1592 position nucleotide sequence of sequence 1 in the sequence table, with the unnamed gene of this PCR product is RsTyrDC, the OFR of this gene is the 69-1592 position nucleotide sequence of sequence 1 in the sequence table, and the albumen called after RsTyrDC of this genes encoding, this proteic aminoacid sequence are the sequence 2 in the sequence table.

Table 1 RsTyrDC gene cDNA total length primer (restriction enzyme site is used for the structure of plant expression vector)

5, the bioinformatic analysis of RsTyrDC gene

The full length cDNA sequence of the gene that obtains is submitted GenBank (sequential reception number: DQ471943) by the Bankit instrument of NCBI.Utilize DNAman software that the full length cDNA sequence of the RsTyrDC gene of acquisition is analyzed (sequence 1).The RsTyrDCcDNA total length 1715bp that is obtained, comprising the complete open reading frame of 1524bp (open reading frame, ORF (sequence 1 is from 5 ' terminal 69-1592 position Nucleotide), infer 508 amino acid whose polypeptide of coding, molecular weight (MW) is 56.88kDa, and iso-electric point (pI) value is 6.15; 5 ' non-district (5 ' UTR) and 123bp 3 ' non-district (3 ' UTR) and the poly-A tail of translating of translating that contains 67bp simultaneously.Before 5 ' the UTR district initiator codon 1 terminator codon is arranged, and match with the coding region reading frame, the cDNA that illustrative experiment obtained is a total length.

6, (known gene thinks that to write structural domain good in the proteic structure function domain analysis of RsTyrDC.)

(http://www.ncbi.nlm.nih.gov/BLAST/) carries out domain analyses (Fig. 5) with the BLAST server, and the result shows and contains complete TyrDC characteristic structural domain in 508 aminoacid sequences of RsTyrDC.

The RsTyrDC gene can obtain by above method, but also synthetic.

Embodiment 2, commentaries on classics RsTyrDC Radix Rhodiolae plant

CDNA shown in the sequence 1 in the artificial synthesized sequence table.

1, the structure of pBSRsTyrDC expression vector

CDNA with above-mentioned synthetic is a template, uses primer TyrQC5 '-1 and TyrQC3 '-1 to carry out pcr amplification, the small segment that the PCR product that obtains obtains after cutting with BgLII and Spe I enzyme, insert carrier pBRin713 (Ma, L.Q., Liu, B.Y., Gao, D.Y., Pang, X.B., Lu, S.Y., Yu, H.S., Wang, H., Yan, F., Li, Z.Q., Li, Y.F., Ye, H.C., 2007.Molecular cloning and overexpression of a novel UDP-glucosyltransferase elevating salidroside levels in Rhodiola sachalinensis.Plant Cell Rep.26,989-999; The public can obtain from Beijing Agricultural College.) between the BgLII and Spe I recognition site of carrier, make up recombinant vectors, the recombinant vectors that builds is cut with PCR and identified through transforming, carry out after the screening enzyme, identify result such as Fig. 7 (M, DL15000+2000Marker with BgLII and Spe I double digestion; 1-2, positive plasmid.) shown in, as seen from the figure, obtain the dna fragmentation of about 1.5Kb; With RsTyrDC gene specific primer TyrQC3 '-1 and 35S-UP primer (5 '-TGATATCTCCACTGACGTAAGGGATG-3 ') is primer, is that template is carried out PCR with the recombinant vectors, PCR qualification result such as Fig. 8 (M, DL2000Marker; 1-8, positive plasmid.) shown in, obtain the dna fragmentation of about 1.6Kb; Enzyme is cut the recombinant vectors called after pBSRsTyrDC of the dna fragmentation that the dna fragmentation that obtains about 1.5Kb and PCR obtain about 1.6Kb, and its building process as shown in Figure 6.PBSRsTyrDC is sent to order-checking, and the result proves further that for pBSRsTyrDC inserts sequence 1 from 5 ' terminal 69-1592 position Nucleotide (the OFR frame of RsTyrDC gene) between the BgLII of pBRin713 and Spe I recognition site this vector construction is correct.

