CN102337279A - Method of increasing content of tanshinone in hairy roots of salvia miltiorrhiza bunge through cotransformation of SmHMGR and SmDXR double genes - Google Patents
Method of increasing content of tanshinone in hairy roots of salvia miltiorrhiza bunge through cotransformation of SmHMGR and SmDXR double genes Download PDFInfo
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Abstract
The invention relates to the field of biological technology and discloses a method of increasing the content of tanshinone. According to the method, the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene SmHMGR and the 1-deoxy-D-xylosone-5-phosphate reductase gene SmDXR of salvia miltiorrhiza bunge are used to construct a recombinant vector, and genetic transformation is carried out on leaves of salvia miltiorrhiza bunge so as to obtain hairy roots of salvia miltiorrhiza bunge with transgenic SmHMGR and SmDXR genes. The content of tanshinone in the obtained transgenic hairy roots of salvia miltiorrhiza bunge in the invention substantially increases, and total tanshinone content in a line (HD34) of cotransformed SmHMGR and SmDXR double genes is 5.30 times of that of a comparison hairy root of salvia miltiorrhiza bunge. The invention provides the method for increasing content of tanshinone in the hairy roots of salvia miltiorrhiza bunge, providing a novel high-quality drug source for producing tanshinone in critical clinical demand and having a positive promotion effect in alleviating shortage of drug sources for tanshinone.
Description
Technical field
The invention belongs to biological technical field, specifically, relate to a kind of metabolic engineering strategy that utilizes, improve the method for Hairy Root Cultures of Salvia miltiorrhiza TANSHINONES content with two key gene cotransformations.
Background technology
Cardiovascular and cerebrovascular diseases is " the No.1 killer " who threatens universe's health and life at present.According to statistics, annual nearly 1,700 ten thousand people in the whole world die from cardiovascular and cerebrovascular diseases, account for 1/3 of the total death toll in the whole world; Annual nearly 2,600,000 people of China die from cardiovascular and cerebrovascular diseases.Therefore the clinical medicine of efficient, low toxicity of active research and exploitation and cheap treatment cardiovascular and cerebrovascular diseases has very profound significance to improving level of human health.
The red sage root (Salvia miltiorrhiza Bunge) has another name called Radix Salviae Miltiorrhizae, red, red ginseng, the ginseng etc. of invigorating blood circulation, and is under the jurisdiction of dicotyledonous Labiatae Salvia per nnial herb.As a kind of traditional Chinese medicine, the red sage root is mainly used in the treatment to cardiovascular disorder clinically, and promoting blood flow to regulate menstruation is also arranged, stasis-dispelling and pain-killing, the cool blood carbuncle that disappears, the relieving restlessness that clears away heart-fire, the effect of nourishing blood to tranquillize the mind.The main pharmaceutical compound of the red sage root comprises fat-soluble diterpene quinone (tanshinone component) and water miscible phenolic acid compound, also contains flavonoid, other compositions such as triterpenes sterol.It is antitumor that modern study shows that the contained tanshinone material of the red sage root has, and Azelaic Acid is anti-oxidant, and therefore multiple pharmacologically active such as atherosclerosis has great demand.Yet because wild resource reduces day by day, the red sage root is a per nnial herb in addition, and growth cycle is longer, and active pharmaceutical ingredients content is low, under traditional cultivation mode, is faced with quality serious degradation and breed breeding cost and crosses high many drawbacks.Therefore, the demand that how to make the supply of this raw medicinal material of the red sage root on quality and quantity, satisfy clinical application has better become a research focus.The complex structure of TANSHINONES causes its chemosynthesis process very loaded down with trivial details, and cost is higher and be prone to cause environmental pollution; Its activeconstituents accumulation volume of cell that cell culture processes under isolated condition obtains is very low and stable very poor.Engineeredly develop increasingly mature with the hairly root culture technique rapidly, solve TANSHINONES medicine source property in short supply problem new approaches are provided for improving the content of TANSHINONES composition in the Hairy Root Cultures of Salvia miltiorrhiza.
According to the literature search analysis; 3-hydroxy-3-methyl glutaryl-CoA reductase gene (SmHMGR) and 1-deoxy-D-xylulose-5-phosphoric acid reduction enzyme gene (SmDXR) is two crucial rate-limiting enzyme genes in the TANSHINONES biosynthetic pathway; They lay respectively in cytoplasmic MVA approach and the intravital DXP approach of tenuigenin, are the important target spots of TANSHINONES metabolic engineering.Adopt genetic engineering means; With above-mentioned two the key gene SmHMGR and the SmDXR cotransformation red sage root; To break the ink-bottle effect of TANSHINONES biosynthetic pathway; Obtain the Hairy Root Cultures of Salvia miltiorrhiza of high yield TANSHINONES, novel high-quality medicine source is provided, do not find to improve the relevant report of Hairy Root Cultures of Salvia miltiorrhiza TANSHINONES content at present as yet with the mentioned two key gene cotransformation strategies of theme of the present invention for commercially producing TANSHINONES.Therefore, the present invention has crucial meaning on the problem of reality solution TANSHINONES medicine source property in short supply.
Summary of the invention
The objective of the invention is to overcome the deficiency in the routine techniques, a kind of method of effective raising Hairy Root Cultures of Salvia miltiorrhiza TANSHINONES content is provided.
The present invention realizes through following technical scheme: the encoder block sequence of clone SmHMGR and SmDXR gene from the red sage root; Structure contains the plant bivalent efficient expression vector of the full-length cDNA molecule of above-mentioned two kinds of genes; (like pBiA4 etc.) is mediation with Agrobacterium rhizogenes; Genetic transformation red sage root blade imports SmHMGR and SmDXR gene in the red sage root cell simultaneously, obtains to change the Hairy Root Cultures of Salvia miltiorrhiza of SmHMGR and SmDXR gene; The integration situation of PCR testing goal gene SmHMGR and SmDXR, QRT-PCR analyzes and inserts goal gene SmHMGR and the expression of SmDXR in hairly root, TANSHINONES composition (dihydrotanshinone in the high-performance liquid chromatogram determination transgenic hairly root; VSZ3505; Tanshinone I, Tanshinone II A) content, DPPH removes radical and 1; 2, the anti-oxidant activity of TANSHINONES in the 3-pyrogallol autoxidation measuring transgenic hairly root.
