CN101928308A

CN101928308A - Method for preparing salidroside by using RsTyrDC

Info

Publication number: CN101928308A
Application number: CN2010102619643A
Authority: CN
Inventors: 师光禄; 王有年; 马兰青; 于寒松; 张继星
Original assignee: Beijing University of Agriculture
Current assignee: Beijing University of Agriculture
Priority date: 2010-08-24
Filing date: 2010-08-24
Publication date: 2010-12-29

Abstract

The invention discloses a method for preparing salidroside by using RsTyrDC. The method for preparing the salidroside comprises the following steps of: 1) importing coding genes of RSTYRDC protein into an explant of a target plant to obtain transgenic callus; and 2) extracting salidroside from the transgenic callus to obtain the salidroside, wherein the amino acid sequence of the RSTYRDC protein is a sequence 2 in a sequence table. The invention provides the innovative and practical related technical method for systematically over-pressing the biosynthetic pathway key enzyme, namely tyrosine decarboxylase (RSTyrDC) of the salidroside by using the transgenic callus of the rhodiola sachalinensis and application.

Description

A kind of RsTyrDC of utilization prepares 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

The purpose of this invention is to provide the method that a kind of RsTyrDC of utilization prepares rhodioloside.

A kind of method for preparing rhodioloside provided by the invention comprises the steps: 1) with the explant of the proteic encoding gene importing of RSTYRDC purpose plant, obtain transgenic callus; 2) from described transgenic callus, extract rhodioloside, obtain rhodioloside; The proteic aminoacid sequence of described RSTYRDC is the sequence 2 in the sequence table.

Above-mentioned 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 tumefaciens 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 explant of purpose plant, the method that obtains transgenic calli comprises the steps: to infect the explant of described purpose plant with the reorganization agrobacterium tumefaciens, carries out common cultivation, antibacterial cultivation, inducing culture, resistance screening cultivation and subculture resistance screening more successively and cultivates.

Describedly infect in the step of explant of described purpose plant with the reorganization agrobacterium tumefaciens, the described time of infecting 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 1.5mg/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 1.0mg/L, the concentration of described 1-naphthylacetic acid in the used substratum of described antibacterial cultivation is 0.1mg/L, and the concentration of described cephamycin in the used substratum of described antibacterial cultivation is 500mg/L;

The used substratum of described inducing culture is prepared as follows: add 6-benzyl aminopurine in the MS substratum, the 1-naphthylacetic acid, Totomycin and cephamycin, obtain the used substratum of described inducing culture, the concentration of described 6-benzyl aminopurine in the used substratum of described inducing culture is 1.0mg/L, the concentration of described 1-naphthylacetic acid in the used substratum of described inducing culture is 0.1mg/L, the concentration of described Totomycin in the used substratum of described inducing culture is 2.5mg/L, and the concentration of described cephamycin in the used substratum of described inducing culture is 500mg/L;

The substratum that described resistance screening is cultivated is prepared as follows: add 6-benzyl aminopurine in the MS substratum, the 1-naphthylacetic acid, Totomycin and cephamycin, obtain the substratum that described resistance screening is cultivated, the concentration of described 6-benzyl aminopurine in the substratum that described resistance screening is cultivated is 1.0mg/L, the concentration of described 1-naphthylacetic acid in the substratum that described resistance screening is cultivated is 0.1mg/L, the concentration of described Totomycin in the substratum that described resistance screening is cultivated is 30mg/L, and described cephamycin is 500mg/L in the concentration of the substratum that described resistance screening is cultivated;

Every 1000ml is described to be cultivated used substratum altogether and prepares as follows: add 1.5ml6-benzyladenine mother liquor, 0.15ml1-naphthylacetic acid mother liquor and 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 1.0ml6-benzyladenine mother liquor, 0.1ml1-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 inducing culture of every 1000ml is prepared as follows: after adding 1.0ml 6-benzyl aminopurine mother liquor, 0.1ml 1-naphthylacetic acid mother liquor, 0.25ml Totomycin 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 inducing culture;