The recombinant vectors pBSRsTyrDC that builds is imported Agrobacterium (Agrobacterium tumefaciens) EHA105 (Ma, L.Q., Liu by freeze-thaw method, B.Y., Gao, D.Y., Pang, X.B., Lu, S.Y., Yu, H.S., Wang, H., Yan, F., Li, Z.Q., Li, Y.F., Ye, H.C., 2007.Molecular cloning and overexpression of a novel UDP-glucosyltransferase elevating salidroside levels in Rhodiola sachalinensis.Plant Cell Rep.26,989-999; The public can obtain from Beijing Agricultural College.) in, obtain the bacterium of recombinating, identify through BgLII and Spe I double digestion behind the extraction plasmid, obtain the positive reorganization of the reorganization bacterium bacterium of the dna fragmentation of about 1.5Kb, called after EHA105/pBSRsTyrDC.

2, change the acquisition of RsTyrDC Radix Rhodiolae plant

Table 2 Radix Rhodiolae agrobacterium mediation converted is as follows with substratum

Be seeded on the solid medium (YEB) EHA105/pBSRsTyrDC of above-mentioned acquisition streak culture, activate 3 times down in 27 ℃, single bacterium colony on the picking flat board, be seeded in 50ml YEB liquid nutrient medium, in 27 ℃, 120rpm min, dark concussion is down cultivated, and subculture 3 times is cultivated EHA105/pBSRsTyrDC to OD ₆₀₀Be to be used to infect Radix Rhodiolae (Rhodiola sachalinensis A.Bor at 0.5 o'clock; Ma, L.Q., Liu, B.Y., Gao, D.Y., Pang, X.B., Lu, S.Y., Yu, H.S., Wang, H., Yan, F., Li, Z.Q., Li, Y.F., Ye, H.C., 2007.Molecular cloning and overexpression of a novel UDP-glucosyltransferase elevating salidroside levels in Rhodiola sachalinensis.Plant Cell Rep.26,989-999; The public can obtain from Beijing Agricultural College.) the leaf dish, time of infection is 9 minutes.

The Radix Rhodiolae leaf dish that Agrobacterium EHA105/pBSRsTyrDC was infected places on the common substratum, after cultivating 3 days altogether under 25 ℃ of illumination conditions, transfer to only to contain and suppress Agrobacterium growth microbiotic (cephamycin, Cef 500mg/L) carries out 5 days antibacterial cultivation on the antibacterial substratum, culture temperature is 25 ℃, continuous illumination; To receive on the substratum of inducing culture through the explant of antibacterial cultivation, under 25 ℃ of continuous illumination conditions, inducing culture of 3 all subcultures obtains callus.After treating that callus forms, a part of callus is received on the division culture medium under 25 ℃ of continuous illumination conditions an evoking adventive bud differentiation of 4 all subcultures.After about 8 weeks, produce many indefinite buds (Fig. 9 B callus is being selected differentiation and seedling emergence on the substratum) around can observing callus, treat to downcut when indefinite bud grows to the 2cm left and right sides, move to and contain Totomycin (Hyg) 1.0mg/L, carry out root culture (Fig. 9 C, indefinite bud is emerged) on the root media of cephamycin 500mg/L on the selection substratum, incubation time was 6 weeks, culture temperature is 25 ℃, and continuous illumination obtains 20 strain T0 for changeing RsTyrDC Radix Rhodiolae plant.

The composition and the compound method of described MS substratum are as follows:

Table 1 is a MS substratum mother liquor

Table 2 is a MS substratum compound method (1000ml)

Mother liquor

In a large number

Trace

Calcium salt

Molysite

Organic I

Organic II

Consumption

20ml

10ml

Compound method: place the distilled water about 200ml in the big graduated cylinder earlier, with adding in the big graduated cylinder after the above-mentioned mother liquor weighing, add 400--500ml distilled water again, constant volume is poured in the triangular flask to 1000ml after the adding 30 gram sucrose dissolved.With PH instrumentation pH value to 5.80--5.85.Melt bottle sterilization after adding 7--7.5g agar.

Described 1/2MS ₀The preparation method of substratum and above-mentioned MS substratum are basic identical, unique different be that concentration with a large amount of, the trace in the MS substratum, calcium salt, molysite, organic I and organic II all reduces by half.

