CN101914122A

CN101914122A - Method for preparing salidroside by utilizing UGT72B14

Info

Publication number: CN101914122A
Application number: CN 201010261967
Authority: CN
Inventors: 马兰青; 王有年; 师光禄; 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

Abstract

The invention discloses a method for preparing salidroside by utilizing UGT72B14. The method provided by the invention includes the following steps: 1) UGT72B14 protein encoding gene is imported into a target plant, so as to obtain transgenic callus; 2) salidroside is extracted from the transgenic callus; and the amino acid sequence of the UGT72B14 protein is shown as the sequence 2 in sequence list. The UGT71B14 protein encoding gene is imported into the target plant by a recombinant plant expression vector; the recombinant plant expression vector is obtained by inserting the UGT72B14 protein encoding gene into the multiple cloning site of vector pCAMBIA1301. The invention provides relevant technical approach with novelty and practicability and application thereof for expressing key enzyme UGT72B14 in biosynthesis of salidroside by utilizing rhodiola sachalinensis transgenic callus.

Description

Utilize UGT72B14 to prepare the method for rhodioloside

Technical field

The present invention relates to the method that a kind of UGT72B14 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.

Summary of the invention

The purpose of this invention is to provide the method that a kind of UGT72B14 of utilization prepares rhodioloside.

A kind of method for preparing rhodioloside provided by the invention comprises the steps: the explant of the proteic encoding gene importing of UGT72B14 purpose plant is obtained transgenic calli; 2) from described transgenic calli, extract rhodioloside, obtain rhodioloside; The proteic aminoacid sequence of described UGT72B14 is the sequence 2 in the sequence table.

Above-mentioned sequence 2 is made up of 473 amino acid.

The proteic encoding gene of described UGT72B14 is by the explant of the Agrobacterium tumefaciens mediated importing purpose plant of reorganization;

The proteic encoding gene of described UGT72B14 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 UGT72B14 carrier pCAMBIA1301 and Bst EII recognition site.

The proteic encoding gene of described UGT72B14 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; Described reorganization agrobacterium tumefaciens is for importing the reorganization agrobacterium tumefaciens that the host Agrobacterium obtains with described recombinant plant expression vector.

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 1.5mg/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 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 1.5mg/L, the concentration of described 1-naphthylacetic acid in the used substratum of described inducing culture is 0.15mg/L, the concentration of described Totomycin in the used substratum of described inducing culture is 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 1.5mg/L, the concentration of described 1-naphthylacetic acid in the substratum that described resistance screening is cultivated is 0.15mg/L, the concentration of described Totomycin in the substratum that described resistance screening is cultivated is 20mg/L, and the concentration of described cephamycin in the substratum that described resistance screening is cultivated is 500mg/L;

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, 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 1.5ml6-benzyladenine 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 inducing culture of every 1000ml is prepared as follows: after adding 1.5ml6-benzyladenine mother liquor, 0.15ml1-naphthylacetic acid mother liquor, 0.05ml 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 1.5ml6-benzyladenine mother liquor, 0.15ml1-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 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 2 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, 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 1.5mg/L, the concentration of described 1-naphthylacetic acid in the used substratum of I is 0.15mg/L, the concentration of described Totomycin in the used substratum of I is 15mg/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, 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 1.5mg/L, the concentration of described 1-naphthylacetic acid in the used substratum of II is 0.15mg/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.

I: the used substratum of every 1000ml is prepared as follows: after adding 1.5ml6-benzyladenine mother liquor, 0.15ml1-naphthylacetic acid mother liquor, 0.15ml 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 I; In the described step I, described subculture 3 times carries out subculture according to every 10-13 days interval;

II: the used substratum of every 1000ml is prepared as follows: after adding 1.5ml6-benzyladenine mother liquor, 0.15ml1-naphthylacetic acid mother liquor, 0.1ml 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 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 10ml 1M, 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;

The proteic encoding gene of described UGT72B14 be in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 53rd to the 1474th Nucleotide.Above-mentioned sequence 1 is made up of 1671 Nucleotide, its OFR be sequence 1 from the dna molecular shown in 5 ' terminal the 53rd to the 1474th 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 UGT72B14 gene is placed 35S promoter+Ω enhanser downstream of high efficiency plant expression vector pCAMBIA1301, transform Radix Rhodiolae by the Agrobacterium rhizogenes mediated method, by the enhancement expression and the gene transformation medium optimization of potent promotor, the efficiently expressing of UGT72B14 cause in the transgenosis Radix Rhodiolae callus salidroside content contrast improve 3.3 times.The present invention provides related art method and the application with novelty and practicality for utilizing the Radix Rhodiolae transgenic calli to cross expression rhodioloside biosynthetic pathway key enzyme-UGT72B14.

