CN102827866B - Method for increasing content of rosmarinic acid in red-rooted salvia through transgenosis - Google Patents
Method for increasing content of rosmarinic acid in red-rooted salvia through transgenosis Download PDFInfo
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Abstract
The invention discloses a method for increasing the content of rosmarinic acid in red-rooted salvia through transgenosis. The method comprises the following four steps: 1) cloning the Antirrhinum majus Rosea1 gene; 2) constructing a plant expression vector containing the Antirrhinum majus Rosea1 gene; 3) preparing an Agrobacterium tumefaciens strain of the plant expression vector containing the Antirrhinum majus Rosea1 gene; and 4) preparing a transgenic red-rooted salvia plant. The real-time fluorescent quantitative reverse transcription-polymerase chain reaction is adopted to detect the expression of the Antirrhinum majus Rosea1 gene in transgenic red-rooted salvia; and high performance liquid chromatography is performed to measure the content of rosmarinic acid in the transgenic red-rooted salvia expressing the Antirrhinum majus Rosea1 gene and the transgenic red-rooted salvia plant with higher rosmarinic acid content is obtained through screening. The content of rosmarinic acid in the 90-day dry roots of the transgenic red-rooted salvia plant obtained by the method is up to 129.37+/-3.47mg/g, which is 2.16 times of the content of rosmarinic acid in the dry roots of the common non-transgenic red-rooted salvia.
Description
Technical field
The invention belongs to the Gene Engineering of Medicinal Plants technical field, be specifically related to transform the method that Common Snapdragon Rosea1 gene improves rosmarinic acid contents in the red sage root.
Background technology
The red sage root is China's large famous-region drug commonly used, and the dry root and rhizome for the Labiatae salvia red sage root (Salvia miltiorrhiza Bunge), have significant clinical treatment effect to cardiovascular and cerebrovascular diseases, cancer and various inflammation.Along with the further investigation of effective component in red sage and pharmacologically active thereof, the medicine that the red sage root of take in recent years is main raw material, preparation, makeup and serial health product emerge in an endless stream, and supply falls short of demand for red sage root resource.Be accompanied by the minimizing gradually of expanding day and the wild resource of red rooted salvia demand, the artificial growth area increases year by year.Yet as often cross-pollinated plant, in red sage root kind, make a variation larger, the proterties complexity.Planted rooted salvia is only planted the situation of not selecting and is made each place of production red sage root quality uneven, and deterioration of strains is serious, and active constituent content is unstable, becomes one of biggest obstacle that the red sage root cracks the international market on a large scale as high-quality plant amedica.Therefore, improve the content of content, the especially rosmarinic acid of effective constituent in the red sage root, significant to cultivating excellent red sage root resource.
In order to improve quality, to meet the need of market, the content that utilizes in recent years the modern biotechnology means such as genetically engineered, cell engineering to be improved to improve its medicinal material effective constituent to the red sage root more and more is subject to people's attention.Among all multi-methods, utilize genetic engineering technique to be transformed the hereditary property of medicinal plant secondary metabolism approach, improve active components in medicinal plant content, cultivation can accumulate the new variety of target secondary metabolite in a large number, more and more receives people's concern.The modern molecular breeding technology that the transgenic technology of take is core at the improvement medicinal plant, enrich natural resources of Chinese medicinal materials, improve disease resistance and resistance, tempting application prospect arranged in the novel transgenosis medicinal material of the high natural drug content of cultivation.But the method for utilizing at present transgenic technology to improve the content, particularly rosmarinic acid contents of salvianolic acid constituents in the red sage root there is no report.
The plant metabolism approach is the polystep reaction participated in by plurality of enzymes, is subject to the impact of the factors such as growth, environment, and is difficult to prove effective when individual gene is modified with.Transcriptional regulator can regulate and control the expression of a plurality of key genes in the Plant Secondary Metabolites route of synthesis, effectively activate or suppress the whole piece pathways metabolism, thereby regulate specific secondary metabolites whether synthesize and accumulation volume the number.The expression that changes transcription factor often can cause proterties that this transcription factor is controlled in plant that larger change occurs, and therefore, utilizing engineered method regulative transcription factor is the plant genetic operational means with using value.The present invention, by building the transcription factor over-express vector, imports Common Snapdragon Rosea1 gene in the red sage root, improves the content of rosmarinic acid in the red sage root, can fill up the blank of this area research, thereby this technological line has stronger novelty.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiency in above-mentioned technology, and a kind of novel method that improves rosmarinic acid contents in the red sage root is provided.