The substratum that the described resistance screening of every 1000ml is cultivated is prepared as follows: after adding 1.0ml 6-benzyl aminopurine mother liquor, 0.1ml 1-naphthylacetic acid mother liquor, 0.3ml Totomycin mother liquor, 5.0ml cephamycin mother liquor in the 700mlMS liquid nutrient medium, distilled water is settled to 1000ml, obtains the substratum that described resistance screening is cultivated;

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 time of described inducing culture was 3 weeks, and the temperature of described inducing culture is 25 ℃, and described inducing culture is continuous illumination;

The time that described resistance screening is cultivated was 10 weeks, and the temperature that described resistance screening is cultivated is 25 ℃, and described resistance screening is cultivated and is continuous illumination.

Described subculture resistance screening cultured method is for cultivating according to step shown in following I and the II successively:

I: continuous illumination, 25 ℃, subculture is cultivated for 3 times in following substratum, and the used substratum of I is prepared as follows: add 6-benzyl aminopurine, 1-naphthylacetic acid, Totomycin and cephamycin in the MS substratum, obtain the used substratum of I; The concentration of described 6-benzyl aminopurine in the used substratum of I is 1.0mg/L, the concentration of described 1-naphthylacetic acid in the used substratum of I is 0.1mg/L, the concentration of described Totomycin in the used substratum of I is 20mg/L, and the concentration of described cephamycin in the used substratum of I is 500mg/L;

II: continuous illumination, 25 ℃, subculture is cultivated for 2 times in following substratum, and the used substratum of II is prepared as follows: add 6-benzyl aminopurine, 1-naphthylacetic acid, Totomycin and cephamycin in the MS substratum, obtain the used substratum of II; The concentration of described 6-benzyl aminopurine in the used substratum of II is 1.0mg/L, the concentration of described 1-naphthylacetic acid in the used substratum of II is 0.1mg/L, the concentration of described Totomycin in the used substratum of II is 10mg/L, and the concentration of described cephamycin in the used substratum of II is 500mg/L.

The used substratum of I: 1000ml is prepared as follows: after adding 1.0ml6-benzyladenine mother liquor, 0.1ml1-naphthylacetic acid mother liquor, 0.2ml Totomycin 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 I; In the described step I, described subculture 3 times carries out subculture according to every 10-13 days interval;

The used substratum of II: 1000ml is prepared as follows: after adding 1.0ml6-benzyladenine mother liquor, 0.1ml1-naphthylacetic acid mother liquor, 1.0ml Totomycin 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 II; In the described step II, described subculture 2 times carries out subculture according to per 15 days interval.

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 after the 1000mg cephamycin is dissolved in the water, add the water constant volume to 10ml, obtain described cephamycin mother liquor;

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

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) 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.

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, salidroside content improves doubly 4.3 times than the wild-type contrast in the transgenosis Radix Rhodiolae callus of acquisition.Content of the present invention provides related art method and the application with novelty and practicality for utilizing Radix Rhodiolae transgenic calli 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 influence of Totomycin to the leaf growth state

Figure 10 is the Radix Rhodiolae genetic transformation process (agrobacterium-mediated transformation) of RsTyrDC

Figure 11 is the PCR detected result of part transgenosis Radix Rhodiolae callus

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

Figure 13 is the real-time fluorescence RT-PCR detected result of part transgenosis Radix Rhodiolae

Figure 14 is that transgenosis Radix Rhodiolae callus 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 5: 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, 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 callus

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 fragment that the PCR product that obtains obtains after cutting with BgLII and Spe I enzyme, be inserted into 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, will build recombinant vectors and cut with PCR and identify 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, DL2000 Marker; 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 sends to order-checking with high efficiency plant expression vector, the result proves further that for this carrier is to insert 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 expression vector 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, obtaining the bacterium of recombinating, the bacterium of should recombinating is identified through BgLII and Spe I double digestion after extracting plasmid, obtains 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 callus

