CN102876713A - Method for improving content of rosmarinic acid and salvianolic acid B in salvia miltiorrhiza bunge while transforming genes - Google Patents
Method for improving content of rosmarinic acid and salvianolic acid B in salvia miltiorrhiza bunge while transforming genes Download PDFInfo
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- CN102876713A CN102876713A CN2012103526306A CN201210352630A CN102876713A CN 102876713 A CN102876713 A CN 102876713A CN 2012103526306 A CN2012103526306 A CN 2012103526306A CN 201210352630 A CN201210352630 A CN 201210352630A CN 102876713 A CN102876713 A CN 102876713A
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Abstract
The invention relates to a method for improving the content of rosmarinic acid and salvianolic acid B in salvia miltiorrhiza bunge while transforming genes, comprising the following steps: cloning snapdragon Rosea1 and Delila genes, constructing plant expression vectors comprising the snapdragon Rosea1 and Delila genes, preparing agrobacterium tumefaciens comprising the plant expression vectors, and preparing the transgenic salvia miltiorrhiza bunge. The expression of snapdragon Rosea1 and Delila genes in the salvia miltiorrhiza bunge is detected by adopting the real-time fluorescent quantitative reverse transcription and polymerase chain reaction, the content of the rosmarinic acid and the salvianolic acid B in the salvia miltiorrhiza bunge is detected at the same time by adopting the high performance liquid chromatography, and the transgenic salvia miltiorrhiza bunge with improved content of rosmarinic acid and salvianolic acid B are obtained by screening. The content of the rosmarinic acid in the dried root with a growing period of 45 days of the obtained transgenic salvia miltiorrhiza bunge is 26.96+/-0.90mg/g, the content of the salvianolic acid B is 63.64+/-1.94mg/g, and the rosmarinic acid and salvianolic acid B in the salvia miltiorrhiza bunge are respectively 1.60 times and 2.12 times the content of the rosmarinic acid and the salvianolic acid B in the non-transgenic oridinary salvia miltiorrhiza bunge in the corresponding period.
Description
Technical field
The invention belongs to the Gene Engineering of Medicinal Plants technical field, be specifically related in the red sage root method that combination cotransformation Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene improve liposoluble ingredient rosmarinic acid and content of danshinolic acid B in the red sage root simultaneously.
Background technology
The red sage root (Salvia miltiorrhiza Bunge) is Labiatae (Labiatae) Salvia per nnial herb, is used as medicine with its root and rhizome, is one of China's bulk medicinal materials commonly used.The main active ingredient of the red sage root is fat-soluble tanshinone and water miscible phenolic acids, has promoting blood circulation and removing blood stasisly, inducing meastruation to relieve menalgia, and the effects such as relieving restlessness that clear away heart-fire are widely used in the treatment of cardiovascular and cerebrovascular diseases, cancer and various inflammation clinically.Be accompanied by the expanding day of red sage root crude drug demand and the demand of GAP, cultivating the high good salvia new variety of active component content has become one of key issue that needs to be resolved hurrily in the production of red sage root crude drug.Based on the application method of multiplex its water decoction of traditional Chinese medicine, the soluble salvianolic acid constituents take rosmarinic acid and salvianolic acid B as representative is generally considered the main active substances basis of red sage root performance drug effect, is subject to especially investigator's common concern.Salvia-soluble liposoluble ingredient main manifestations is antioxygenation, and rosmarinic acid is put on market in Germany in nineteen ninety as anti-inflammation analgesia medicine; Salvianic acidA and rancinamycin IV are the main effective constituent of Treated with Radix Salviae Miltiorrhizae coronary heart disease; Salvianolic acid B is one of the strongest natural product of present known antioxygenation; it also is the compound that content is the highest in the red sage root, anti-oxidant activity is the strongest; be one of the water-soluble index composition of the red rooted salvia quality control of the version Pharmacopoeia of the People's Republic of China in 2010 regulation, a plurality of organs such as the heart, brain, liver, kidney, stomach are had provide protection.