Solving the problems of the technologies described above adopted technical scheme comprises the steps:
1, the clone of Common Snapdragon Rosea1 gene
Extract the total RNA of Common Snapdragon reverse transcription and become cDNA standby, upstream primer, the downstream primer of design Common Snapdragon Rosea1 genes encoding frame, and introduce BglII restriction enzyme site and protection bases G A on upstream primer
aGATCT,introduce BstEII restriction enzyme site and protection base CAG on downstream primer
gGTCACC, the sequence of upstream primer is 5 '-GA
aGATCTaTGGAAAAGAATTGTCGTGGAGT-3 ', the sequence of downstream primer is 5 '-CAG
gGTCACCtTAATTTCCAATTTGTTGGGCCT-3 ', take Common Snapdragon cDNA as template, through polymerase chain reaction (PCR) amplification, the amplified production obtained carries out electrophoretic separation, reclaiming amplified production is connected with the T4DNA ligase enzyme with pMD19-T Simple carrier, connecting product adopts the heat shock conversion method to proceed to bacillus coli DH 5 alpha, the volume ratio that connects product and bacillus coli DH 5 alpha is 1:10, connecting product proceeds in 100 μ L bacillus coli DH 5 alpha competent cells, random choose gained positive colony, through the bacterium colony polymerase chain reaction, detect, it is as follows that sequence verification obtains the encoding sequence of described gene:
atggaaaaga attgtcgtgg agtgagaaaa ggtacttgga ccaaagaaga 50
agacactctc ttgaggcaat gtatagaaga gtatggtgaa gggaaatggc 100
atcaagttcc acacagagca gggttgaacc ggtgtaggaa gagttgcagg 150
ctgaggtggt tgaattatct gaggccaaat atcaaaagag gtcggttttc 200
gagagatgaa gtggacctaa ttgtgaggct tcataagctg ttgggtaaca 250
aatggtcgct gattgctggt agaattcctg gaaggacagc taatgacgtg 300
aagaactttt ggaatactca tgtggggaag aatttaggcg aggatggaga 350
acgatgccgg aaaaatgtta tgaacacaaa aaccattaag ctgactaata 400
tcgtaagacc ccgagctcgg accttcaccg gattgcacgt tacttggccg 450
agagaagtcg gaaaaaccga tgaattttca aatgtccggt taacaactga 500
tgagattcca gattgtgaga agcaaacgca attttacaat gatgttgcgt 550
cgccacaaga tgaagttgaa gactgcattc agtggtggag taagttgcta 600
gaaacaacgg aggatgggga attaggaaac ctattcgagg aggcccaaca 650
aattggaaat taa 663
2, build the plant expression vector that contains Common Snapdragon Rosea1 gene
The pMD 19-T Simple carrier and the pCAMBIA-1302 plant expression vector that contain Common Snapdragon Rosea1 gene with BglII and BstEII double digestion, the enzyme reclaimed is cut product, with the T4DNA ligase enzyme, connect, connecting product adopts the heat shock conversion method to transform bacillus coli DH 5 alpha, the picking mono-clonal, carry out the detection of bacterium colony polymerase chain reaction and extract the plasmid enzyme restriction screening positive clone, obtain the plant expression vector that contains Common Snapdragon Rosea1 gene, this expression vector sequence is as nucleotides sequence list<210 > 2.
The agrobacterium tumefaciens bacterial strain of the plant expression vector that 3, preparation contains Common Snapdragon Rosea1 gene
The plant expression vector that will contain Common Snapdragon Rosea1 gene transforms agrobacterium tumefaciens EHA105 competent cell, adopt the frozen-thawed method to be transformed, product after conversion is inverted and is cultivated 18～36 hours in 28 ℃ of constant incubators, the single bacterium colony of picking resistance carries out the screening of bacterium colony polymerase chain reaction, obtains the agrobacterium tumefaciens bacterial strain that contains Common Snapdragon Rosea1 gene plant expression vector.
4, preparation transgenosis red sage root plant
Adopt the conventional organization cultural method to obtain aseptic red sage root test-tube plantlet, Agrobacterium tumefaciens mediated red sage root genetic conversion system adopts Ye Panfa, being about to the succeeding transfer culture red sage root aseptic seedling blade of 15 days is cut into small pieces, proceed in every 1L substratum and contain the 6-benzyl aminoadenine that 10mL concentration is 1.0mg/mL, on the MS substratum of the a-naphthylacetic acid that 1mL concentration is 1.0mg/mL, preculture is 1 day, contaminate with the agrobacterium tumefaciens bacterial strain that contains Common Snapdragon Rosea1 gene plant expression vector the red sage root blade that preculture is crossed, the dip-dye method is that 100 rev/mins of 28 ℃ of constant temperature are contaminated 25～30 minutes, the blade of contaminating is placed in every 1L substratum and contains the 6-benzyl aminoadenine that 10mL concentration is 1.0mg/mL, on the MS substratum of the a-naphthylacetic acid that 1mL concentration is 1.0mg/mL, dark the cultivation has Agrobacterium to grow to blade edge in 2～3 days, blade takes out from substratum, be transferred in every 1L substratum in the MS substratum that contains the cephamycin that Totomycin that 10mL concentration is 3mg/mL and 10mL concentration are 200mg/mL and cultivated, grow after resistant buds it to be transferred in every 1L substratum on the 1/2MS substratum that contains the cephamycin that Totomycin that 10mL concentration is 3mg/mL and 10mL concentration are 200mg/mL and take root, obtain the regeneration red sage root plant of hygromycin resistance, DNA with the specific detection primer polymerase chain reaction (PCR) amplification of Common Snapdragon Rosea1 gene regeneration red sage root plant, the positive strain of observing 663bp purpose band under the ultraviolet ray that wavelength is 302nm is transgenosis red sage root plant.
In the plant expression vector step 2 that contains Common Snapdragon Rosea1 gene at structure of the present invention, BglII and BstEII double digestion are: get the pMD19-T Simple carrier that contains Common Snapdragon Rosea1 gene, 10 * H Buffer, redistilled water, restriction enzyme BglII was in 37 ℃ of reactions 1 hour 20 minutes, add again restriction enzyme BstEII, 60 ℃ are reacted 1 hour, restriction enzyme BglII and restriction enzyme BstEII, the pMD19-T Simple carrier that contains Common Snapdragon Rosea1 gene, redistilled water, the volume ratio of 10 * HBuffer is 3:3:20:19:5.Get pCAMBIA-1302 plant expression vector, 10 * H Buffer, redistilled water, restriction enzyme BglII in 37 ℃ of reactions 1 hour 20 minutes, add again restriction enzyme BstEII, 60 ℃ are reacted 1 hour, and the volume ratio of restriction enzyme BglII and restriction enzyme BstEII, pCAMBIA-1302 plant expression vector, redistilled water, 10 * H Buffer is 3:3:20:19:5.