The present invention includes following concrete steps:
(1) salvia 3-hydroxy-3-methylglutaryl A reductase gene SmHMGR and 1-deoxy-D-xylulose-5-phosphoric acid reduction enzyme gene SmDXR are inserted plant expression vector, make up recombinant vectors; Described salvia 3-hydroxy-3-methylglutaryl A reductase gene and 1-deoxy-D-xylulose-5-phosphoric acid reduction enzyme gene order is respectively the dna sequence dna of SEQ ID:1 and SEQ ID:2;
Plant expression vector is pCAMBIAl304 plasmid and the pCAMBIAl304 of pBI121 plasmid through obtaining after making up
+Carrier comprises CaMV35S promotor and terminator, MCS, replication origin and kalamycin resistance site;
(2) recombinant vectors that step (1) is obtained changes Agrobacterium rhizogenes over to, makes up the Agrobacterium rhizogenes that contains recombinant vectors;
Agrobacterium rhizogenes can be selected agrobacterium rhizogene strain C58C1, agrobacterium rhizogene strain pBiA4 or agrobacterium rhizogene strain LBA9402 for use;
(3) contain the Agrobacterium rhizogenes mediation salvia 3-hydroxy-3-methylglutaryl A reductase gene and the 1-deoxy-D-xylulose-5-phosphoric acid reduction enzyme gene transformation red sage root of recombinant vectors with step (2) gained; Obtain hairly root: can the Agrobacterium rhizogenes that contain recombinant vectors and red sage root explant (like young leaflet tablet) be cultivated altogether, grow degerming behind the hairly root;
(4) salvia 3-hydroxy-3-methylglutaryl A reductase gene and the 1-deoxy-D-xylulose-5-phosphoric acid reduction enzyme gene in detection step (3) hairly root got the positive transgenic hairly root of The selection result;
Detection method is PCR, obtains the positive clone's of PCR qualification result hairly root; Quantitatively QRT-PCR detects, the expression of goal gene SmHMGR and SmDXR in the transgenic hairly root; Through high-performance liquid chromatogram determination red sage root transgenic hairly root TANSHINONES content, screening obtains the high strain system of TANSHINONES content again.
DPPH removes the anti-oxidant activity of TANSHINONES in the radical measuring red sage root transgenic hairly root; 1,2, the anti-oxidant activity of TANSHINONES in the 3-pyrogallol autoxidation measuring red sage root transgenic hairly root.
During above-mentioned PCR detects used Auele Specific Primer be respectively be positioned at composition type expression promoter CaMV35S inside upstream primer and be positioned at SmHMGR and the downstream primer of SmDXR gene inside; Behind pcr amplification, the strain system that the purpose band occurs is transgenic positive hairly root clone strain system.
Described quantitative QRT-PCR measures TANSHINONES biosynthesis related genes (SmHMGR in the red sage root transgenic hairly root; The method of expression SmDXR) is: carry out the extraction of total RNA to be accredited as male hairly root strain system through PCR; And be inverted to cDNA article one chain system as template with total RNA of same amount; Utilizing software to design respectively to cross over an intron to be positioned on two exons is the primer of goal gene primer and house-keeping gene ubiquitin; The above-mentioned cDNA first chain system of getting same amount is that template is carried out the QRT-PCR amplification, analyzes genes involved (SmHMGR, relative expression quantity SmDXR).
The condition of described high-performance liquid chromatogram determination TANSHINONES content is following: chromatographic column C-18 reverse phase silica gel post, moving phase are acetonitrile: water (65: 35), detect wavelength 270nm, 30 ℃ of column temperatures, flow velocity 1mL/min, sample size 20 μ L.
The method that described DPPH removes the anti-oxidant activity of TANSHINONES in the free radical method mensuration red sage root transgenic hairly root is: will from red sage root transgenic hairly root, extract gradient (the 1 μ L that the TANSHINONES sample of collecting is got different amounts; 2 μ L; 4 μ L, 8 μ L, 16 μ L) react as the DPPH solution (methanol solution of 0.2mM) in radical source with 2mL; And under room temperature (25 ℃), being incubated 30min, sample is surveyed absorption value under spectrophotometer 517nm.
The method that described anti-pyrogallol oxidation experiment is measured the anti-oxidant activity of TANSHINONES in the red sage root transgenic hairly root is: will from red sage root transgenic hairly root, extract the Tris-HCl damping fluid (PH8.0 that the TANSHINONES sample of collecting is got 10 μ L and 2.8mL0.05M; Comprise 1mM EDTA) and the pyrogallol of 0.2mL6mM at room temperature mix; 325nm is every under spectrophotometer measures light absorption value at a distance from 30s, measures 4min.The resistance of oxidation comparison that has proved the TANSHINONES in the Hairy Root Cultures of Salvia miltiorrhiza that obtains through transgenic method through experiment is taken a picture to wanting high, and the strategy that therefore obtains the Hairy Root Cultures of Salvia miltiorrhiza of high yield TANSHINONES through method of the present invention is efficient feasible.
The present invention uses conventional biological experimental method such as vector construction, genetic transformation, Molecular Detection, quantitatively QRT-PCR analyzes, TANSHINONES extraction and assay, DPPH remove radical and 1; 2; Anti-oxidant activity of TANSHINONES etc. has been set up a kind of method that improves TANSHINONES content in the Hairy Root Cultures of Salvia miltiorrhiza in the 3-pyrogallol autoxidation measuring transgenic hairly root.Adopt corotation SmHMGR; The metabolic engineering strategy of SmDXR gene has obtained the red sage root transgenic hairly root strain system of high yield TANSHINONES; Total tanshinone content significantly improves in the transgenic Hairy Root Cultures of Salvia miltiorrhiza that is obtained; The total tanshinone content of one of them strain system (HD34) is 5.30 times of contrast, and wherein the Tanshinone I raising is the most obvious, is 18.42 times (0.383mg/g DW) of control group.The present invention provides a kind of novel high-quality medicine source for the TANSHINONES of producing tool important clinical demand; For commercialization mass production TANSHINONES and reduction drug price provide possibility; Property in short supply to alleviating TANSHINONES medicine source plays positive promoter action, also for the heavy demand of large-scale production TANSHINONES clinical medicine important source is provided.
Description of drawings
Fig. 1 is pCAMBIAl304
+-SmHMGR, pCAMBIAl304
+-SmDXR and pCAMBIAl304
+-SmHMGR-SmDXR vector construction figure.