Along with the further investigation of effective component in red sage and pharmacologically active thereof, the medicine take the red sage root as main raw material, preparation, makeup and serial health product emerge in an endless stream in recent years, and supply falls short of demand for red sage root resource.Be accompanied by the gradually minimizing 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, make a variation in the red sage root kind larger, proterties is complicated.Planted rooted salvia only plants the situation of not selecting so that each place of production red sage root quality is 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.In addition, species diversity is poor in the ecotope of field, richness is low because the artificial cultivation condition puts in poison, and causes that red sage root disease and pest dominant population is outstanding, disease and pest takes place frequently, and has had a strong impact on output and the quality of medicinal material.The problems referred to above become the bottleneck of salviamiltiorrhizabung medicinal material industry development.Because red sage root wild resource almost exhausts, the present medicinal red sage root is take artificial culture as main, and people pay attention to causing the red sage root quality good and the bad to differ greatly not to the seed selection of red sage root improved seeds in the cultivation.Therefore, the content of effective constituent, especially rosmarinic acid and salvianolic acid B in the raising red sage root, the red sage root resource of cultivating excellence is significant.
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 that the red sage root is improved to improve its medicinal material effective constituent more and more is subject to people's attention.Among all multi-methods, utilize genetic engineering technique that the hereditary property of medicinal plant secondary metabolism approach is transformed, 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.Modern molecular breeding technology take transgenic technology as 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 utilize at present the content, particularly rosmarinic acid of salvianolic acid constituents in the transgenic technology raising red sage root and the method for content of danshinolic acid B to there is no report.
Summary of the invention
Technical problem to be solved by this invention is to overcome the deficiency of above-mentioned technology, provides a kind of transgenosis to improve simultaneously the method for rosmarinic acid and content of danshinolic acid B in the red sage root.
Solving the problems of the technologies described above the technical scheme that adopts is that it comprises the steps:
1, Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene cloning
Extracting the total RNA of Common Snapdragon and reverse transcription becomes cDNA for subsequent use; adopt Primer Premier 5.0 softwares; upstream primer, the downstream primer of design Common Snapdragon Delila and Rosea1 genes encoding frame, and before the upstream primer of Common Snapdragon Delila gene, introduce BamH I restriction enzyme site and protection bases G A
GGATCC, before downstream primer, introduce Sac I restriction enzyme site and protection base CAG
GAGCTC, Common Snapdragon Delila upstream region of gene primer sequence is Delila-F:5 '-GA
GGATCCATGGCTACTGGTATCCAAAACCAAAAG-3 ', the downstream primer sequence is Delila-R:5 '-CAG
GAGCTCAACTTCAAGACTTCATAGTAACTTTCTG-3 '; Before the upstream primer of Common Snapdragon Rosea1 gene, introduce BglII restriction enzyme site and protection bases G A
AGATCT, before downstream primer, introduce BstEII restriction enzyme site and protection base CAG
GGTAACC, the upstream primer of Common Snapdragon Rosea1 gene is Rosea1-F:5 '-GA
AGATCTATGGAAAAGAATTGTCGTGGAGT-3 ', downstream primer are Rosea1-R:5 '-CAG
GGTAACCTTAATTTCCAATTTGTTGGGCCT-3 '; Take Common Snapdragon cDNA as template, Common Snapdragon Delila gene and Rosea1 gene increase respectively through the polymerase chain reaction, the amplified production that obtains carries out electrophoretic separation, reclaiming amplified production is connected with the T4DNA ligase enzyme with the pMD19-TSimple carrier, connecting product adopts the heat shock conversion method to change bacillus coli DH 5 alpha over to, the volume ratio that connects product and bacillus coli DH 5 alpha is 1:10, connecting product changes in the 100 μ L bacillus coli DH 5 alpha competent cells, random choose gained positive colony, detect through the bacterium colony polymerase chain reaction, sequence verification obtains the encoding sequence of described gene, and sequence is as follows:
Common Snapdragon Rosea1 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
Common Snapdragon Delila gene:
atggctactg gtatccaaaa ccaaaagata gtgcctgaga atttgaggaa 50
gcaacttgct attgctgtga gaagtatcca atggagttat gcaattttct 100
ggtccaattc agttgcacaa ccaggggtct tggagtgggg tgatgggttc 150
tacaatggag atattaaaac tcgaaaaact gtacaatctg tcgaattgaa 200
tcaagatcag ctgggattgc agagaagtga tcaattgaga gaactttatg 250
agtctctttc acttggtgaa accaacacac aagctaaaag gcctactgct 300
gcattatcac cagaagacct cactgatgct gagtggtttt tcttggtttg 350
catgtctttc atattcaata ttggccaagg gttgcctgga agaacattag 400
cacgaaatca agcagtatgg ctatgcaacg ctcatcgtgc ggacaccaaa 450
gttttctcgc gttctttgct tgcaaagagt gcgtcaattc agacagttgt 500
gtgctttcca tattcagaag gtgtagttga gctgggagca acagagctag 550
taccggagga tttgaatcta atccagcata taaaaacttc attcttggac 600
agtcctgcca ccgttcccaa gattcccaac tatgtctcca acagtattac 650
aaacaacaat gacctcattt gtgaagcgct tgaacatgct aatataccag 700
aaaacgatct tgatcagctt ttgaattgtc cagacacgaa catatgttct 750
cctgataaca gtttggatga ctttgcagac aatttactca tagacgaatc 800
gaatttggca gaaggcatca atggggaggt tcctcaaaca caaagctggc 850
ctttcatgga tgatgcaatc agcaattgtc tcaatagttc tatgaattct 900
agtgactgta tatctcaaac tcatgaaaat ctagagtctt ttgctccact 950
ttctgatgga aaagggccac cggagacgaa taattgtatg cacagcactc 1000
aaaaatgcaa tcagcagata gaaaacacgg gtgtccaagg cgatgaggtc 1050
cattatcaag gggtactttc caatcttttg aagagttccc atcagttggt 1100
tcttggtccc tacttcagaa atgggaatag agaatcaagc ttcgttagtt 1150
ggaacaagga tggatcgtcg ggtactcatg ttccccgaag cggaacctca 1200
caaagatttc tgaagaaagt actttttgaa gtagctagaa tgcatgaaaa 1250
ctccaggctt gatgctggta aacaaaaggg caacagtgac tgccttgcaa 1300
agccaacggc tgatgaaatt gatagaaacc acgtcttgtc agagagaaaa 1350
cgcagagaga aaataaacga acggtttatg attcttgcat ccctagtccc 1400
atccggtggc aaggttgaca aagtatcaat actagaccat acaatagatt 1450
acttgagagg gcttgagagg aaagtcgacg agctggaatc taacaaaatg 1500
gtaaagggcc gggggcggga atcaactaca aaaactaaac tacacgatgc 1550
cattgagagg acctctgata attatggcgc aacaaggaca agtaacgtca 1600
agaaaccgtt gacaaacaag agaaaggctt ctgatacgga caagattgga 1650
gccgtaaata gcagaggtcg attgaaagat tccttaacag ataatataac 1700
tgtgaacatt acaaacaagg atgtgttgat tgtcgtgact tgttcttcca 1750
aggagtttgt attgcttgaa gtgatggaag ccgtaagacg actaagtttg 1800
gattccgaaa ctgttcaatc ttccaacaga gatggaatga tatctattac 1850
cataaaagcc aagtgcaagg gattgaaggt tgcatcagca agtgtgatca 1900
aacaagctct tcagaaagtt actatgaagt cttgaagtt 1939
2, make up the plant expression vector that contains Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene
With EcoR I and HindIII double digestion plant expression vector pBI121 and pCAMBIA-1302, the enzyme that reclaims is cut product and is connected with the T4DNA ligase enzyme, connecting product adopts the heat shock conversion method to transform bacillus coli DH 5 alpha, the picking mono-clonal, detect and extract plasmid through the bacterium colony polymerase chain reaction and do enzyme and cut the detection screening positive clone, obtain the pCAMBIA-1302+ intermediate carrier; The pMD19-T Simple carrier and the pCAMBIA1302+ intermediate carrier that contain Common Snapdragon Delila gene with BamH I and Sac I double digestion, the enzyme that reclaims is cut product and is connected with the T4DNA ligase enzyme, connecting product adopts the heat shock conversion method to transform bacillus coli DH 5 alpha, the picking mono-clonal carries out the bacterium colony polymerase chain reaction and detects and extract plasmid enzyme restriction checking acquisition pCAMBIA1302
+-DEL recombinant plasmid; The pMD19-T Simple carrier and the pCAMBIA1302 that contain Common Snapdragon Rosea1 gene with BglII and BstEII double digestion
+-DEL recombinant plasmid, the enzyme that reclaims is cut product utilization T4DNA ligase enzyme and is connected, connecting product adopts the heat shock conversion method to transform bacillus coli DH 5 alpha, the picking mono-clonal, detect and extract the plasmid enzyme restriction checking through the bacterium colony polymerase chain reaction, obtain containing the plant expression vector of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene, this expression vector sequence is shown in Nucleotide tabulation＜210〉3.