The present invention proceeds to red sage root plant by Common Snapdragon Rosea1 gene, and screening has obtained the transgenosis red sage root plant that rosmarinic acid contents significantly improves.In the growth genetically modified red sage root dry root of 90 days, the content of rosmarinic acid is up to 129.37 ± 3.47mg/g, is 2.16 times of rosmarinic acid contents (59.76 ± 0.50mg/g) in non-transformed common red sage root dry root of contemporaneously.
The accompanying drawing explanation
Fig. 1 be preparation contain Common Snapdragon Rosea1 gene the agrobacterium tumefaciens bacterial strain of plant expression vector the time bacterium colony polymerase chain reaction the selection result electrophorogram.
Fig. 2 is the DNA electrophorogram of the transgenosis red sage root plant of polymerase chain reaction detection.
Fig. 3 is that Common Snapdragon Rosea1 genetic expression spirogram in transgenosis red sage root plant is detected in real time fluorescent quantitative reverse transcription-polymerase chain reaction.
Fig. 4 is the color atlas of rosmarinic acid contents in high-performance liquid chromatogram determination transgenosis red sage root plant.
Fig. 5 is the color atlas of rosmarinic acid contents in high-performance liquid chromatogram determination contrast red sage root plant.
Embodiment
Below in conjunction with drawings and Examples, the present invention is described in more detail, but the invention is not restricted to these embodiment.
1, the clone of Common Snapdragon Rosea1 gene
(1) extract the total RNA of Common Snapdragon
Choose fresh, healthy and strong Common Snapdragon seedling complete stool 100mg, utilize the E.Z.N.A. of OMEGA company
tMplant RNA Kit extracts the total RNA of test kit separation and Extraction Common Snapdragon.Extracting method is with reference to the E.Z.N.A. of OMGA company
tMplant RNA Kit extracts the test kit specification sheets.
(2) reverse transcription Common Snapdragon cDNA
Adopt the PrimeScript RT reagent Kit of Takara company reverse transcription test kit to carry out, reaction system is: 5 * PrimeScript
tM buffe 2 μ L, PrimeScript
tMrT Enzyme Mix 10.5 μ L, Oligo dT Primer 0.5 μ L, Random 6mers 0.5 μ L, the total RNA 1 μ L of Common Snapdragon; RNase free dH
2o 5.5 μ L, the reverse transcription reaction condition is: hatch 30 minutes for 37 ℃, 85 ℃ of reactions 5 seconds cDNA, by 10 times of redistilled water dilutions for cDNA ,-20 ℃ of Refrigerator stores are standby.
(3) polymerase chain reaction (PCR) amplification of Common Snapdragon Rosea1 gene
With reference to the Common Snapdragon Rosea1 gene that in GENBANK, oneself announces; with Premier Primer 5.0 softwares; design can amplify upstream primer, the downstream primer of Common Snapdragon Rosea1 genes encoding frame, and introduces BglI restriction enzyme site and protection bases G A on upstream primer
aGATCT, introduce BstEII restriction enzyme site and protection base CAG on downstream primer
gGTCACC, so that construction of expression vector.The upstream primer sequence is 5 '-GA
aGATCTaTGGAAAAGAATTGTCGTGGAGT-3 ', the downstream primer sequence is 5 '-CAG
gGTCACCtTAATTTCCAATTTGTTGGGCCT-3 '.Take Common Snapdragon cDNA as template, through polymerase chain reaction (PCR) amplification, the reaction amplification system is: Common Snapdragon cDNA2 μ L, dNTP(is purchased from Takara company) 4 μ L, upstream primer 1 μ L, downstream primer 1 μ L, LA Taq Polymerase (purchased from Takara company) 0.5 μ L, 10 * LA PCR BufferII(is purchased from Takara company) 5 μ L, redistilled water 36.5 μ L; The method of polymerase chain reaction (PCR) amplification is: 94 ℃, 5 minutes; 94 ℃, 30 seconds, 60 ℃, 30 seconds, 72 ℃, 45 seconds, 35 circulations; 72 ℃, 10 minutes.The agarose gel electrophoresis that is 1% with massfraction by the amplified production of acquisition is separated, reclaim target fragment, with pMD19-T Simple carrier (purchased from Takara company), with T4DNA ligase enzyme (purchased from Takara company), be connected, linked system is: the PCR purifying reclaims product 7.5 μ L, 10 * T4 ligase enzyme Buffer(is purchased from Takara company) 1 μ L, T4DNA ligase enzyme 1 μ L, pMD19-T Simple carrier 0.5 μ L; Condition of contact is: 4 ℃ of connections spend the night (12～16 hours).10 μ L connect product and adopt the heat shock conversion method to proceed in 100 μ L bacillus coli DH 5 alpha competent cells, and random choose gained positive colony detects through the bacterium colony polymerase chain reaction.Send the order-checking of Shenzhen Hua Da genome company after detection, sequencing result is:
atggaaaaga attgtcgtgg agtgagaaaa ggtacttgga ccaaagaaga 50
agacactctc ttgaggcaat gtatagaaga gtatggtgaa gggaaatggc 100
atcaagttcc acacagagca gggttgaacc ggtgtaggaa gagttgcagg 150
ctgaggtggt tgaattatct gaggccaaat atcaaaagag gtcggttttc 200
gagagatgaa gtggacctaa ttgtgaggct tcataagctg ttgggtaaca 250
aatggtcgct gattgctggt agaattcctg gaaggacagc taatgacgtg 300
aagaactttt ggaatactca tgtggggaag aatttaggcg aggatggaga 350
acgatgccgg aaaaatgtta tgaacacaaa aaccattaag ctgactaata 400
tcgtaagacc ccgagctcgg accttcaccg gattgcacgt tacttggccg 450
agagaagtcg gaaaaaccga tgaattttca aatgtccggt taacaactga 500
tgagattcca gattgtgaga agcaaacgca attttacaat gatgttgcgt 550
cgccacaaga tgaagttgaa gactgcattc agtggtggag taagttgcta 600
gaaacaacgg aggatgggga attaggaaac ctattcgagg aggcccaaca 650
aattggaaat taa 663
Sequencing result shows, the encoder block consensus nucleic acid sequence of the Common Snapdragon Rosea1 gene of reporting in the sequence of cloning and GENBANK (accession number of Common Snapdragon Rosea1 gene in GENBANK is DQ275529).