The PCR of Fig. 2 transgenic hairly root identifies.A, SmHMGR gene (432bp) and rolB gene thereof; B, the PCR of its rolB gene of SmDXR gene (666bp) detects; C, SmHMGR gene (432bp) and SmDXR gene (666bp) and rolB gene thereof.M, and DL-2000Marker (100-2,000bp); 1304+, positive control (pCAMBIAl304
+-SmHMGR-SmDXR plasmid); NC1, negative control (the wild-type red sage root); NC2, the water contrast; BC, empty map (pCAMBIAl304
+The hairly root that the plasmid genetic transformation red sage root obtains); D, H and HD are the different transgenic lines of SmHMGR/SmDXR single-gene and dual-gene genetic transformation red sage root acquisition.
Fig. 3 transgenic Hairy Root Cultures of Salvia miltiorrhiza QRT-PCR.BC is empty map (pCAMBIAl304
+The Hairy Root Cultures of Salvia miltiorrhiza that the plasmid genetic transformation obtains); H is the SmHMGR expression amount; D is the SmDXR expression amount; HD is the different transgenic lines of SmHMGR and the dual-gene genetic transformation red sage root acquisition of SmDXR.
Fig. 4 red sage root list changes and corotation SmHMGR, and the strain of SmDXR gene is that average TANSHINONES contains spirogram.HT: dihydrotanshinone; CT: VSZ3505; T1: Tanshinone I; T2A: Tanshinone II A; TT: total tanshinone; H: single hairly root strain system that changes SmHMGR; D: single hairly root strain of changeing SmDXR is HD: corotation SmHMGR, SmDXR gene Hairy Root Cultures of Salvia miltiorrhiza strain system; BC: empty map (pCAMBIAl304
+The hairly root that the plasmid genetic transformation red sage root obtains).
Fig. 5 and Fig. 6 for mix mark article (A) and sample 1304
+(B), singly change D 6 (D) and the corotation SmHMGR of SmHMGRH28 (C), single SmDXR of commentaries on classics, the HD34 of SmDXR (E) TANSHINONES assay peak figure.1～4 represents dihydrotanshinone, VSZ3505, Tanshinone I and Tanshinone II A respectively.
Fig. 7 is that the DPPH free radical scavenging activity is with TANSHINONES concentration change trend map.Sample is that HD30 and HD34 and SmHMGR single-gene and the SmHMGR of H12 and H28, corotation SmHMGR and SmDXR of D5 and D6, the SmHMGR of BC, single SmDXR of commentaries on classics clones with the hairly root that the dual-gene genetic transformation red sage root of SmDXR obtains; BC, empty map (pCAMBIAl304
+The hairly root that the plasmid genetic transformation red sage root obtains).
Fig. 8 is anti-pyrogallol autoxidation figure.Sample is that HD30 and HD34 and SmHMGR single-gene and the SmHMGR of H12 and H28, corotation SmHMGR and the SmDXR of D5 and the D6 of BC, single SmDXR of commentaries on classics, single SmHMGR of commentaries on classics clones with the hairly root that the dual-gene genetic transformation red sage root of SmDXR obtains; BC, empty map (pCAMBIAl304
+The hairly root that the plasmid genetic transformation red sage root obtains).
Embodiment
Set forth the present invention in detail below in conjunction with specific embodiment.Be interpreted as: these embodiment only be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, molecular cloning (Sambrook etc.) for example, or according to the reagent that manufacturer provided or the condition of the incidental specification proposes of test kit.
1.1. the extraction of the total RNA of the red sage root and cDNA first chain is synthetic
The RNA prep pure plant kit that uses TIANGEN company to provide extracts total RNA (specification sheets in the extraction step reference reagent box) from red sage root transgenic hairly root.The fresh weight amount that is used to extract the red sage root transgenic hairly root of total RNA is about 0.1g, and the DNA in leaching process in the sample removes with DNase I working fluid.The RNA that extracts is measured relevant light absorption value on spectrophotometer, calculate purity and the concentration of the RNA that extracts.Through after calculating, is initial amount with 0.5 μ g RNA according to the concentration of different RNA sample respectively, carries out synthesize (the related description book that operation steps provides with reference to TaKaRa company) of the first chain cDNA with ThermoScript II XL (AMV).
1.2.SmHMGR with the design of SmDXR encoding sequence special primer and the acquisition of target gene fragment
According to the encoding sequence (SEQ ID NO.1) of said SmHMGR gene and the encoding sequence (SEQ ID NO.2) of SmDXR gene; Design amplifies the upstream and downstream special primer of complete encoder block respectively; And on the upstream and downstream primer, introduce restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively, so that make up plant expression vector.With the described first chain cDNA is template, behind pcr amplification, checks order.Determined dna sequence is given birth to worker's biotechnology ltd by Shanghai and is accomplished.Sequencing result shows that the encoding sequence of red sage root SmHMGR gene that sequence of being cloned and our laboratory are logined (SEQ ID NO.1) and SmDXR gene (SEQ ID NO.2) is in full accord on GenBank.
2.1. intermediate carrier pCAMBIAl304
+Structure
With pBI121 and pCAMBIAl304 is material, makes up plant expression vector pCAMBIAl304
+Particularly, HindIII/EcoRI double digestion pBI121 and pCAMBIAl304; Reclaim pBIl21-GUS expression cassette and the big fragment of pCAMBIAl304; Connect and transform the checking of picking mono-clonal bacterium colony extracting plasmid enzyme restriction.The result shows, plant expression vector pCAMBIAl304
+Make up successfully, comprise CaMV35S promotor and terminator, MCS, replication origin and kalamycin resistance site.
2.2. plant expression vector pCAMBIAl304
+The structure (see figure 1) of-SmHMGR
At the successful pCAMBIAl304 of above-mentioned structure
+On the basis, use the SmHMGR gene of from the red sage root, being cloned into to replace gus gene.Particularly, the BglII/BstEII enzyme is cut double digestion pMD18T-SmHMGR and pCAMBIAl304
+Reclaim SmHMGR gene and pCAMBIAl304
+Big fragment; Connect and transform picking mono-clonal bacterium colony PCR screening positive clone; Extract the further enzyme of plasmid and cut checking.The result shows that the SmHMGR gene successfully is building up to plant expression vector pCAMBIAl304
+In (comprising CaMV35S promotor and terminator, MCS, replication origin and kalamycin resistance site), thereby obtain to contain the plant expression vector pCAMBIAl304 of SmHMGR gene
+-SmHMGR.