3, T0 is for the evaluation of changeing RsTyrDC Radix Rhodiolae plant

1) Radix Rhodiolae transfer-gen plant PCR and real-time fluorescence PCR are identified

Is template to obtain 20 strain T0 in the above-mentioned steps 2 for the nuclear gene group DNA that the blade that changes RsTyrDC Radix Rhodiolae plant (the positive seedling of Hyg) extracts, and PCR that carries out and real-time fluorescence PCR detect.Interference for fear of native gene, one of PCR primer is the 3 ' primer (TyrQC3 '-1) of target gene, another primer is one section nucleotide sequence (35S-UP corresponding to CaMV35S promotor upstream, 5 '-TGATATCTCCACTGACGTAAGGGATG-3 '), this primer does not have complementary sequence on wild-type Radix Rhodiolae genome.Figure 10 (PCK: plasmid pBSRsTyrDC; RCK: wild-type; 1-9:T0 is for changeing RsTyrDC Radix Rhodiolae plant) be the PCR detected result that transforms the part transgenosis Radix Rhodiolae plant of RsTyrDC, the result shows that positive control is identical for changeing the dna segment that amplification obtains in the RsTyrDC Radix Rhodiolae plant with T0, for about 1.6Kb, negative control (wild-type Radix Rhodiolae) is this amplified production not, illustrates to obtain the positive T0 of 20 strains for changeing RsTyrDC Radix Rhodiolae plant.

The result of real-time fluorescence PCR such as Figure 11 (PCK: plasmid pBSRsTyrDC; RCK: wild-type; TGP: positive T0 is for changeing RsTyrDC Radix Rhodiolae plant; BCK1,2: blank .) shown in.T0 is identical with positive control for changeing RsTyrDC Radix Rhodiolae plant, has amplified production to generate, and all obtain nearly " S " type curve, and wild-type plant and blank 1,2 unanimity does not have amplified production to generate, and obtain being nearly horizontal linear.

PCR and real-time fluorescence PCR qualification result explanation RsTyrDC gene have been incorporated in the Radix Rhodiolae genomic dna.

2) Radix Rhodiolae transfer-gen plant real-time fluorescence RT-PCR is identified

In order to understand fully that RsTyrDC at the transcriptional level expression, has carried out real-time fluorescence RT-PCR.Material adopts the positive T0 of above-mentioned evaluation for changeing RsTyrDC Radix Rhodiolae plant leaf and corresponding wild type plant leaf, extracts its RNA, carries out real-time fluorescence PCR.If positive plasmid and wild-type and blank 1,2 (blank 1-is template with the sterilized water, when blank 2-extracts RNA, with the plant tissue after the sterilized water replacement grinding).

(contrast of PCK-positive plasmid, the positive T0 of TGP-is for changeing RsTyrDC Radix Rhodiolae plant, BCK1,2 blanks, RCK-wild-type plant to the results are shown in Figure 12.), can judge by figure, RsTyrDC obtains in transfer-gen plant and the consistent result of positive plasmid contrast, illustrates at transcriptional level and expresses.Wild-type and blank 1,2 unanimity.

Comprehensive aforementioned Molecular Detection illustrates that the RsTyrDC gene not only successfully is incorporated in the Radix Rhodiolae genomic dna, and obtains the transcriptional level expression.

Adopting uses the same method changes empty carrier pBRin713 in the Radix Rhodiolae over to, obtains T0 for changeing pBRin713 Radix Rhodiolae plant.

Adopting uses the same method imports pCAMBIA1301-RsTyrDC in the Radix Rhodiolae, obtains to change pCAMBIA1301-RsTyrDC Radix Rhodiolae plant.

PCAMBIA1301 is at (Ge Y, Cheng X, Hopkins A, Wang ZY.Generation of transgenic Lolium temulentum plants by Agrobacterium tumefaciens-mediated transformation.Plant Cell Rep.200726 (6): 783-789) record.

Construction process and the pBSRsTyrDC of pCAMBIA1301-RsTyrDC are basic identical, obtain for sequence in the sequence table 1 is inserted between the BgLII of pCAMBIA1301 carrier and Bst EII recognition site from 5 ' terminal 69-1592 position Nucleotide.