2, build the plant expression vector that contains Common Snapdragon Rosea1 gene
Take pCAMBIA-1302 as plant expression vector, the biomaterial that pCAMBIA-1302 is market sale, by Australian CAMBIA company, produced, with the green fluorescence protein gene on Common Snapdragon Rosea1 Gene Replacement pCAMBIA-1302, with BglII and BstEII double digestion contains Common Snapdragon Rosea1 gene respectively pMD19-T Simple carrier and pCAMBIA-1302 plant expression vector, the system method of the pMD19-T Simple carrier that double digestion contains Common Snapdragon Rosea1 gene is: get the pMD19-T Simple carrier 10 μ L that contain Common Snapdragon Rosea1 gene, 10 * HBuffer2.5 μ L, redistilled water 9.5 μ L, restriction enzyme BglII1.5 μ L, under 37 ℃, reaction is 1 hour 20 minutes, add again 1.5 μ L restriction enzyme BstEII, under 60 ℃, reaction is 1 hour, the recovery purifying obtains Common Snapdragon Rosea1 gene enzyme and cuts product, restriction enzyme BglII and restriction enzyme BstEII, the pMD19-T Simple carrier that contains Common Snapdragon Rosea1 gene, redistilled water, the volume ratio of 10 * H Buffer is 3:3:20:19:5.
The system method of double digestion pCAMBIA-1302 plant expression vector is: pCAMBIA-1302 plant expression vector 10 μ L, 10 * H Buffer, 2.5 μ L, restriction enzyme BglII 1.5 μ L, redistilled water 9.5 μ L, under 37 ℃, reaction is 1 hour 20 minutes, add again 1.5 μ L restriction enzyme BstEII to react 1 hour under 60 ℃, the recovery purifying obtains the pCAMBIA-1302 klenow fragment and cuts product, restriction enzyme BglII and restriction enzyme BstEII, the pCAMBIA-1302 plant expression vector, redistilled water, the volume ratio of 10 * HBuffer is 3:3:20:19:5.
The Common Snapdragon Rosea1 gene enzyme reclaimed cuts product and the pCAMBIA-1302 klenow fragment is cut product, with the T4DNA ligase enzyme, connects; Linked system is: Common Snapdragon Rosea1 gene enzyme is cut product 6 μ L, the pCAMBIA-1302 klenow fragment is cut product 2 μ L, 10 * T4 ligase enzyme Buffer, 1 μ L, T4DNA ligase enzyme 1 μ L; Condition of contact is: 4 ℃ of connections spend the night (12～16 hours), connecting product adopts the heat shock conversion method to transform bacillus coli DH 5 alpha, the picking mono-clonal, carry out the bacterium colony polymerase chain reaction and extract the plasmid enzyme restriction screening positive clone, obtain the plant expression vector that contains Common Snapdragon Rosea1 gene, this expression vector sequence is as nucleotides sequence list<210 > 2.
The above-mentioned plant expression vector that contains Common Snapdragon Rosea1 gene, can be used for improving by gene engineering strategy the content of rosmarinic acid in the red sage root.
The agrobacterium tumefaciens bacterial strain of the plant expression vector that 3, preparation contains Common Snapdragon Rosea1 gene
The plant expression vector that will contain Common Snapdragon Rosea1 gene transforms agrobacterium tumefaciens EHA105 competent cell, adopt the frozen-thawed method to be transformed, agrobacterium tumefaciens EH4105 competent cell is the biomaterial that sell in market, by Australian CAMBIA company, produced, the frozen-thawed method for transformation is: get 100 μ L agrobacterium tumefaciens EHA105 competent cells and join that centrifuge tube is built-in to be dissolved on ice, to join in the competent cell after dissolving containing Common Snapdragon Rosea1 gene plant expression vector 30ng, ice bath 40 minutes, liquid nitrogen flash freezer 1 minute, 37 ℃ of water-bath heat shocks 3 minutes, add 400 μ L LB liquid nutrient mediums in centrifuge tube, 100 rev/mins of renewal cultivations of 28 ℃ of constant temperature 3～4 hours, to transform after product evenly coats on 10mL LB solid medium (kantlex that the rifomycin that is 20mg/mL containing 40 μ L concentration and 50 μ L concentration are 20mg/mL), be inverted and cultivate 18～36 hours in 28 ℃ of constant incubators, the single bacterium colony of picking resistance, be inoculated in (kantlex that the rifomycin that is 20mg/mL containing 20 μ L concentration and 25 μ L concentration are 20mg/mL) in 10mL LB liquid nutrient medium, 180 rev/mins of shaking culture of 28 ℃ of constant temperature 16 hours, carry out the screening of bacterium colony polymerase chain reaction, the agrobacterium tumefaciens bacterial strain of the plant expression vector that acquisition contains Common Snapdragon Rosea1 gene.The single bacterium colony of picking resistance carries out bacterium colony polymerase chain reaction the selection result and sees Fig. 1, in Fig. 1, M is DNA molecular amount standard, be followed successively by from top to bottom 100,250,500,750,1000,2000bp, the first swimming lane+expression positive control contains the plant expression vector of Common Snapdragon Rosea1 gene, the second swimming lane 1 and the 3rd swimming lane 2 mean different strains, and the 4th swimming lane-expression does not connect the plant expression vector pCAMBIA-1302 of described gene.As seen from Figure 1, use the polymerase chain reaction special primer, can amplify the specific DNA fragment of 663bp, illustrate that containing Common Snapdragon Rosea1 gene plant expression vector successfully is transformed in the agrobacterium tumefaciens bacterial strain.