Description of drawings

Fig. 1 is a glycosyl transferase activity variation tendency in time in the root of Rhodiola sachalinensis

Fig. 2 is the electrophoresis result of the total RNA of root of Rhodiola sachalinensis

Fig. 3 is root of Rhodiola sachalinensis 3 ' RACE result

Fig. 4 is by being obtained gene 5 ' RACE result

Fig. 5 is the pcr amplification of UGT72B14 full-length cDNA

Fig. 6 is the proteic structure function domain analysis of UGT72B14

Fig. 7 is that the BgLII and the Bst EII double digestion of pBSUGT72B14 expression vector identified

Fig. 8 is that the PCR of pBSUGT72B14 expression vector identifies

Fig. 9 is the influence of Totomycin to the leaf growth state

Figure 10 is one of Radix Rhodiolae genetic transformation process (agrobacterium-mediated transformation) of UGT72B14

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

Figure 12 is the PCR-Southern detected result of part transgenosis Radix Rhodiolae callus

Figure 13 is the Northern detected result of part transgenosis Radix Rhodiolae

Figure 14 is a transgenosis Radix Rhodiolae HPLC analytical results

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, UGT72B14 gene isolation

Use RACE (rapid-amplification of cDNA ends) and successfully obtain 1 new UDP-glucosyl transferase gene cDNA complete sequence, international glycosyltransferase Professional Committee called after UGT72B14 from the separation of Radix Rhodiolae root tissue in conjunction with design of CODEHOP (consensus-degenerate hybrid oligonucleotide primers) degenerated primer and methyl jasmonate (MeJA) inductive method.

Detailed process is as follows:

1, glycosyl transferase activity variation tendency in time in the root of Rhodiola sachalinensis

For the RNA of optimized Separation tyrosol glucosyl transferase gene extracts period, the glycosyl transferase activity that this research is induced the generation of Radix Rhodiolae root to MeJA variation tendency is in time analyzed.Research is substrate with the tyrosol, has measured the ratio vigor (U/mg FW) of glycosyltransferase in different time Radix Rhodiolae callus and the root crude enzyme liquid, the results are shown in Figure 1.As seen from the figure, the specific activity of enzyme of catalysis tyrosol observed value in the time of the 12nd day is the highest in 6 times in the root, will lag behind transcription because albumen is synthetic, therefore chooses 10 days root tissue and extracts material as RNA.

2, the extraction of total RNA

Use improved CTAB method to extract root of Rhodiola sachalinensis RNA, improved RNA is extracted and is added strong reductant beta-mercaptoethanol and sequestrant PVP40 in the damping fluid, has effectively reduced the influence of polyphenols; The selective precipitation of LiCl has then been got rid of the interference of a large amount of glucides to extracting in the Root of Kirilow Rhodiola.Purity detecting result is the average OD of the total RNA of root ₂₆₀/ OD ₂₈₀Value is about 1.85, illustrates that the total RNA that extracts is purer, and this method is a kind of effective root of Rhodiola sachalinensis method for extracting total RNA (Fig. 2).

3, the separation of UGT72B14 gene 3 ' terminal sequence

With the total RNA of Radix Rhodiolae root tissue is template, with QT (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, and (Block H:5 '-CAGCTGTTGGAGTTTTTGTTACTCAytgyggntgga-3 ' .N is any base to the degenerated primer that designs according to CODEHOP; Y is C or T), carry out the sleeve type PCR amplification with the anchor primer that designs for combination, the PCR electrophoresis result shows the specific band (Fig. 3: M:DL2000marker about 1 about 500bp of size of acquisition; 1-3: be respectively 16d, 12d, the pcr amplification result of 0d RNA), the base length of the gene that obtains conforms to the possible length of expectation.

The cDNA fragment that obtained is found through back behind the dna sequencing and after carrying out bioinformatic analysis, its aminoacid sequence and registered plant UDP-glucanotransferase C-terminal sequence have higher homology (being up to 65%), illustrate that this sequence is a 3 ' end of 1 UDP-glucosyl transferase gene of Radix Rhodiolae, length is 530bp, and is a new gene.