3, preparation contains the agrobacterium tumefaciens bacterial strain of the plant expression vector of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene
The plant expression vector that will contain Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene transforms agrobacterium tumefaciens EHA105 competent cell, adopt the frozen-thawed method to transform, product after the conversion is inverted in 28 ℃ of constant incubators and was cultivated 18～36 hours, the single bacterium colony of picking resistance carries out the screening of bacterium colony polymerase chain reaction, obtains to contain the agrobacterium tumefaciens bacterial strain of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene plant expression vector.
4, preparation transgenosis red sage root plant
Adopt conventional organization culture technique 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 15 days red sage root aseptic seedling blade of succeeding transfer culture is cut into small pieces, change over to and contain the 6-benzyl aminoadenine that 10mL concentration is 1.0mg/mL in every 1L substratum, 1mL concentration is preculture 1 day on the MS substratum of a-naphthylacetic acid of 1.0mg/mL, contaminate the red sage root blade that preculture is crossed with the agrobacterium tumefaciens bacterial strain that contains Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene plant expression vector, the dip-dye method is that 100 rev/mins of 28 ℃ of constant temperature were contaminated 25～30 minutes, place every 1L substratum to contain the 6-benzyl aminoadenine that 10mL concentration is 1.0mg/mL in the blade of contaminating, 1mL concentration is that dark the cultivation had Agrobacterium to grow to blade edge in 2～3 days on the MS substratum of a-naphthylacetic acid of 1.0mg/mL, blade is taken out from substratum, be transferred in the MS substratum that contains the cephamycin that Totomycin that 10mL concentration is 3mg/mL and 10mL concentration is 200mg/mL in every 1L substratum and cultivate, grow behind the resistant buds it to be transferred on the 1/2MS substratum that contains the cephamycin that Totomycin that 10mL concentration is 3mg/mL and 10mL concentration is 200mg/mL in every 1L substratum and take root, obtain the regeneration red sage root plant of hygromycin resistance, with the DNA of the specific detection primer polymerase chain reaction (PCR) amplification of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene regeneration red sage root plant, wavelength is that the positive strain that can observe simultaneously 663bp and 1939bp purpose band under the ultraviolet ray of 302nm is transgenosis red sage root plant.
Contain in the step 2 of plant expression vector of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene at structure of the present invention, describedly with EcoRI and HindIII double digestion be: get plant expression vector pBI121,10 * MBuffer, restriction enzyme EcoRI, restriction enzyme HindII, redistilled water in 37 ℃ of reactions hours 10 minutes, the volume ratio of restriction enzyme EcoR I and restriction enzyme HindII, 10 * M Buffer, redistilled water, plant expression vector pBI121 is 3:3:5:19:20.Get plant expression vector pCAMBIA-1302,10 * M Buffer, restriction enzyme EcoR I, restriction enzyme HindII, redistilled water in 37 ℃ of reactions hours 10 minutes, the volume ratio of restriction enzyme EcoR I and restriction enzyme HindII, 10 * M Buffer, redistilled water, plant expression vector pCAMBIA-1302 is 3:3:5:19:20.
Contain in the step 2 of plant expression vector of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene at structure of the present invention, describedly with BamH I and Sac I double digestion be: get the pMD19-T Simple carrier that contains Common Snapdragon Delila gene, 10 * K Buffer, restriction enzyme BamH I, restriction enzyme Sac I, redistilled water is in 37 ℃ of reactions 2 hours 20 minutes, restriction enzyme BamH I and restriction enzyme Sac I, 10 * K Buffer, redistilled water, the volume ratio that contains the pMD19-TSimple carrier of Common Snapdragon Delila gene is 3:3:5:19:20.Get pCAMBIA1302+ intermediate carrier, 10 * KBuffer, restriction enzyme BamH I, restriction enzyme Sac I, redistilled water in 37 ℃ of reactions hours 20 minutes, the volume ratio of restriction enzyme BamH I and restriction enzyme SacI, 10 * KBuffer, redistilled water, pCAMBIA1302+ intermediate carrier is 3:3:5:19:20.