4, preparation transgenosis red sage root plant
(1) the Agrobacterium tumefaciens mediated Common Snapdragon Rosea1 gene transformation red sage root
The acquisition of aseptic red sage root test-tube plantlet can adopt the conventional organization cultural method, and concrete grammar is as follows:
Salvia seeds is rinsed with flowing water, by volume fraction, be 75% aqueous ethanolic solution surface sterilization 20 seconds, redistilled water rinses 2 times, each 4 minutes, the mercuric chloride solution surface sterilization that is 0.1% with massfraction 10 minutes, redistilled water rinses 5 times, seed blots the remaining moisture in surface with filter paper and is placed on the MS minimum medium and sprouts, 25 ℃, 16 hours/8 hours 3000Lux light/dark cultivations, the in vitro cuttings of acquisition, cut off from internode, will be with the stem segment cuttage of 2 axillalry buds on the 1/2MS substratum, every surrounding successive propagation is once.Transform the red sage root test-tube plantlet that material used is subculture 2～4 times, wherein the succeeding transfer culture seedling of 15 days is for gene transformation, and the succeeding transfer culture seedling of 30 days is for nucleic acid extraction.
Agrobacterium tumefaciens mediated red sage root genetic conversion system adopts Ye Panfa, and concrete grammar is as follows:
Choose the succeeding transfer culture red sage root aseptic seedling of 15 days, its blade is cut into to the fritter of 0.5 centimetre of 0.5 cm x, proceed to that in the preculture substratum, (pre-culture medium is that every 1LMS substratum contains the 6-benzyl aminoadenine that 10mL concentration is 1.0mg/mL, the substratum of the a-naphthylacetic acid that 1mL concentration is 1.0mg/mL), under normal culture condition, preculture is 1 day, a part of blade that preculture is crossed proceeds in the agrobacterium tumefaciens bacterium liquid that contains Common Snapdragon Rosea1 gene plant expression vector diluted, 100 rev/mins of dip-dyes of 28 ℃ of constant temperature 25～30 minutes, take out blade, with aseptic filter paper, blot, transferring on the preculture substratum dark the cultivation has Agrobacterium to grow to blade edge in 2～3 days, the pre-incubated blade of another part does not carry out the dip-dye of Agrobacterium, directly on the preculture substratum, cultivates as blank, after sprouting, bud is peeled off, and is placed on the 1/2MS substratum and takes root, cultivate after 2～3 days, blade is taken out from former substratum, be transferred to and select on substratum (selecting substratum is the substratum that contains the cephamycin that Totomycin that 10mL concentration is 3mg/mL and 10mL concentration are 200mg/mL in 1L MS substratum), within every 10～15 days, change and once select substratum, when resistant buds grows to approximately 0.5～1 centimetre of left and right, resistant buds is cut, on the 1/2MS substratum proceeded to, take root, screening the plant that can normally take root after 3～4 times moves and receives succeeding transfer culture on the MS substratum, complete plant is cut off from internode, stem segment cuttage succeeding transfer culture on the MS substratum with 2 axillalry buds, within 4～6 weeks, a subculture is changed in left and right, the positive transformation plant of screening and contrast strain are transplanted to equal conditions in greenhouse and are cultivated, and transplant the transgenosis red sage root plant of the cultivating 90 days mensuration for rosmarinic acid contents.
(2) polymerase chain reaction of transgenosis red sage root plant is detected
Adopt Bioer company DNA of plants to extract test kit and extract contrast red sage root plant, transform described gene red sage root strain and transform the DNA that does not contain described gene red sage root strain, adopt the upstream primer of Common Snapdragon Rosea1 genes encoding frame, downstream primer carries out the polymerase chain reaction detection to Common Snapdragon Rosea1 gene, detect electrophorogram and see Fig. 2, in Fig. 2, M is DNA molecular amount standard, be followed successively by from top to bottom 100, 250, 500, 750, 1000, 2000bp, the first swimming lane+expression positive control contains the plant expression vector of Common Snapdragon Rosea1 gene, the second swimming lane-expression is not containing the DNA profiling contrast, it is non-transformed red sage root strain that the 3rd swimming lane CK-means to contrast red sage root plant, the zero load contrast strain that it is not conversion of plant expression vector pCAMBIA1302 containing described gene red sage root strain that the 4th swimming lane CK+ means to transform, all the other swimming lane 1-20 mean different transgenic lines, as seen from Figure 2, the conversion red sage root genomic dna of take is template amplification, can observe 663bp purpose band under the ultraviolet ray that is 302nm at wavelength, do not amplify any fragment and take when contrast red sage root plant does not contain described gene red sage root genomic dna as template with conversion, illustrate that Common Snapdragon Rosea1 gene has inserted in red sage root genome.