4, the clone of UGTC2 and UGTR gene 5 ' terminal sequence

The test-results of 5 ' of the gene that obtains-RACE such as Fig. 4 (M:DL 2000marker; The 1:PCR amplification) shown in.With specific shown in Fig. 4, with estimate that the band that size (conserved regions to 5 ' end base length) conforms to reclaims, is connected T carrier, conversion, evaluation, order-checking, analysis, order-checking after, found that institute's calling sequence and other plant UDP-glucosyl transferase gene homology are not very high, best result does not reach 62%, and mrna length is 1241bp.

5, Full Length cDNA Cloning

Utilize DNAman software that the cDNA3 ' terminal sequence of isolating gene and 5 ' terminal sequence sequencing result are carried out electronics and merge, obtain total length and be 1671 sequence, called after UGT72B14.The nucleotides sequence of UGT72B14 is classified the sequence 1 in the sequence table as, the OFR sequence be in the sequence table sequence 1 from 5 ' terminal the 53rd to the 1474th Nucleotide.UGT72B14 encoded protein called after UGT72B14, its aminoacid sequence are the sequence 2 in the sequence table.

Corresponding primer in the table 1, cDNA first chain with the root reverse transcription is a template, amplifying length is the interior sequence of complete opening code-reading frame of the UGT72B14 gene of 1433bp (guarantor draws together the sequence of restriction enzyme site and protection base), the results are shown in Figure 5 (M:DL2000 marker; 1:UGTR full length gene pcr amplification result), the electrophoresis showed sequence length conforms to the expection size.Equally, this band order-checking after recovery, T carrier connect, transform, identify.Sequencing result conforms to the result that electronics merges.

Table 1UGT72B14 cDNA total length primer (restriction enzyme site is used for the structure of plant expression vector)

6, the bioinformatic analysis of UGT72B14 gene

The full length cDNA sequence of the UGT72B14 gene that obtains is submitted GenBank by the Bankit instrument on NCBI website (http://www.ncbi.nlm.nih.gov/BankIt/index.html), and acceptance number is EU567325.

Utilize DNAman software that the UGT72B14 full length gene cDNA sequence that obtains is analyzed, the UGT72B14 cDNA total length 1671bp (sequence 1) that is obtained that is obtained, comprising complete open reading frame (the open reading frame of 1422bp (sequence 1 is from 5 ' terminal the 53rd to the 1474th Nucleotide), ORF), infer 473 amino acid whose polypeptide of coding, contain 5 ' non-district (5 ' UTR) and 97bp 3 ' non-district (3 ' UTR) and the poly-A tail of translating of translating of 53bp simultaneously.Terminator codon appears before 5 ' the UTR district initiator codon, and the sequence alignment by BLASTp and known other plant glycosyltransferase, the result proves that the cDNA of the UGT72B14 gene of acquisition is complete.

7, the 26S Proteasome Structure and Function domain analysis of UGT72B14 gene

With the protein blast in the BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/) UGT72B14 gene supposition amino acid sequence coded is carried out domain analyses, the results are shown in Figure 6, analysis revealed UGT72B14 contains a typical UDP-glucanotransferase characteristic structural domain between 261-440, and a typical C OG1819 is arranged between 255-442 also is the glycosyltransferase structural domain, sees Fig. 6.

The UGT72B14 gene can obtain by aforesaid method, but also synthetic.

Embodiment 2. changes the UGT72B14 Radix Rhodiolae

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

1, the structure of pCAUGT72B14 carrier

With containing BgLII and Bst EII restriction enzyme site pUGTRup and pUGTRdown (table 1) is primer, is template with the CDNA of synthetic, amplifies the PCR product.The fragment that obtains after this PCR product cut through BgLII and Bst EII enzyme is inserted into pCAMBIA1301 carrier (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.) BgLII and Bst EII recognition site between obtain recombinant vectors, with recombinant vectors transformed into escherichia coli DH5 α, obtain the bacterium of recombinating through behind the antibiotic-screening, the extraction plasmid carries out enzyme and cuts evaluation and PCR evaluation, qualification result shows, enzyme is cut band and carrier strap (M, the DL15000+2000 Marker as shown in Figure 7, about back generation 1.5kb; 1-8, positive plasmid.), and band (Fig. 8, M, DL2000 Marker about 1.5kb also appear in the electrophoresis result of PCR; 1-8, positive plasmid) plasmid called after pCAUGT72B14.PCAUGT72B14 is served the sea gives birth to the further sequence verification of worker biotechnology Services Co., Ltd, the result for pCAUGT72B14 be between the BgLII of pCAMBIA1301 carrier and Bst EII recognition site in the insertion sequence table sequence 1 from 5 ' end the 53rd to the 1474th Nucleotide (the ORF sequence of UGT72B14).