Contain in the step 2 of plant expression vector of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene at structure of the present invention, describedly with Bgl II and BstEII double digestion be: get the pMD19-T Simple carrier that contains Common Snapdragon Rosea1 gene, 10 * H Buffer, restriction enzyme BglII, redistilled water in 37 ℃ the reaction 1 hour 20 minutes after, add again restriction enzyme BstEII at 60 ℃ of reactions 1 hour, restriction enzyme BglII and restriction enzyme BstEII, 10 * H Buffer, redistilled water, the volume ratio that contains the pMD19-T Simple carrier of Common Snapdragon Rosea1 gene is 3:3:5:19:20.Get pCAMBIA1302
+-DEL recombinant plasmid, 10 * H Buffer, restriction enzyme BglII, redistilled water are in 37 ℃ of reactions after 1 hour 20 minutes, add again restriction enzyme BstEII at 60 ℃ of lower reactions 1 hour, restriction enzyme BglII and restriction enzyme BstEII, 10 * H Buffer, redistilled water, pCAMBIA1302
+The volume ratio of-DEL recombinant plasmid is 3:3:5:19:20.
The present invention changes Common Snapdragon Rosea1 gene and the Common Snapdragon Delila assortment of genes over to red sage root plant, and screening has obtained the transgenosis red sage root plant that the content of rosmarinic acid and salvianolic acid B significantly improves simultaneously.In 45 days genetically modified red sage root dry root of growth, the content of rosmarinic acid is 26.96 ± 0.90mg/g, the content of salvianolic acid B reaches 63.64 ± 1.94mg/g, it is respectively rosmarinic acid in the non-transformed common red sage root dry root of contemporaneously (16.90 ± 0.48mg/g) and (30.04 ± 0.89mg/g) 1.60 times and 2.12 times of salvianolic acid Bs, rosmarinic acid contents provides a kind of novel method in the red sage root in order to improve, and can promote the use of in the cultivation of the red sage root.
Description of drawings
Electrophorogram is detected in bacterium colony polymerase chain reaction when Fig. 1 is the agrobacterium tumefaciens for preparing the plant expression vector that contains Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene.
Fig. 2 is the DNA electrophorogram that transgenosis red sage root plant is detected in the polymerase chain reaction.
Fig. 3 is the expression spirogram that real time fluorescent quantitative reverse transcription-polymerase chain reaction is detected Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene in the transgenosis red sage root plant simultaneously.
Fig. 4 is the color atlas of rosmarinic acid and content of danshinolic acid B in the high-performance liquid chromatogram determination transgenosis red sage root plant.
Fig. 5 is the color atlas of rosmarinic acid and content of danshinolic acid B in the high-performance liquid chromatogram determination contrast ginseng plant.
Embodiment
The present invention is described in more detail below in conjunction with drawings and Examples, but the invention is not restricted to these embodiment.
1, Common Snapdragon Rosea1 gene and Delila gene cloning
(1) extraction of the total RNA of Common Snapdragon
Choose fresh, healthy and strong Common Snapdragon seedling complete stool, utilize the E.Z.N.A. of OMEGA company
TMPlant RNA Kit extracts its total RNA of test kit separation and Extraction.Extracting method is with reference to the E.Z.N.A. of OMEGA company
TMPlant RNA Kit extracts the test kit specification sheets.
(2) cDNA the first chain is synthetic
Adopt the Takara PrimeScriptRT reagent Kit of company reverse transcription test kit to carry 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 Common Snapdragon; RNase free dH
2O 5.5 μ L, the reverse transcription reaction condition is: hatched 30 minutes for 37 ℃, 85 ℃ of reactions got cDNA in 5 seconds, and cDNA is diluted 10 times with redistilled water, and-20 ℃ save backup.