The selected marker resistance screening is the key that obtains transgenic plant material.If screening pressure is too high, can cause the transgenic line growth conditions low even dead; Otherwise a large amount of false positive materials then can appear, for follow-up screening operation increases difficulty.Contain hygromycin phosphotransferase gene Hpt in the plant expression vector that this experiment is adopted and to decompose Totomycin in the substratum.The Totomycin of different concns (Hyg) is handled Fig. 9 is seen in the influence of Radix Rhodiolae leaf explant.When Totomycin concentration was 0mg/L, the explant growth was vigorous; When Totomycin concentration is 5mg/L, the explant decreased growth, lethality rate reaches 50.17%; When Totomycin concentration was 10mg/L, lethality rate was up to 93.0%; When Totomycin concentration was 15mg/L, blade was all dead.,, when reaching 10mg/L, Hyg concentration can suppress fully Totomycin susceptibility result of experiment from the Radix Rhodiolae blade from the formation of blade evoked callus.If select but explant experiences high pressure at the very start, may cause the death of transformant.So this experiment determines that transgenosis initial stage antibiotic-screening concentration Hyg is 5mg/L.For obtaining positive transgenosis resistant calli, be that 5mg/L acquisition raised growth state very screens in transgenosis initial stage Totomycin concentration, obtain the ideal experimental result.

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 treated the OD of EHA105/pBSRsTyrDC 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 (the Radix Rhodiolae leaf dish that Figure 10 A infected Agrobacterium places on the common substratum) 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 C ef carries out 5 days antibacterial cultivation on antibacterial culture medium 500mg/L); To receive through the explant of antibacterial cultivation and contain on Totomycin Hyg2.5mg/L, the Cef500mg/L inducing culture substratum, under 25 ℃ of continuous illumination conditions, 3 all subcultures once carry out inducing culture (Figure 10 B transforms the leaf dish and selecting to produce callus on the substratum).After treating that callus that inducing culture obtains forms, a part of callus is received on the resistance screening culture medium, carried out resistance screening cultivation screening transgenosis resistant calli (Figure 10 C callus places high the selection to press on the substratum) under high density Totomycin (30mg/L) condition; Being taken at the callus that survives on the resistance screening culture medium takes to reduce gradually the mode of Totomycin concentration and carries out the subculture resistance screening shown in I and the II successively and cultivate, be specially behind subculture on the Totomycin 20mg/L substratum 3 times, changing on the substratum of Totomycin 10mg/L subculture over to preserves and cultivates, form the good resistant calli (kanamycin-resistant callus tissue that Figure 10 D filters out) of growth conditions, carry out Molecular Detection and index determining as changeing RsTyrDC Radix Rhodiolae callus.

The used substratum of the described inducing culture of every 1000ml is prepared as follows: after adding 1.0ml 6-benzyl aminopurine mother liquor, 0.1ml1-naphthalene second enzyme mother liquor, 0.25ml Totomycin 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 inducing culture;

The time of described inducing culture is 2-3 week, and the temperature of described inducing culture is 25 ℃, and described inducing culture is continuous illumination;

The time that described resistance screening is cultivated was 10 weeks, and the temperature that described resistance screening is cultivated is 25 ℃, and described resistance screening is cultivated and is continuous illumination;

The used substratum of I: 1000ml is prepared as follows: after adding 1.0ml6-benzyladenine mother liquor, 0.1ml1-naphthylacetic acid mother liquor, 0.2ml Totomycin 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 I; Incubation time is 40d, subculture 3 times, 25 ℃, continuous illumination;

The used substratum of II: 1000ml is prepared as follows: after adding 1.0ml6 benzyladenine mother liquor, 0.1ml1-naphthylacetic acid mother liquor, 1.0ml Totomycin 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 II; Incubation time is 30d, every 15d subculture 1 time, 25 ℃, continuous illumination;

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 10ml 1M, obtain described 1-naphthylacetic acid mother liquor;

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.