By freeze-thaw method recombinant plasmid pCAUGT72B14 is transformed into Agrobacterium (Agrobacterium tumefaciens) EHA105 (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.) in the competent cell, obtain the reorganization bacterium, extract plasmid enzyme restriction and identify, produce the positive EHA105/pCAUGT72B14 of reorganization bacterium of the plasmid correspondence of band about 1.5kb and 9.5kb left and right sides band.

2, change the acquisition of UGT72B14 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 (Hyg) 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 0, 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, in transgenosis initial stage Totomycin concentration is that 5mg/L obtains for improving screening efficiency, Totomycin concentration to be increased to 20mg/L on the raised growth callus in good condition basis, carry out the resistant calli screening, obtain the ideal experimental result.

Be seeded on the solid medium (YEB) the positive EHA105/pCAUGT72B14 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 the OD of positive EHA105/pCAUGT72B14 ₆₀₀To 0.5, be used to infect Radix Rhodiolae (Rhodiola sachalinensis A.Bor; 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.

Earlier the aseptic seedling blade of Radix Rhodiolae is cut into the fritter of 0.5 * 0.5cm, the blade face is connected on downwards on the pre-culture medium, secretly cultivates 2d (table 2) for 25 ℃, uses the D of above-mentioned acquisition again ₆₀₀It is the aseptic seedling blade that 0.5 positive EHA105/pCAUGT72B14 infects Radix Rhodiolae on the pre-culture medium, the Radix Rhodiolae leaf dish that positive EHA105/pCAUGT72B14 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 on the antibacterial substratum that suppresses Agrobacterium growth microbiotic (cephamycin, Cef 500mg/L) and carry out 5 days antibacterial cultivation; To receive through the explant of antibacterial cultivation and contain on Totomycin 5mg/L, the Cef 500mg/L inducing culture substratum, under 25 ℃ of continuous illumination conditions, 2 all subcultures once, (Figure 10 B transforms the leaf dish and is selecting to produce callus on the substratum to carry out inducing culture; ).After treating that callus that inducing culture obtains forms, a part of callus is received on the resistance screening culture medium, screening transgenosis resistant calli under high density Totomycin Hyg (20mg/L) condition (Figure 10 C callus places high the selection to press on the substratum); 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, being specially on the substratum that changes Totomycin 10mg/L behind subculture on the Totomycin 15mg/L substratum 3 times over to subculture 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 UGT72B14 Radix Rhodiolae callus.

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;

I: used substratum is prepared as follows: after adding 1.5ml6-benzyladenine mother liquor, 0.15ml1-naphthylacetic acid mother liquor, 0.15ml 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 I; Incubation time is 40d, subculture 3 times, 25 ℃, continuous illumination;

II: used substratum is prepared as follows: after adding 1.5ml6-benzyladenine mother liquor, 0.15ml1-naphthylacetic acid mother liquor, 0.1ml 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 II; Incubation time is 30d, every 15d subculture 1 time, 25 ℃, continuous illumination;

Described 2,4-chlorophenoxyacetic 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 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, changeing UGT72B14 Radix Rhodiolae callus identifies

1) PCR identifies

Commentaries on classics UGT72B14 Radix Rhodiolae callus with above-mentioned acquisition is a 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 (pUGTRdown) 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 of the part transgenic calli of conversion UGT72B14; PCK is plasmid pCAUGT72B14; RCK is a wild-type; 1-20 changes UGT72B14 Radix Rhodiolae callus for part, and positive control is a template with plasmid pCAUGT72B14.The result shows with primer pUGTRdown identical with positive control (PCK) with the 35S-UP dna segment that amplification obtains from change UGT72B14 Radix Rhodiolae callus, for about 1.6Kb, negative control (wild-type Radix Rhodiolae, RCK) this amplified production not.The PCR detected result is all positive in 20 commentaries on classics UGT72B14 Radix Rhodiolae callus that detect.The efficient that the high density hygromycin selection is described is high.