(3) Common Snapdragon Rosea1 gene and Delila gene pyramiding PCR amplification
1. the polymerase chain reaction (PCR) amplification of Common Snapdragon Rosea1 gene
With reference to the Common Snapdragon Rosea1 gene that oneself announces among the GENBANK; with Premier Primer 5.0 softwares; design can amplify upstream primer, the downstream primer of Common Snapdragon Rosea1 genes encoding frame, and introduces BglII restriction enzyme site and protection bases G A at upstream primer
AGATCT, introduce BstEII restriction enzyme site and protection base CAG on the downstream primer
GGTCACC, so that construction of expression vector.The upstream primer sequence is Rosea1-F:5 '-GA
AGATCTATGGAAAAGAATTGTCGTGGAGT-3 ', the downstream primer sequence is Rosea1-R: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 available from Takara company) 4 μ L, upstream primer Rosea1-F 1 μ L, downstream primer Rosea1-R 1 μ L, LA Taq Polymerase (available from Takara company) 0.5 μ L, 10 * LA PCR BufferII(is available 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.Be that 1% agarose gel electrophoresis separates with the amplified production massfraction that obtains, reclaim target fragment, be connected with T4DNA ligase enzyme (available from Takara company) with pMD19-TSimple carrier (available from Takara company), linked system is: the PCR purifying reclaims product 7.5 μ L, 10 * T4 ligase enzyme Buffer(is available from Takara company) 1 μ L, T4DNA ligase enzyme 1 μ L, pMD19-TSimple 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 change in the 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 the large genome company of Shenzhen China after the 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
2. Common Snapdragon Delila gene pyramiding PCR amplification
With reference to the Common Snapdragon Delila gene that oneself announces among the GENBANK; use PremierPrimer5.0 software; design can amplify upstream primer, the downstream primer of Common Snapdragon Delila genes encoding frame, and introduces BamH I restriction enzyme site and protection bases G A before upstream primer
GGATCC, before downstream primer, introduce Sac I restriction enzyme site and protection base CAG
GAGCTC, so that construction of expression vector.Upstream primer sequence D elila-F:5 '-GA
GGATCCATGGCTACTGGTATCCAAAACCAAAAG-3 ', the downstream primer sequence is Delila-R:5 '-CAG
GAGCTCAACTTCAAGACTTCATAGTAACTTTCTG-3 '.Take Common Snapdragon cDNA as template, through polymerase chain reaction (PCR) amplification, the reaction amplification system is: Common Snapdragon cDNA 2 μ L, dNTP 4 μ L, upstream primer Delila-F 1 μ L, downstream primer Delila-R 1 μ L, LA Taq Polymerase 0.5 μ L, 10 * LA PCR BufferII5 μ L, redistilled water 36.5 μ L; The method of polymerase chain reaction (PCR) amplification is: 94 ℃, and 5 minutes; 94 ℃, 35 seconds, 62 ℃, 30 seconds, 72 ℃, 3 minutes, 38 circulations; 72 ℃, 10 minutes.Be that 1% agarose gel electrophoresis separates with the amplified production massfraction that obtains, reclaiming target fragment is connected with the T4DNA ligase enzyme with the pMD19-TSimple carrier, linked system is: the PCR purifying reclaims product 7.5 μ L, 10 * T4 ligase enzyme Buffer, 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 change in the 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 the large genome company of Shenzhen China after the detection, sequencing result is:
atggctactg gtatccaaaa ccaaaagata gtgcctgaga atttgaggaa 50
gcaacttgct attgctgtga gaagtatcca atggagttat gcaattttct 100
ggtccaattc agttgcacaa ccaggggtct tggagtgggg tgatgggttc 150
tacaatggag atattaaaac tcgaaaaact gtacaatctg tcgaattgaa 200
tcaagatcag ctgggattgc agagaagtga tcaattgaga gaactttatg 250
agtctctttc acttggtgaa accaacacac aagctaaaag gcctactgct 300
gcattatcac cagaagacct cactgatgct gagtggtttt tcttggtttg 350
catgtctttc atattcaata ttggccaagg gttgcctgga agaacattag 400
cacgaaatca agcagtatgg ctatgcaacg ctcatcgtgc ggacaccaaa 450
gttttctcgc gttctttgct tgcaaagagt gcgtcaattc agacagttgt 500
gtgctttcca tattcagaag gtgtagttga gctgggagca acagagctag 550
taccggagga tttgaatcta atccagcata taaaaacttc attcttggac 600
agtcctgcca ccgttcccaa gattcccaac tatgtctcca acagtattac 650
aaacaacaat gacctcattt gtgaagcgct tgaacatgct aatataccag 700
aaaacgatct tgatcagctt ttgaattgtc cagacacgaa catatgttct 750
cctgataaca gtttggatga ctttgcagac aatttactca tagacgaatc 800
gaatttggca gaaggcatca atggggaggt tcctcaaaca caaagctggc 850
ctttcatgga tgatgcaatc agcaattgtc tcaatagttc tatgaattct 900
agtgactgta tatctcaaac tcatgaaaat ctagagtctt ttgctccact 950
ttctgatgga aaagggccac cggagacgaa taattgtatg cacagcactc 1000
aaaaatgcaa tcagcagata gaaaacacgg gtgtccaagg cgatgaggtc 1050
cattatcaag gggtactttc caatcttttg aagagttccc atcagttggt 1100
tcttggtccc tacttcagaa atgggaatag agaatcaagc ttcgttagtt 1150
ggaacaagga tggatcgtcg ggtactcatg ttccccgaag cggaacctca 1200
caaagatttc tgaagaaagt actttttgaa gtagctagaa tgcatgaaaa 1250
ctccaggctt gatgctggta aacaaaaggg caacagtgac tgccttgcaa 1300
agccaacggc tgatgaaatt gatagaaacc acgtcttgtc agagagaaaa 1350
cgcagagaga aaataaacga acggtttatg attcttgcat ccctagtccc 1400
atccggtggc aaggttgaca aagtatcaat actagaccat acaatagatt 1450
acttgagagg gcttgagagg aaagtcgacg agctggaatc taacaaaatg 1500
gtaaagggcc gggggcggga atcaactaca aaaactaaac tacacgatgc 1550
cattgagagg acctctgata attatggcgc aacaaggaca agtaacgtca 1600
agaaaccgtt gacaaacaag agaaaggctt ctgatacgga caagattgga 1650
gccgtaaata gcagaggtcg attgaaagat tccttaacag ataatataac 1700
tgtgaacatt acaaacaagg atgtgttgat tgtcgtgact tgttcttcca 1750
aggagtttgt attgcttgaa gtgatggaag ccgtaagacg actaagtttg 1800
gattccgaaa ctgttcaatc ttccaacaga gatggaatga tatctattac 1850
cataaaagcc aagtgcaagg gattgaaggt tgcatcagca agtgtgatca 1900
aacaagctct tcagaaagtt actatgaagt cttgaagtt 1939
Sequencing result shows, the Common Snapdragon Rosea1 gene of reporting among the sequence of cloning and the GenBank and the encoder block nucleotide sequence consistent (accession number of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene is respectively among the GenBank: DQ275529 and M84913) of Common Snapdragon Delila gene.Described Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene cloning, rosmarinic acid and content of danshinolic acid B provide important transcription factor gene in the red sage root in order to improve simultaneously by transgenosis.