2.3. plant expression vector pCAMBIAl304
+The structure (see figure 1) of-SmDXR
At the successful pCAMBIAl304 of above-mentioned structure
+On the basis, use the SmDXR gene of from the red sage root, being cloned into to replace the hygromycin gene.Particularly, the XholI/XholI enzyme is cut double digestion pMD18T-SmDXR and pCAMBIAl304
+Reclaim SmdR gene and pCAMBIAl304
+Big fragment; Connect and transform picking mono-clonal bacterium colony PCR screening positive clone; Extract the further enzyme of plasmid and cut checking.The result shows that the SmDXR gene successfully is building up to plant expression vector pCAMBIAl304
+In (comprising CaMV35S promotor and terminator, MCS, replication origin and kalamycin resistance site), thereby obtain to contain the plant expression vector pCAMBIAl304 of SmDXR gene
+-SmDXR.
2.4. plant expression vector pCAMBIAl304
+The structure (see figure 1) of-SmHMGR-SmDXR
With above-mentioned pCAMBIAl304
+-SmHMGR is the basis, uses the SmDXR gene replacement pCAMBIAl304 that from the red sage root, clones
+The hygromycin gene of-SmHMGR.Particularly, two XhoI double digestion pMD 18T-SmDXR and pCAMBIAl304 simultaneously
+-SmHMGR; Reclaim SmHMGR gene and pCAMBIAl304
+The big fragment of-SmHMGR; Connect and transform picking mono-clonal bacterium colony PCR screening positive clone; Extract the further enzyme of plasmid and cut checking.The result shows that the SmDXR gene successfully is building up to plant expression vector pCAMBIAl304
+(comprise CaMV35S promotor and terminator, MCS, replication origin and kalamycin resistance site) among-the SmHMGR, thereby obtain to contain the plant expression vector pCAMBIAl304 of SmHMGR and SmDXR
+-SmHMGR-SmDXR.
Present embodiment is connected in expression regulation sequence with TANSHINONES biosynthetic pathway key gene SmHMGR and SmDXR operability ground, forms the plant expression vector pCAMBIAl304 that contains SmHMGR and SmDXR gene
+-SmHMGR-SmDXR, this expression vector can be used for improving TANSHINONES content in the red sage root through the metabolic engineering strategy, and structure is as shown in Figure 1.
3.1. contain plant expression vector pCAMBIAl304
+The acquisition of-SmHMGR/SmDXR Agrobacterium rhizogenes engineering bacteria
With the plant expression vector pCAMBIAl304 that contains SmHMGR and/or SmDXR gene among the embodiment 2
+-SmHMGR/SmDXR changes among the Agrobacterium rhizogenes pBiA4, and picking mono-clonal bacterium colony carries out the PCR checking.The result shows that the plant expression vector that contains SmHMGR and SmDXR gene successfully is building up among the agrobacterium rhizogene strain pBiA4.
3.2. Agrobacterium rhizogenes mediation SmHMGR, the SmDXR gene genetic transforms the red sage root
3.2.1. the preparatory cultivation of explant
The healthy and strong aseptic seedling blade of the clip red sage root (0.5cm
2), be inoculated on the preparatory culture medium (1/2MS) 25 ℃ of dark 2d that cultivate.
3.2.2. the common cultivation of Agrobacterium and explant
With above-mentioned through pre-incubated red sage root leaf explant (like young leaflet tablet); After putting into the 1/2MS suspension that contains the good above-mentioned Agrobacterium rhizogenes engineering bacteria of activation and soaking 10min (jiggle explant is fully contacted with bacterium liquid); The red sage root blade that takes out after contaminating blots surperficial bacterium liquid with aseptic thieving paper; Forward among the common culture medium 1/2MS, secretly cultivate 3-4d.
3.2.3. inducing and succeeding transfer culture of hairly root
The red sage root explant of above-mentioned common cultivation 3-4d is transferred in the degerming solid medium (1/2MS+cb300mg/L), and 25 ℃ of dark cultivations about 2 weeks can grow hairly root from the explant wound.The red sage root explant of root of hair is transferred on the degerming solid medium (1/2MS+Cef 500mg/L); 25 ℃ dark cultivate treated in 2 weeks hairly root grow to 3cm when above the single hairly root of clip as a clone; Continue to be inoculated in except that dark the cultivation for two weeks in the bacterium culture medium (1/2MS+Cef300mg/L), fall again afterwards and anti-ly go to dark cultivation of 1/2MS+Cef 100mg/L and overflow until no Agrobacterium.
3.3. the PCR of transgenic Hairy Root Cultures of Salvia miltiorrhiza detects
3.3.1. the extraction of transgenic hairly root genomic dna
The present invention adopts the CTAB method to extract transgenic hairly root genomic dna.Put into the 1.5mL centrifuge tube about the transgenic hairly root 5cm that degerming finishes among the clip 3.2.3, add an amount of silica sand and 600 μ LCTAB lysates (65 ℃ of preheatings contain 1% beta-mercaptoethanol), material is fully ground with grinding rod.Place 65 ℃ of water-bath 40-50min, mixing sample (15min/ time) repeatedly therebetween adds the phenol that waits volume after being cooled to room temperature; Put upside down mixing emulsification 10min gently, the centrifugal 20min of 12000rpm carefully draws supernatant in new EP pipe; Add equal-volume phenol/chloroform (1: 1), mixing gently, the centrifugal 20min of 12000rpm; Slowly draw supernatant in new EP pipe, add isopyknic chloroform mixing, the centrifugal 20min of 12000rpm; Slowly draw supernatant in new EP pipe, add the absolute ethyl alcohol of 2 times of volume precoolings, separate out deposition.With the rifle head deposition is chosen in the new EP pipe, add 4 ℃ of washings of 75% ethanol and spend the night.Inferior daily 75% ethanol is washed twice again, the sucking-off supernatant, and room temperature is dried, and adds 30-50 μ L water dissolution deposition, and is frozen, subsequent use in-20 ℃ of Ultralow Temperature Freezers after handling with the RNA enzyme.