3, change the Molecular Identification of RsTyrDC Radix Rhodiolae callus

To obtain changeing RsTyrDC Radix Rhodiolae callus (the positive callus of Totomycin) in the above-mentioned steps 2 is material, and the extraction genomic dna is a template, carries out PCR and detects.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 CaMV 35S promoter upstream, 5 '-TGATATCTCCACTGACGTAAGGGATG-3 '), this primer does not have complementary sequence on wild-type Radix Rhodiolae genome.Figure 11 is the PCR detected result (PCK: plasmid pCARsTyrDC of the part transgenic calli of conversion RsTyrDC; RCK: wild-type; 1-15: part is changeed RsTyrDC Radix Rhodiolae callus.), positive control is a template with plasmid pBSRsTyrDC.The result shows that to use primer TyrQC3 '-1 and the 35S-UP dna segment that amplification obtains from change RsTyrDC Radix Rhodiolae callus identical with positive control (PCK), for about 1.6Kb, negative control (wild-type Radix Rhodiolae, RCK) this amplified production not.The PCR detected result is all positive in 25 commentaries on classics RsTyrDC Radix Rhodiolae callus systems detecting.The efficient height of high density hygromycin selection is described.

Based on transgenic line in for some time with the Agrobacterium syntrophism, get a part of callus induce, screening, subculture added cephamycin (Cef 500mg/L) in 5 months always and obtain to take off the bacterium transgenic calli in the experimentation, stop to take off bacterium (stopping to add cephamycin C ef 500mg/L) another part 4 weeks before detection and obtain to stop to take off the bacterium transgenic calli, stop to take off the regeneration that does not have Agrobacterium in the material culturing process of bacterium.The genomic dna that extraction takes off the bacterium transgenic calli, stop to take off bacterium transgenic calli and wild-type callus is a template, carries out real-time fluorescence PCR.Primer is TyrQC3 '-1 and 35S-UP, positive control is a template with plasmid pBSRsTyrDC, blank (be template with the sterilized water when blank 1-real-time fluorescence PCR is identified, when blank 2 extracts DNA, RNA, the plant tissue after replacing grinding with sterilized water) (as follows).As Figure 12 (PCK: plasmid pCARsTyrDC; DBTC: taking off bacterium changes RsTyrDC Radix Rhodiolae callus, ODBTC: stop to take off bacterium and change RsTyrDC Radix Rhodiolae callus, RCK: wild-type callus, the BCK1:2-blank) shown in, take off bacterium and not take off bacterium transgenosis callus identical with positive control, there is amplified production to generate, all obtains nearly " S " type curve, illustrate that the cDNA of RsTyrDC really has been incorporated among the Radix Rhodiolae nuclear gene group DNA.And wild-type callus and blank 1,2 unanimity do not have amplified production to generate, and obtain being nearly horizontal linear.

In order to understand fully that RsTyrDC at the transcriptional level expression, has carried out real-time fluorescence RT-PCR.Extract the RNA of the positive RsTyrDC of commentaries on classics Radix Rhodiolae callus and the RNA of wild-type callus, obtain the cDNA of positive RsTyrDC Radix Rhodiolae callus cDNA of commentaries on classics and wild-type callus respectively through reverse transcription, be template with cDNA separately respectively, primer is TyrQC3 '-1 and 35S-UP, carries out real-time fluorescence PCR.If positive plasmid and wild-type and blank 1,2 the results are shown in Figure 13 (PCK: positive plasmid contrast, TPC: positive RsTyrDC Radix Rhodiolae callus, BCK1:2-blank, the RCK: the wild-type callus) of changeing.Can be judged by figure, the positive commentaries on classics obtains in the RsTyrDC Radix Rhodiolae callus and the consistent result of positive plasmid contrast, illustrates in transcriptional level and expresses.