5, the expression of Common Snapdragon Rosea1 gene in transgenosis red sage root plant is detected in real time fluorescent quantitative reverse transcription-polymerase chain reaction
(1) design of primer is with synthetic
With Premier Primer 5.0 softwares, the synthetic required Common Snapdragon Rosea1 gene of real time fluorescent quantitative reverse transcription-polymerase chain reaction (PCR) amplification and the primer of red sage root house-keeping gene Actin muscle-β gene of being applicable to of design.Rosea1 gene and red sage root house-keeping gene Actin muscle-β gene real time fluorescent quantitative reverse transcription-polymerase chain reaction (PCR) amplification detects primer and is respectively qRos1-F, qRos1-R; SmACT-F, SmACT-R, flag F be upstream primer, mark R's is downstream primer.Primer sequence is respectively:
qRos1-F：5’-AAATGGTCGCTGATTGCTGGTA-3’，
qRos1-R：5’-CGTTCTCCATCCTCGCCTAAAT-3’；
SmACT-F：5’-AGGAACCACCGATCCAGACA-3’，
SmACT-R：5’-GGTGCCCTGAGGTCCTGTT-3’。
(2) extraction of the total RNA of the red sage root
Adopt the E.Z.N.A. of OMEGA company
tMplant RNA Kit extracts test kit to carry out according to the test kit specification sheets;
(3) cDNA the first chain is synthetic
The synthetic employing Takara PrimeScript RT reagent Kit of the company reverse transcription test kit of cDNA the first chain carries out.Reaction system is: 5 * PrimeScript
tMbuffe 2 μ L, PrimeScript
tMrT Enzyme Mix 10.5 μ L, Oligo dT Primer 0.5 μ L, Random 6mers 0.5 μ L, the total RNA 1 μ L of the red sage root; RNase free dH
2o 5.5 μ L, reaction conditions is 37 ℃ hatches 30 minutes, and 85 ℃ of reactions obtain cDNA in 5 seconds, and by 50 times of the cDNA of gained dilutions ,-20 ℃ save backup.
(4) expression of Common Snapdragon Rosea1 gene in transgenosis red sage root plant is detected in real time fluorescent quantitative reverse transcription-polymerase chain reaction
The non-transformed red sage root strain of take is blank, transforms the negative contrast of plant expression vector red sage root strain that does not contain described gene, measures the expression amount of Common Snapdragon Rosea1 gene with real time fluorescent quantitative reverse transcription-polymerase chain reaction.Real time fluorescent quantitative reverse transcription-polymerase chain reaction (PCR) amplification system is: cDNA 5 μ L, 2 * SYBR Premix Ex Taq
tMiI(is purchased from Takara company) 10 μ L, fluorescent quantitation reverse transcription-polymerase chain reaction (PCR) amplification detects upstream primer 0.2 μ L, fluorescent quantitation reverse transcription-polymerase chain reaction (PCR) amplification detects downstream primer 0.2 μ L, redistilled water 4.6 μ L.Real time fluorescent quantitative reverse transcription-polymerase chain reaction (PCR) amplification condition is: 94 ℃ of denaturations, 1 minute; 40 circulations (94 ℃ of sex change, 10 seconds; 60 ℃ of fluorescent signals of annealing and collect, 25 seconds); 95 ℃, 1 minute; 60 ℃, 1 minute; 60 ℃～95 ℃, within every 30 seconds, raise 0.5 ℃, collect first order fluorescence.After reaction finishes, the fluorescent signal value adopts " the relatively relative quantification method of Ct value " to carry out gene expression analysis.Concrete treatment process is according to " iQ
tM5 multiple real time fluorescence quantifying PCR specification sheetss " carry out.The results are shown in Figure 3, in Fig. 3,1-20, represent different transgenic lines; CK-, be non-transformed red sage root strain, and CK+, for transforming the strain of the plant expression vector that does not contain described gene; *, * *, * * * mean that respectively experimental group and control group relatively have the significance difference opposite sex (P<0.05,0.01,0.001).As seen from Figure 3, except the 9th strain, the relative expression quantity of Rosea1 gene has significant difference (P<0.05) compared with the control, shows described goal gene overexpression in the red sage root.
6, use the content of rosmarinic acid in high-performance liquid chromatogram determination transgenosis red sage root strain
(1) preparation of high-efficient liquid phase chromatogram condition and system suitability and standardized solution
1. high-efficient liquid phase chromatogram condition
Adopt the Japanese SHIMADZU LC-2010 of company high performance liquid chromatograph, chromatographic column is Phenomenex silica matrix post (5 μ mC
18reverse post, 4.6mm * 250mm), aqueous acetic acid, acetonitrile and the methyl alcohol that the massfraction of take is 0.4% is the eluent gradient wash-out, and the eluent gradient elution program is 0.01～5 minute, the aqueous acetic acid volume that massfraction is 0.4% reduces to 90% by 95%, and the acetonitrile volume rises to 10% by 5%; 5～25 minutes, the aqueous acetic acid volume that massfraction is 0.4% reduced to 67% by 90%, and the acetonitrile volume rises to 30% by 10%, and the methyl alcohol volume rises to 3% by 0; 25～40 minutes, the aqueous acetic acid volume that massfraction is 0.4% reduced to 60% by 67%, and the acetonitrile volume rises to 35% by 30%, and the methyl alcohol volume rises to 5% by 3%; 30 ℃ of column temperatures, flow velocity 1.0mL/ minute, detect wavelength 280nm, sample size 20 μ L.