3.3.2. design of primers and PCR detect
At pCAMBIAl304
+-SmHMGR-SmDXR is last to be started the composition type expression promoter CaMV35S that inserts destination gene expression and inserts the difference designs specificity upper reaches and downstream primer on the gene:
35S promterF252 5 '-ATGGAGGTCATAAGCAACCAC-3 ' and SmHMGR 171R 5 '-GTAGGCGACGGAGAAGAACA-3 ';
And SmDXR 414R 5 '-TAATGACTCGTCTCTTACGGA-3 ', with PCR method total DNA of above-mentioned hairly root is carried out Molecular Detection.Experimental result shows, utilizes above-mentioned special primer, in a part of transgenic hairly root, can detect and positive control (pCAMBIAl304
+-SmHMGR-SmDXR plasmid is a template) quite big or small segmental PCR product.And with pCAMBIAl304
+When the genomic dna of hairly root that the empty carrier genetic transformation red sage root is sent and wild-type red sage root plant root and water are template, do not amplify any fragment, see Fig. 2.
Wherein, A1, B1 and C1:SmHMGR gene (432bp), SmDXR gene (666bp), SmHMGR gene (432bp)+SmDXR gene (666bp) PCR detect.M, and DL-2000Marker (100-2,000bp); Positive contrast (the pCAMBIAl304 of PC
+-SmHMGR-SmDXR plasmid); The negative contrast of NC1 (the wild-type red sage root); NC2 is the water contrast; BC: empty map (pCAMBIAl304
+The hairly root that the plasmid genetic transformation red sage root obtains); H transforms the strain system that Hairy Root Cultures of Salvia miltiorrhiza obtained for the SmHMGR monogenic inheritance; D transforms the strain system that hairly root obtained for the SmDXR monogenic inheritance; HD is the different transgenic lines of SmHMGR and the dual-gene genetic transformation red sage root acquisition of SmDXR; A2, B2 and C2: corresponding hairly root rolB gene identification figure.
Presentation of results, SmHMGR, the SmDXR gene has been incorporated in the red sage root genome.
Present embodiment is with described plant expression vector transforming agrobacterium rhizogenes; Acquisition is used to transform the agrobacterium rhizogene strain pBiA4 that contains SmHMGR and SmDXR gene plant expression vector of the red sage root; Utilize constructed agrobacterium rhizogene strain genetic transformation red sage root cell, obtain transgenic hairly root through PCR test positive clone.The acquisition of transgenic Hairy Root Cultures of Salvia miltiorrhiza provides direct material for the hairly root that screening obtains the high yield TANSHINONES.Agrobacterium rhizogene strain pBiA4 also can use Agrobacterium rhizogenes to replace as agrobacterium rhizogene strain C58C1 or agrobacterium rhizogene strain LBA940.
4.1. hairly root liquid culture
Get in the foregoing description 3.2 and be accredited as male and the hairly root 2-3cm rapidly that grows through PCR, in Bechtop with its put into contain the 100mL1/2MS liquid nutrient medium shake in the bottle 25 ℃; 100rpm; Dark cultivate 80 days results, a subculture is changed in the centre, after getting the proper amount of fresh hairly root and blotting surface-moisture with thieving paper; Wrap to immerse with masking foil and be stored in-80 ℃ in the liquid nitrogen after freezing and be used for RNA and extract, be used for TANSHINONES content after all the other hairly root oven dry and extract.
4.2.RNA extraction and cDNA first chain synthetic
Method is with 1.1
4.3. primer design is with synthetic
Encoding sequence according to TANSHINONES biosynthetic metabolism pathway key enzyme gene SmHMGR and SmDXR is used for detecting Hairy Root Cultures of Salvia miltiorrhiza SmHMGR and SmDXR expression of gene situation with Express3.0 design primer respectively; Set primer is to cross over an intron; Being positioned on two exons to avoid red sage root source DNA is the amplification (SmHMGR of template; SmDXR is all red sage root source gene), house-keeping gene ubiquitin is used as confidential reference items.The primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized.
The primer of quantitative PCR is:
4.4. quantitatively QRT-PCR detects SmHMGR, SmDXR and house-keeping gene ubiquitin expression of gene
Above-mentioned cDNA (diluting 10 times) first chain with same amount is a template, carries out the QRT-PCR amplification with above-mentioned designed primer respectively.The specification sheets of the Biosystem StepOne instrument of producing with reference to U.S. Applied Biosystem company carries out the quantitative PCR operation.The quantitative PCR reaction system is following:
PCR reaction conditions: 94 ℃ of 5min, 40 circulations (72 ℃ are extended 20sec for 94 ℃ of sex change 20sec, 55 ℃ of annealing 20sec), 72 ℃ of 10min.Goal gene and house-keeping gene ubiquitin respectively do three repetitions.Interpretation of result shows; SmHMGR; SmDXR gene overexpression and all be better than its expression in control group all in changing the hairly root clone of corresponding gene over to; (see A～C of Fig. 3, wherein BC is a blank, and H is different transgenic lines and the expression amount thereof that the SmHMGR genetic transformation red sage root obtains but the expression amount between the different clones has certain difference; D is different transgenic lines and the expression amount thereof that the SmDXR genetic transformation red sage root obtains; HD is the different transgenic lines and the corresponding expression amount thereof of SmHMGR and the dual-gene genetic transformation red sage root acquisition of SmDXR).
Present embodiment adopts SmHMGR in the quantitative QRT-PCR technical measurement transgenic Hairy Root Cultures of Salvia miltiorrhiza, SmDXR expression of gene situation, and for analyzing SmHMGR, SmDXR gene role in the TANSHINONES building-up process provides certain foundation.
Embodiment 5 utilizes HPLC to measure TANSHINONES content in the transgenic Hairy Root Cultures of Salvia miltiorrhiza
5.1. the preparation of chromatographic condition and standard substance stock solution
Chromatographic column is a C-18 reverse phase silica gel post, and moving phase is acetonitrile: water (65: 35), detect wavelength 270nm, 30 ℃ of column temperatures, flow velocity 1mL/min, sample size 20 μ L.
Precision takes by weighing dihydrotanshinone, and it is 280 μ g/mL that VSZ3505, Tanshinone I, Tanshinone II A standard substance are configured to concentration respectively, 300 μ g/mL, 28 μ g/mL, 290 μ g/mL standard substance stock solutions, be stored in-20 ℃ subsequent use.