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 callus over to, obtains to change pBRin713 Radix Rhodiolae callus.

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

PCAMBIA1301-RsTyrDC 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, the public can obtain from Beijing Agricultural College.) 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 salidroside content in the RsTyrDC Radix Rhodiolae callus

To be changeed RsTyrDC Radix Rhodiolae callus, wild-type contrast (Radix Rhodiolae callus), carried out high-performance liquid chromatogram determination by the commentaries on classics pBRin713 Radix Rhodiolae callus of embodiment 2 acquisitions with by the salidroside content in the commentaries on classics pCAMBIA1301-RsTyrDC Radix Rhodiolae callus of embodiment 2 acquisitions by the positive that embodiment 2 obtains, concrete grammar be as follows:

From the positive commentaries on classics RsTyrDC Radix Rhodiolae callus that 2 steps by embodiment 2 obtain, extract rhodioloside, concrete extracting method is as follows: with the Radix Rhodiolae transgenic calli in baking oven 60 ℃ dry to constant weight, pulverize the back and cross 80 order mesh screens, accurately take by weighing the 0.8g sample and add methyl alcohol 8ml, 30min (ultrasonic power 100w, time 30min) is extracted in excusing from death, hatch 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 5.61min.The retention time of the rhodioloside in the wild-type callus contrast that HPLC detects is 5.61min, and the retention time of changeing the rhodioloside in the RsTyrDC Radix Rhodiolae callus is 5.61min.

The experiment triplicate, results averaged.

Rhodioloside sample retention time is 5.61min.Result such as Figure 14 (A: callus, B: change pBRin713 callus C: change RsTyrDC Radix Rhodiolae callus; ) shown in, as seen from the figure: the salidroside content in the wild-type callus is the 1.55mg/g dry weight, the salidroside content that changes in the RsTyrDC Radix Rhodiolae callus is the 8.22mg/g dry weight, the salidroside content that changes in the RsTyrDC Radix Rhodiolae callus improves 4.3 times than the wild-type contrast, the conversion of RsTyrDC has obviously improved the accumulating level of rhodioloside, has direct using value.And the rhodioside content that changes in the pCAMBIA1301-RsTyrDC Radix Rhodiolae callus is 3.77mg/g DW, far below changeing RsTyrDC Radix Rhodiolae callus (using the pBRin713 carrier).

The result who changes the contrast of pBRin713 Radix Rhodiolae callus and wild-type callus does not have significant difference.

Claims

1. a method for preparing rhodioloside comprises the steps: 1) with the explant of the proteic encoding gene importing of RSTYRDC purpose plant, obtain transgenic callus; 2) from described transgenic callus, 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 explant that the proteic encoding gene of described RSTYRDC is imported the purpose plant, the method that obtains transgenic calli comprises the steps: to infect the explant of described purpose plant with the reorganization agrobacterium tumefaciens, carries out common cultivation, antibacterial cultivation, inducing culture, resistance screening cultivation and subculture resistance screening more successively and cultivates.

5. according to arbitrary described method among the claim 1-4, it is characterized in that: describedly infect in the step of explant of described purpose plant with the reorganization agrobacterium tumefaciens, the described time of infecting is 9 minutes.

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:

In the described step I, described subculture 3 times carries out subculture according to every 10-13 days interval;

In the described step II, described subculture 2 times carries out subculture according to per 15 days interval.

9. according to arbitrary described method among the claim 1-8, it is characterized in that: the proteic encoding gene of described RsTyrDC is following 1) or 2) or 3):

3) sequence in the sequence table 1.

10. according to the arbitrary described method of claim 1-9, it is characterized in that: described purpose plant is dicotyledons or monocotyledons, described dicotyledons is preferably the Crassulaceae plant, described Crassulaceae plant optimization is a Rhodida plant, and described Rhodida plant especially is preferably Radix Rhodiolae.