Embodiment 3, the HPLC mensuration of changeing RsTyrDC Radix Rhodiolae plant salidroside content

Respectively the positive T0 that obtained by embodiment 2 has been carried out high-performance liquid chromatogram determination for changeing RsTyrDC Radix Rhodiolae plant, T0 for the salidroside content in the blade of the commentaries on classics pCAMBIA1301-RsTyrDC Radix Rhodiolae plant that changes pBRin713 Radix Rhodiolae plant, wild-type contrast (Radix Rhodiolae) and obtained by embodiment 2, concrete grammar is as follows:

From the positive commentaries on classics RsTyrDC Radix Rhodiolae plant leaf that 2 steps by embodiment 2 obtain, extract rhodioloside, concrete extracting method is as follows: with the Radix Rhodiolae rotaring gene plant blade in baking oven 60 ℃ dry to constant weight, pulverize the back and cross 80 order mesh screens, accurately take by weighing the methyl alcohol 8ml of 0.8g sample adding 100%, supersound extraction 30min, hatch 30min (ultrasonic power 100w, time 30min) in 60 ℃ afterwards,, collect supernatant in the centrifugal 15min of room temperature 11000g; Precipitation is extracted once with 50% methanol aqueous solution again, after supernatant is merged, crosses 0.45 μ m filter membrane.

Get solution after the filtration and carry out HPLC and detect, testing conditions is: chromatographic column: Hypersyl ODS post, 5mm, 4.6 * 150mm; Moving phase: water-methanol-acetonitrile (70: 25: 5, v/v/v); Flow velocity: 0.8ml/min; Detect wavelength 275nm, bandwidth 10nm, reference wavelength 400nm, bandwidth 80nm; External standard method is quantitative.

Rhodioloside standard substance (Shanghai Tongtian Biotechnology Co., Ltd., Catalog E-0069), retention time is 5.72 minutes.The retention time of the rhodioloside in the wild-type plant contrast that HPLC detects is 5.72 minutes, and the retention time of changeing the rhodioloside in the RsTyrDC Radix Rhodiolae plant is: 5.72 minutes.

The experiment triplicate, results averaged.

Result such as Figure 13 (A: wild-type plant, B: positive T0 is for changeing RsTyrDC Radix Rhodiolae plant) shown in, the result shows: the salidroside content in the wild-type plant leaf is the 0.92mg/g dry weight, salidroside content is the 3.4mg/g dry weight to positive T0 in the RsTyrDC Radix Rhodiolae plant leaf for changeing, and positive T0 improves 2.7 times for the salidroside content that changes in the RsTyrDC Radix Rhodiolae plant leaf than the wild-type contrast.And the rhodioside content that changes in the pCAMBIA1301-RsTyrDC Radix Rhodiolae blade is 1.73mg/g DW, far below changeing RsTyrDC Radix Rhodiolae blade (using the pBRin713 carrier).

T0 does not have significant difference for the result who changes pBRin713 empty carrier Radix Rhodiolae plant and wild-type contrast (Radix Rhodiolae).

Claims

1. a method for preparing rhodioloside comprises the steps: 1) the proteic encoding gene of RSTYRDC is imported the purpose plant, obtain transgenic plant; 2) from described transgenic plant, extract rhodioloside, obtain rhodioloside; The proteic aminoacid sequence of described RSTYRDC is the sequence 2 in the sequence table.

2. method according to claim 1 is characterized in that:

3. method according to claim 1 and 2 is characterized in that:

Described recombinant plant expression vector is for obtaining between the multiple clone site of the proteic encoding gene of described RSTYRDC being inserted carrier pBRin713.

4. according to arbitrary described method among the claim 1-3, it is characterized in that: the proteic encoding gene of described RSTYRDC is imported the purpose plant, the method that obtains transgenic plant comprises the steps: to infect the explant of described purpose plant with the reorganization agrobacterium tumefaciens, carries out common cultivation, antibacterial cultivation, inducing culture, differentiation culture and root culture more successively and obtains described transgenic plant.

5. according to arbitrary described method among the claim 1-4, it is characterized in that:

6. according to arbitrary described method among the claim 1-5, it is characterized in that: described explant is the leaf dish.

7. according to arbitrary described method among the claim 1-6, it is characterized in that:

8. according to arbitrary described method among the claim 1-7, it is characterized in that:

The proteic encoding gene of described RsTyrDC is following 1) or 2) or 3):

3) dna molecular shown in the sequence in the sequence table 1.

9. according to arbitrary described method among the claim 1-8, it is characterized in that:

10. cultivate the transgenic plant method that salidroside content improves for one kind, obtain transgenic plant for the proteic encoding gene of RsTyrDC is imported the purpose plant, the salidroside content of described transgenic plant is higher than described purpose plant;