5.2. the making of typical curve
Moving phase acetonitrile among the present invention: water was volume ratio 65: 35 o'clock; The RT of dihydrotanshinone, VSZ3505, Tanshinone I, Tanshinone II A is respectively 12.72min, 19.79min, 20.53min and 34.00min; Three kinds of TANSHINONES compositions separate fully, and the peak type is good.Above-mentioned standard substance stock solution article are got 5 μ L respectively, 10 μ L, 20 μ L, 30 μ L, 40 μ L sample introduction under corresponding chromatographic condition, record collection of illustrative plates and chromatographic parameter carry out regression analysis with peak area (Y) to standard substance concentration (X, μ g/mL) respectively.The result shows that dihydrotanshinone is presenting good linear relationship at 9-72 μ g/mL, Tanshinone I I A at 9-72 μ g/mL, Tanshinone I at 9-72 μ g/mL, VSZ3505 among the present invention in 5.5-44 μ g/mL scope.
5.3. the extraction and the mensuration of transgenic hairly root TANSHINONES content
The transgenic Hairy Root Cultures of Salvia miltiorrhiza of results among the embodiment 4 and oven dry is put into mortar fully grind, get 200mg hairly root dry powder and add methyl alcohol: methylene dichloride (3: 1, v/v) 16mL; Supersound extraction 60min, room temperature is placed and is spent the night, and takes out centrifugal (12000rpm next day; 10min), draw the vacuum-drying of supernatant extraction liquid, resistates is used methyl alcohol (analytical pure) dissolving of 2mL again; Sample is respectively got 20 μ L after with the membrane filtration of 0.22 μ M, injects high performance liquid chromatograph, writes down each component peaks area; The substitution equation of linear regression calculates and promptly gets sample TANSHINONES content.
As shown in Figure 4; The average content of the red sage root transgenic hairly root total tanshinone (dihydrotanshinone, VSZ3505, Tanshinone I, Tanshinone II A) of corotation SmHMGR and SmDXR gene is 2.506mg/g DW in the present invention, is 4.09 times of control group hairly root (0.613mg/g DW).Wherein the average content of dihydrotanshinone, VSZ3505, Tanshinone I, Tanshinone II A is respectively 1.084mg/g DW, 1.025mg/g DW, 0.260mg/g DW and 0.138mg/g DW, is respectively 4.09 times (0.264mg/gDW), 3.50 times (0.293mg/g DW) of control group hairly root, 12.49 times (0.021mg/g DW) and 3.96 times (0.035mg/gDW).One of them clone's dihydrotanshinone, VSZ3505, Tanshinone I; Tanshinone II A and total tanshinone content can reach 5.70 times (1.508mg/g DW) of control group respectively; Doubly 4.05 (1.185mg/g DW), 18.42 times (0.383mg/g DW), 4.91 times (0.171mg/g DW).And average total tanshinone content is 1.131mg/g DW among single commentaries on classics SmHMGR; Be 1.85 times of control group; Wherein the average content of dihydrotanshinone, VSZ3505, Tanshinone I, Tanshinone II A is respectively 0.296mg/g DW, 0.752mg/g DW, 0.030mg/g DW and 0.053mg/g DW, is respectively 1.12 times (0.264mg/gDW), 2.57 times (0.293mg/g DW) of control group hairly root, 1.46 times (0.021mg/g DW) and 1.53 times (0.035mg/gDW).And average total tanshinone content is 1.740mg/g DW among single commentaries on classics SmDXR; Be 2.84 times of control group; Wherein the average content of dihydrotanshinone, VSZ3505, Tanshinone I, Tanshinone II A is respectively 0.668mg/g DW, 0.789mg/g DW, 0.166mg/g DW and 0.116mg/g DW, is respectively 2.53 times (0.264mg/g DW), 2.70 times (0.293mg/g DW) of control group hairly root, 7.99 times (0.021mg/gDW) and 3.33 times (0.035mg/g DW).
In its diagram, HT: dihydrotanshinone; CT: VSZ3505; T1: Tanshinone I; T2A: Tanshinone I I A; TT: total tanshinone; H: single hairly root strain system that changes SmHMGR; D: single hairly root strain system that changes SmDXR; HD: corotation SmHMGR, SmDXR gene Hairy Root Cultures of Salvia miltiorrhiza strain system; BC: empty map (pCAMBIAl304
+The hairly root that the plasmid genetic transformation red sage root obtains).
Fig. 5 and Fig. 6 are for mixing mark article (A) and sample 1304
+(B), singly the hairly root strain of changeing SmHMGR is that the hairly root strain of H28 (C), single SmDXR of commentaries on classics is D6 (D) and corotation SmHMGR, and the strain of SmDXR gene Hairy Root Cultures of Salvia miltiorrhiza is HD34 (E) TANSHINONES assay peak figure.1～4 represents dihydrotanshinone, VSZ3505, Tanshinone I and Tanshinone I I A respectively.
Present embodiment adopts the HPLC method to measure transgenic Hairy Root Cultures of Salvia miltiorrhiza TANSHINONES content.The result shows that the Hairy Root Cultures of Salvia miltiorrhiza total tanshinone content of corotation SmHMGR and SmDXR gene significantly improves than control group, and wherein the raising of Tanshinone I content is the most obvious.
Embodiment 6DPPH removes the anti-oxidant activity that free radical method is measured TANSHINONES in the transgenic hairly root
6.1 sample solution preparation
Get the tanshinone extract 1 μ L of different amounts among HD30 and the HD34 hairly root clone of H12 and H28, corotation SmHMGR and SmDXR of D5 and the D6 of BC, single SmDXR of commentaries on classics, single SmHMGR of commentaries on classics according to institute's test sample article concentration; 2 μ L; 4 μ L, 8 μ L, 16 μ L and 2mL are as radical source 0.2M DPPH (1; 1-phenylbenzene picryl phenylhydrazine) solution, and under room temperature (25 ℃), be incubated 30min.