2. preparing standard solution
Preparing standard solution: take each 10.0mg of rosmarinic acid standard substance, the volume fraction of take is joined to obtain the concentration standard substance mother liquor that is 10.0mg/mL as 75% methanol solution as solvent, be placed in 4 ℃ of refrigerators and save backup.During experiment, get respectively the standard solution that the standard substance mother liquor is diluted to concentration gradient, sample introduction analysis after 0.22 μ m filtering with microporous membrane.
The eluent gradient elution program adopted in the present invention, the rosmarinic acid retention time is 24.82 ± 0.08 minutes, peak shape is good, can guarantee separating of rosmarinic acid and other liposoluble ingredient in the red sage root.
(2) drawing standard curve
Draw respectively rosmarinic acid standard substance mother liquor and be diluted to 2.0mg/mL, 1.0mg/mL, 0.5mg/mL, 0.25mg/mL, 0.125mg/mL, 6 concentration gradients of 0.0625mg/mL, sample introduction analysis after 0.22 μ m filtering with microporous membrane, drawing standard curve.According to above-mentioned corresponding chromatographic condition sample introduction, record collection of illustrative plates and chromatographic parameter, with the rosmarinic acid peak area, standard substance concentration is carried out to regression analysis respectively, the equation of linear regression that obtains rosmarinic acid is:
y=6×10
-8x-0.015(r
2=0.9990)
In formula, x means the peak area of rosmarinic acid in sample, and y means the concentration of rosmarinic acid, the mg/mL of unit, and r means relation conefficient.
(3) preparation sample solution
30 ℃ of red sage root dry root are dried to constant weight, being placed in mortar grinds, take respectively transgenosis and each 25mg of contrast red sage root plant dried powder, be placed in the 1.5mL centrifuge tube, adding 500 μ L volume fractions is 75% methanol aqueous solution, 30 ℃ of ultrasonic extraction with frequency 30KHz 20 minutes, 12000 rev/mins centrifugal 6 minutes.Shift supernatant stand-by to new centrifuge tube, residue extracts twice again by same procedure, merges supernatant liquor three times, after 0.22 μ m filtering with microporous membrane, with high performance liquid chromatograph, is analyzed, and records rosmarinic acid peak area in each sample, the substitution equation of linear regression
y=6×10
-8x-0.015
Calculate and obtain the content of rosmarinic acid, in formula, x means the peak area of rosmarinic acid in sample, and y means the concentration of rosmarinic acid.
Content with rosmarinic acid in high-performance liquid chromatogram determination transgenosis red sage root strain, the results are shown in Figure 4, Fig. 5, from Fig. 4, Fig. 5, in the growth genetically modified red sage root dry root of 90 days, the content of rosmarinic acid is up to 129.37 ± 3.47mg/g, is 2.16 times of rosmarinic acid contents (59.76 ± 0.50mg/g) in non-transformed common red sage root dry root of contemporaneously.
The present embodiment has obtained the transgenosis red sage root plant of rosmarinic acid high yield by the gene engineering strategy that transforms Common Snapdragon Rosea1 gene; with high effective liquid chromatography for measuring the content of rosmarinic acid in the transgenosis red sage root, for large-scale production rosmarinic acid the final red sage root resource scarcity problem that solves provide a kind of Perfected process.
Claims (2)
1. a transgenosis improves the method for rosmarinic acid contents in the red sage root, it is characterized in that the method comprises the steps:
(1) clone of Common Snapdragon Rosea1 gene
Extract the total RNA of Common Snapdragon reverse transcription and become cDNA standby, upstream primer, the downstream primer of design Common Snapdragon Rosea1 genes encoding frame, and introduce BglII restriction enzyme site and protection bases G A on upstream primer
aGATCT,introduce BstEII restriction enzyme site and protection base CAG on downstream primer
gGTCACC, the sequence of upstream primer is 5 '-GA
aGATCTaTGGAAAAGAATTGTCGTGGAGT-3 ', the sequence of downstream primer is 5 '-CAG
gGTCACCtTAATTTCCAATTTGTTGGGCCT-3 ', take Common Snapdragon cDNA as template, through polymerase chain reaction (PCR) amplification, the amplified production obtained carries out electrophoretic separation, reclaiming amplified production is connected with the T4DNA ligase enzyme with pMD19-T Simple carrier, connecting product adopts the heat shock conversion method to proceed to bacillus coli DH 5 alpha, the volume ratio that connects product and bacillus coli DH 5 alpha is 1:10, connecting product proceeds in 100 μ L bacillus coli DH 5 alpha competent cells, random choose gained positive colony, through the bacterium colony polymerase chain reaction, detect, it is as follows that sequence verification obtains the encoding sequence of described gene:
atggaaaaga attgtcgtgg agtgagaaaa ggtacttgga ccaaagaaga 50
agacactctc ttgaggcaat gtatagaaga gtatggtgaa gggaaatggc 100
atcaagttcc acacagagca gggttgaacc ggtgtaggaa gagttgcagg 150
ctgaggtggt tgaattatct gaggccaaat atcaaaagag gtcggttttc 200
gagagatgaa gtggacctaa ttgtgaggct tcataagctg ttgggtaaca 250
aatggtcgct gattgctggt agaattcctg gaaggacagc taatgacgtg 300