2) PCR-Southern identifies

With the UGT72B14 cDNA of Digoxigenin mark is probe (the ORF sequence of UGT72B14, sequence 1 from 5 ' terminal 53-1474 position Nucleotide in the sequence table.) the positive UGT72B14 Radix Rhodiolae callus amplified production that changes of part PCR is carried out PCR-Southern detection, result such as Figure 12, PCK-plasmid pCAUGT72B14; The RCK-wild-type; 7,20-partly changes UGT72B14 Radix Rhodiolae callus.

Amplified production all produces hybridization signal with probe.Illustrate that the UGT72B14 gene integration is to genomic dna.

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 in the experimentation, another part 4 weeks before detection stop to take off bacterium, stop to take off the regeneration that does not have Agrobacterium in the material culturing process of bacterium, the transgenic calli that adds Cef and stop to take off bacterium has all obtained positive test symbol.

3) Northern identifies

Extract changeing UGT72B14 Radix Rhodiolae callus, and total RNA of corresponding wild-type contrast (Radix Rhodiolae) respectively, be transferred on the nylon membrane behind the denaturing formaldehyde gel electrophoresis, is that probe carries out Northern and detects with the UGT72B14 of Digoxigenin mark.Because the UGT72B14 gene is to separate to go back to again wherein from Radix Rhodiolae, should distinguish the UGT72B14 native gene in the experiment and transcribe interference experimental result, serve as to contrast to distinguish therefore from the transcriptional expression amount with the wild-type callus.The Northern detected result show (among Figure 13,1, the wild-type callus; 5,7,9 for changeing UGT72B14 Radix Rhodiolae callus.), changeing UGT72B14 Radix Rhodiolae callus transcriptional level contrasts apparently higher than wild-type, comprehensive aforementioned Molecular Detection and Northern experimental result, illustrate that the UGT72B14 gene not only successfully is incorporated in the Radix Rhodiolae genomic dna, and obtain that transcriptional level is expressed and transcriptional level is higher.

Adopting uses the same method also changes empty carrier pCAMBIA1301 carrier in the Radix Rhodiolae callus, obtains to change pCAMBIA1301 Radix Rhodiolae callus.

The HPLC of the rhodioloside of embodiment 3, commentaries on classics UGT72B14 Radix Rhodiolae callus detects

The Northern results of hybridization confirms, UGT72B14 transform obtain behind the Radix Rhodiolae and transcriptional level higher, for further the UGT72B14 function being analyzed, salidroside content in transgenosis Radix Rhodiolae callus and the wild-type contrast (callus of Radix Rhodiolae) thereof has been carried out high-performance liquid chromatogram determination, and concrete grammar is as follows:

From the described commentaries on classics UGT72B14 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 4.13 minutes.The retention time of the rhodioloside in the wild-type contrast callus that HPLC detects is 4.13 minutes, and the retention time of changeing the rhodioloside in the UGT72B14 Radix Rhodiolae callus is 4.13 minutes.

The commentaries on classics UGT72B14 Radix Rhodiolae callus that is obtained by embodiment 2 has been carried out HPLC mensuration, and reaching the commentaries on classics pCAMBIA1301 Radix Rhodiolae callus that is obtained by embodiment 2 with wild-type contrast (callus of Radix Rhodiolae) is contrast.The experiment triplicate, results averaged.

The data analysis of the salidroside content measurement result in the callus such as Figure 14 (C: callus, TC: transgenic calli), the result shows: the salidroside content in the wild-type contrast callus (C) is the 1.75mg/g dry weight, the salidroside content that changes in the UGT72B14 Radix Rhodiolae callus (TC) is the 7.53mg/g dry weight, improves 3.3 times than its wild-type contrast.

The wild-type contrast does not have significant difference with the result who changes pCAMBIA1301 empty carrier Radix Rhodiolae callus.

Illustrate that the conversion of UGT72B14 has significantly improved the accumulating level of rhodioloside.

Claims

1. a method for preparing rhodioloside comprises the steps: 1) with the explant of the proteic encoding gene importing of UGT72B14 purpose plant, obtain transgenic calli; 2) from described transgenic calli, extract rhodioloside, obtain rhodioloside; The proteic aminoacid sequence of described UGT72B14 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 UGT72B14 being inserted carrier pCAMBIA1301.

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 UGT72B14 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 UGT72B14 be in the sequence table sequence 1 from 5 ' terminal the 53rd to the 1474th Nucleotide.

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.