6.2 the conversion of the absorbance measurement of sample and DPPH free radical scavenging activity
With the sample in 1 respectively under spectrophotometer 517nm measure and write down its absorption value, convert absorbance to the DPPH free radical scavenging activity according to following formula:
DPPH free radical scavenging activity (%)=[(A
0-A
1)/A
0* 100]
A
0It is the control sample absorbance
A
1Be sample or standard substance absorbance
The result is as shown in Figure 7.Present embodiment has proved the unit TANSHINONES in the Hairy Root Cultures of Salvia miltiorrhiza that obtains through transgenic method through experiment resistance of oxidation and contrast quite, the strategy that therefore obtains the Hairy Root Cultures of Salvia miltiorrhiza of high yield TANSHINONES through method of the present invention is efficient feasible.
7.1 sample solution preparation
Getting different tanshinone extract sample 10 μ L and 2.8mL0.05M Tris-HCl damping fluid (PH8.0 comprises 1mM EDTA) and the pyrogallol of 0.2mL 6mM at room temperature mixes.Sample is D5 and D6, the H12 of single SmHMGR of commentaries on classics and HD30 and the HD34 of H28, corotation SmHMGR and SmDXR of single SmDXR of commentaries on classics.
7.2 reaching, the absorbance measurement of sample resists 1,2, the conversion of 3-pyrogallol autoxidation
With the sample in 1 respectively under spectrophotometer 325nm measure and whenever measure light absorption value at a distance from 30s, measure 4min, blank (not adding sample) done contrast
Clearance rate=(1-sample slope/control group slope) * 100
7.3 anti-pyrogallol autoxidation ability relatively
The result shows: this step calculates the oxidation-resistance of sample D5, D6, H12, H28 and HD34 according to the anti-pyrogallol autoxidation of gained, and wherein HD34 is 52.55%, and control group is 41.38%, apparently higher than control group BC (like Fig. 8).
Present embodiment has proved that through experiment the resistance of oxidation comparison of the TANSHINONES in the Hairy Root Cultures of Salvia miltiorrhiza that obtains through transgenic method takes a picture to wanting high, and the strategy that therefore obtains the Hairy Root Cultures of Salvia miltiorrhiza of high yield TANSHINONES through method of the present invention is efficient feasible.
The present invention adopts corotation SmHMGR, and the metabolic engineering strategy of SmDXR gene has obtained the red sage root transgenic hairly root strain system of high yield TANSHINONES, is the large-scale production TANSHINONES, and the medicine source property in short supply problem of alleviating TANSHINONES provides a kind of novel effective ways.
Claims (5)
1.SmHMGR the method with TANSHINONES content in the dual-gene cotransformation raising of the SmDXR Hairy Root Cultures of Salvia miltiorrhiza is characterized in that, comprises the steps:
(1) salvia 3-hydroxy-3-methylglutaryl A reductase gene SmHMGR and 1-deoxy-D-xylulose-5-phosphoric acid reduction enzyme gene SmDXR are inserted plant expression vector, make up recombinant vectors; Described salvia 3-hydroxy-3-methylglutaryl A reductase gene and 1-deoxy-D-xylulose-5-phosphoric acid reduction enzyme gene order is respectively the dna sequence dna of SEQ ID:1 and SEQ ID:2;
(2) recombinant vectors that step (1) is obtained changes Agrobacterium rhizogenes over to, makes up the Agrobacterium rhizogenes that contains recombinant vectors;
(3) contain the Agrobacterium rhizogenes mediation salvia 3-hydroxy-3-methylglutaryl A reductase gene and the 1-deoxy-D-xylulose-5-phosphoric acid reduction enzyme gene transformation red sage root of recombinant vectors with step (2) gained, obtain hairly root;
(4) salvia 3-hydroxy-3-methylglutaryl A reductase gene and the 1-deoxy-D-xylulose-5-phosphoric acid reduction enzyme gene in detection step (3) hairly root got the positive transgenic hairly root of The selection result.
2. the dual-gene cotransformation of said SmHMGR of claim 1 and SmDXR improves the method for TANSHINONES content in the Hairy Root Cultures of Salvia miltiorrhiza; It is characterized in that; The described plant expression vector of step (1) comprises CaMV35S promotor and terminator, MCS, replication origin and kalamycin resistance site, is pCAMBIAl304 plasmid and the pCAMBIAl304 of pBI121 plasmid through obtaining after making up
+Carrier.
3. the dual-gene cotransformation of said SmHMGR of claim 1 and SmDXR improves the method for TANSHINONES content in the Hairy Root Cultures of Salvia miltiorrhiza; It is characterized in that the described Agrobacterium rhizogenes of step (2) is agrobacterium rhizogene strain C58C1, agrobacterium rhizogene strain pBiA4 or agrobacterium rhizogene strain LBA9402.
4. the dual-gene cotransformation of said SmHMGR of claim 1 and SmDXR improves the method for TANSHINONES content in the Hairy Root Cultures of Salvia miltiorrhiza, it is characterized in that, the described detection method of step (4) is that PCR detects and quantitatively QRT-PCR detection.
5. a recombinant vectors that is used for improving Hairy Root Cultures of Salvia miltiorrhiza TANSHINONES content is characterized in that, contains salvia 3-hydroxy-3-methylglutaryl A reductase gene and 1-deoxy-D-xylulose-5-phosphoric acid reduction enzyme gene.