aagaactttt ggaatactca tgtggggaag aatttaggcg aggatggaga 350
acgatgccgg aaaaatgtta tgaacacaaa aaccattaag ctgactaata 400
tcgtaagacc ccgagctcgg accttcaccg gattgcacgt tacttggccg 450
agagaagtcg gaaaaaccga tgaattttca aatgtccggt taacaactga 500
tgagattcca gattgtgaga agcaaacgca attttacaat gatgttgcgt 550
cgccacaaga tgaagttgaa gactgcattc agtggtggag taagttgcta 600
gaaacaacgg aggatgggga attaggaaac ctattcgagg aggcccaaca 650
aattggaaat taa 663
(2) build the plant expression vector that contains Common Snapdragon Rosea1 gene
The pMD19-T Simple carrier that contains Common Snapdragon Rosea1 gene with BglII and BstEII double digestion and pCAMBIA-1302 plant expression vector, the enzyme reclaimed is cut product, with the T4DNA ligase enzyme, connect, connecting product adopts the heat shock conversion method to transform bacillus coli DH 5 alpha, the picking mono-clonal, carry out the detection of bacterium colony polymerase chain reaction and extract the plasmid enzyme restriction screening positive clone, obtaining the plant expression vector that contains Common Snapdragon Rosea1 gene, this expression vector sequence is as SEQ ID NO:2;
(3) the agrobacterium tumefaciens bacterial strain of the plant expression vector that preparation contains Common Snapdragon Rosea1 gene
The plant expression vector that will contain Common Snapdragon Rosea1 gene transforms agrobacterium tumefaciens EHA105 competent cell, adopt the frozen-thawed method to be transformed, product after conversion is inverted and is cultivated 18～36 hours in 28 ℃ of constant incubators, the single bacterium colony of picking resistance carries out the screening of bacterium colony polymerase chain reaction, obtains the agrobacterium tumefaciens bacterial strain that contains Common Snapdragon Rosea1 gene plant expression vector;
(4) prepare transgenosis red sage root plant
Adopt the conventional organization cultural method to obtain aseptic red sage root test-tube plantlet, Agrobacterium tumefaciens mediated red sage root genetic conversion system adopts Ye Panfa, being about to the succeeding transfer culture red sage root aseptic seedling blade of 15 days is cut into small pieces, proceed in every 1L substratum and contain the 6-benzyl aminoadenine that 10mL concentration is 1.0mg/mL, on the MS substratum of the a-naphthylacetic acid that 1mL concentration is 1.0mg/mL, preculture is 1 day, contaminate with the agrobacterium tumefaciens bacterial strain that contains Common Snapdragon Rosea1 gene plant expression vector the red sage root blade that preculture is crossed, the dip-dye method is that 100 rev/mins of 28 ℃ of constant temperature are contaminated 25～30 minutes, the blade of contaminating is placed in every 1L substratum and contains the 6-benzyl aminoadenine that 10mL concentration is 1.0mg/mL, on the MS substratum of the a-naphthylacetic acid that 1mL concentration is 1.0mg/mL, dark the cultivation has Agrobacterium to grow to blade edge in 2～3 days, blade takes out from substratum, be transferred in every 1L substratum in the MS substratum that contains the cephamycin that Totomycin that 10mL concentration is 3mg/mL and 10mL concentration are 200mg/mL and cultivated, grow after resistant buds it to be transferred in every 1L substratum on the 1/2MS substratum that contains the cephamycin that Totomycin that 10mL concentration is 3mg/mL and 10mL concentration are 200mg/mL and take root, obtain the regeneration red sage root plant of hygromycin resistance, DNA with the specific detection primer polymerase chain reaction (PCR) amplification of Common Snapdragon Rosea1 gene regeneration red sage root plant, the positive strain of observing 663bp purpose band under the ultraviolet ray that wavelength is 302nm is transgenosis red sage root plant.
2. transgenosis according to claim 1 improves the method for rosmarinic acid contents in the red sage root, in the plant expression vector step (2) that it is characterized in that containing Common Snapdragon Rosea1 gene at structure, describedly with BglII and BstEII double digestion, be: get the pMD19-T Simple carrier that contains Common Snapdragon Rosea1 gene, 10 * H Buffer, redistilled water, restriction enzyme BglII was in 37 ℃ of reactions 1 hour 20 minutes, add again restriction enzyme BstE II, 60 ℃ are reacted 1 hour, restriction enzyme BglII and restriction enzyme BstE II, the pMD19-T Simple carrier that contains Common Snapdragon Rosea1 gene, redistilled water, the volume ratio of 10 * H Buffer is 3:3:20:19:5,
Get pCAMBIA-1302 plant expression vector, 10 * H Buffer, redistilled water, restriction enzyme BglII in 37 ℃ of reactions 1 hour 20 minutes, add again restriction enzyme BstE II, 60 ℃ are reacted 1 hour, and the volume ratio of restriction enzyme BglII and restriction enzyme BstE II, pCAMBIA-1302 plant expression vector, redistilled water, 10 * H Buffer is 3:3:20:19:5.
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| CN101863767A (en) * | 2010-03-23 | 2010-10-20 | 南京泽朗农业发展有限公司 | Preparation process of rosmarinic acid |
| CN102061301A (en) * | 2010-08-04 | 2011-05-18 | 陕西师范大学 | Hydroxyl cinnamon acyl transfer enzyme gene sequence of salvia miltiorrhiza bunge as well as coded amino acid sequence and application thereof |
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