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Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020168707A1 (en) * | 1998-03-20 | 2002-11-14 | Michael Wayne Graham | Synthetic genes and genetic constructs comprising same i |
| JP2006288210A (en) * | 2005-04-05 | 2006-10-26 | National Institute Of Advanced Industrial & Technology | Technology for in vivo sugar chain extension by transfer of glycosyltransferase gene |
| KR100756889B1 (en) * | 2006-09-29 | 2007-09-07 | 한국생명공학연구원 | Marker free transgenic potato hairy roots using agrobacterium rhizogenes |
| CN101250542A (en) * | 2008-04-08 | 2008-08-27 | 上海师范大学 | Salvia 3-hydroxy-3-methylglutaryl A synthase gene and its coding protein and application |
| CN101250544A (en) * | 2008-04-08 | 2008-08-27 | 上海师范大学 | Salvia 3-hydroxy-3-methylglutaryl A reductase gene and its coding protein and application |
| CN101250541A (en) * | 2008-04-08 | 2008-08-27 | 上海师范大学 | Salvia 1-deoxidation xylulose-5-phosphate synthase gene 1 and its coding protein and application |
| CN101475947A (en) * | 2009-01-16 | 2009-07-08 | 上海师范大学 | Salvia 1-deoxy-D-xylulose-5-phosphate synthetase II gene, and encoding protein and use thereof |
| CN101475946A (en) * | 2009-01-16 | 2009-07-08 | 上海师范大学 | Geranylgeranyl diphosphate synthase gene in salvia root, and encoding protein and use thereof |
| CN101613704A (en) * | 2009-06-30 | 2009-12-30 | 中国中医科学院中药研究所 | The analysis and the application of a kind of salvia miltiorrhiza acetyl-CoA acyltransferase (SmAACT) gene |
| CN101654678A (en) * | 2009-06-30 | 2010-02-24 | 中国中医科学院中药研究所 | Analysis and application of salvia miltiorrhiza bunge isopentenylpyrophosphate isomerase (SmIPPI) gene |
| CN101863894A (en) * | 2010-06-01 | 2010-10-20 | 广东药学院 | Tanshinone II A derivative and preparation method and application thereof |
| CN101928717A (en) * | 2009-06-30 | 2010-12-29 | 中国中医科学院中药研究所 | Salvia miltrorrhiza squalene synthase (SmSQS) gene and encoding protein and application thereof |
| CN101928715A (en) * | 2009-06-30 | 2010-12-29 | 中国中医科学院中药研究所 | Salviamiltiorrhizabge-cytochrome P450 (SmP450) gene as well as coded protein and application thereof |
| CN101928716A (en) * | 2009-06-30 | 2010-12-29 | 中国中医科学院中药研究所 | SmFPS (Salviamiltiorrhizabge Farnesyl Pyrophosphate Synthase) gene as well as coded protein and application thereof |
| CN101942467A (en) * | 2010-08-27 | 2011-01-12 | 上海师范大学 | Method for enhancing content of tanshinone in salvia miltiorrhiza hairy root by double-key enzyme genetic transformation |
| CN101962650A (en) * | 2010-08-26 | 2011-02-02 | 上海师范大学 | Method for increasing tanshinone content of salvia miltiorrhiza hairy roots by transferring SmGGPPS gene |
-
2011
- 2011-06-22 CN CN 201110170196 patent/CN102337279B/en not_active Expired - Fee Related
Patent Citations (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020168707A1 (en) * | 1998-03-20 | 2002-11-14 | Michael Wayne Graham | Synthetic genes and genetic constructs comprising same i |
| JP2006288210A (en) * | 2005-04-05 | 2006-10-26 | National Institute Of Advanced Industrial & Technology | Technology for in vivo sugar chain extension by transfer of glycosyltransferase gene |
| KR100756889B1 (en) * | 2006-09-29 | 2007-09-07 | 한국생명공학연구원 | Marker free transgenic potato hairy roots using agrobacterium rhizogenes |
| CN101250542A (en) * | 2008-04-08 | 2008-08-27 | 上海师范大学 | Salvia 3-hydroxy-3-methylglutaryl A synthase gene and its coding protein and application |
| CN101250544A (en) * | 2008-04-08 | 2008-08-27 | 上海师范大学 | Salvia 3-hydroxy-3-methylglutaryl A reductase gene and its coding protein and application |
| CN101250541A (en) * | 2008-04-08 | 2008-08-27 | 上海师范大学 | Salvia 1-deoxidation xylulose-5-phosphate synthase gene 1 and its coding protein and application |
| CN101475947A (en) * | 2009-01-16 | 2009-07-08 | 上海师范大学 | Salvia 1-deoxy-D-xylulose-5-phosphate synthetase II gene, and encoding protein and use thereof |
| CN101475946A (en) * | 2009-01-16 | 2009-07-08 | 上海师范大学 | Geranylgeranyl diphosphate synthase gene in salvia root, and encoding protein and use thereof |
| CN101613704A (en) * | 2009-06-30 | 2009-12-30 | 中国中医科学院中药研究所 | The analysis and the application of a kind of salvia miltiorrhiza acetyl-CoA acyltransferase (SmAACT) gene |
| CN101654678A (en) * | 2009-06-30 | 2010-02-24 | 中国中医科学院中药研究所 | Analysis and application of salvia miltiorrhiza bunge isopentenylpyrophosphate isomerase (SmIPPI) gene |
| CN101928717A (en) * | 2009-06-30 | 2010-12-29 | 中国中医科学院中药研究所 | Salvia miltrorrhiza squalene synthase (SmSQS) gene and encoding protein and application thereof |
| CN101928715A (en) * | 2009-06-30 | 2010-12-29 | 中国中医科学院中药研究所 | Salviamiltiorrhizabge-cytochrome P450 (SmP450) gene as well as coded protein and application thereof |
| CN101928716A (en) * | 2009-06-30 | 2010-12-29 | 中国中医科学院中药研究所 | SmFPS (Salviamiltiorrhizabge Farnesyl Pyrophosphate Synthase) gene as well as coded protein and application thereof |
| CN101863894A (en) * | 2010-06-01 | 2010-10-20 | 广东药学院 | Tanshinone II A derivative and preparation method and application thereof |
| CN101962650A (en) * | 2010-08-26 | 2011-02-02 | 上海师范大学 | Method for increasing tanshinone content of salvia miltiorrhiza hairy roots by transferring SmGGPPS gene |
| CN101942467A (en) * | 2010-08-27 | 2011-01-12 | 上海师范大学 | Method for enhancing content of tanshinone in salvia miltiorrhiza hairy root by double-key enzyme genetic transformation |
Non-Patent Citations (4)
| Title |
|---|
| HUI XU ET AL.: "Metabolic regulation and genetic engineering of pharmaceutical component tanshinone biosynthesis in Salvia miltiorrhiza", 《JOURNAL OF MEDICINAL PLANTS RESEARCH》 * |
| SHU JINGWU ET AL.: "Cloning and characterization of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase gene for diterpenoid tanshinone biosynthesis in Salvia miltiorrhiza (Chinese sage) hairy roots", 《BIOTECHNOL. APPL. BIOCHEM.》 * |
| YAN,X ET AL.: "GenBank: FJ476255.1 Salvia miltiorrhiza 1-deoxy-d-xylulose 5-phosphate reductoisomerase mRNA, complete cds", 《GENBANK》 * |
| 廖攀: "丹参酮生物合成相关基因的克隆及其代谢调控", 《中国优秀硕士论文全文数据库》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543275A (en) * | 2016-01-21 | 2016-05-04 | 湖南大学 | Double-gene expression vectors and application thereof |
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