2, make up the plant expression vector that contains Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene
Take pCAMBIA-1302 and pBI121 as plant expression vector, pCAMBIA-1302 and pBI121 are the biomaterial of market sale, are produced by Australian CAMBIA company.With the restriction enzyme of using in restriction enzyme EcoRI and HindIII(the present embodiment and Buffer all available from Takara company) respectively double digestion plant expression vector pBI121 and pCAMBIA-1302.System and the method for EcoR I and HindIII double digestion plant expression vector pBI121 are: get plant expression vector pBI12110 μ L, 10 * M Buffer2.5 μ L, restriction enzyme EcoRI 1.5 μ L, restriction enzyme HindII1.5 μ L, redistilled water 9.5 μ L in 37 ℃ of lower reactions 2 hours 10 minutes, the recovery purifying obtains plant expression vector pBI121 enzyme and cuts product, and the volume ratio of restriction enzyme EcoR I and restriction enzyme HindII, 10 * M Buffer, redistilled water, plant expression vector pBI121 is 3:3:5:19:20.System and the method for EcoR I and HindIII double digestion plant expression vector pCAMBIA-1302 are: get plant expression vector pCAMBIA-130210 μ L, 10 * M Buffer, 2.5 μ L, restriction enzyme EcoR I 1.5 μ L, restriction enzyme HindII1.5 μ L, redistilled water 9.5 μ L were in 37 ℃ of lower reactions 2 hours 10 minutes, the recovery purifying obtains plant expression vector pCAMBIA-1302 enzyme and cuts product, restriction enzyme EcoR I and restriction enzyme HindII, 10 * MBuffer, redistilled water, the volume ratio of plant expression vector pCAMBIA-1302 is 3:3:5:19:20.The enzyme that reclaims is cut product and is connected with the T4DNA ligase enzyme, linked system and condition are: the enzyme of getting plant expression vector pBI121 is cut purified product 2 μ L, the enzyme of pCAMBIA-1302 is cut purified product 6 μ L, T4DNA ligase enzyme 1 μ L, behind 10 * T4 ligase enzyme Buffer, the 1 μ L mixing in 4 ℃ of connections spend the night (12～16 hours).Connect product and adopt the heat shock conversion method to transform escherichia coli DH5a, the picking mono-clonal detects and extracts plasmid and do enzyme and cut the detection screening positive clone, thereby obtains pCAMBIA-1302 through the bacterium colony polymerase chain reaction
+Intermediate carrier.
With restriction enzyme BamH I and Sac I respectively double digestion contain pMD19-T Simple carrier and the pCAMBIA1302 of Common Snapdragon Delila gene
+Intermediate carrier.System and method that BamH I and Sac I double digestion contain the pMD19-T Simple carrier of Common Snapdragon Delila gene are: get the pMD19-T Simple carrier 10 μ L, 10 * K Buffer, the 2.5 μ L that contain Common Snapdragon Delila gene, restriction enzyme BamHI 1.5 μ L, restriction enzyme SacI 1.5 μ L, redistilled water 9.5 μ L in 37 ℃ of reactions 2 hours 20 minutes, reclaim the enzyme that purifying obtains containing Common Snapdragon Delila gene and cut product; Restriction enzyme BamH I and restriction enzyme Sac I, 10 * K Buffer, redistilled water, the volume ratio that contains the pMD19-T Simple carrier of Common Snapdragon Delila gene are: 3:3:5:19:20.BamH I and Sac I double digestion pCAMBIA1302
+The system of intermediate carrier and method are: get pCAMBIA1302
+Intermediate carrier 10 μ L, 10 * K Buffer, 2.5 μ L, restriction enzyme BamH I 1.5 μ L, restriction enzyme SacI 1.5 μ L, redistilled water 9.5 μ L reclaim purifying and obtain pCAMBIA1302 in 37 ℃ of reactions 2 hours 20 minutes
+The enzyme of intermediate carrier is cut product, restriction enzyme BamH I and restriction enzyme Sac I, 10 * K Buffer, redistilled water, pCAMBIA1302
+The volume ratio of intermediate carrier is 3:3:5:19:20.The enzyme that reclaims is cut product utilization T4DNA ligase enzyme and is connected, and linked system and condition are: get the enzyme that contains Common Snapdragon Delila gene and cut purified product 2 μ L, pCAMBIA1302
+The enzyme of intermediate carrier is cut purified product 6 μ L, T4DNA ligase enzyme 1 μ L, behind 10 * T4 ligase enzyme Buffer, the 1 μ L mixing in 4 ℃ of connections spend the night (12～16 hours).Connect product and adopt the heat shock conversion method to transform bacillus coli DH 5 alpha, the picking mono-clonal carries out the detection of bacterium colony polymerase chain reaction and extracts the plasmid enzyme restriction checking obtaining pCAMBIA1302
+-DEL recombinant plasmid.
With restriction enzyme BglII and BstEII respectively double digestion contain pMD19-T Simple carrier and the pCAMBIA1302 of Common Snapdragon Rosea1 gene
+-DEL recombinant plasmid.System and method that BglII and BstEII double digestion contain the pMD19-TSimple carrier of Common Snapdragon Rosea1 gene are: get the pMD19-T Simple carrier 10 μ L, 10 * HBuffer, the 2.5 μ L that contain Common Snapdragon Rosea1 gene, restriction enzyme BglII 1.5 μ L, redistilled water 9.5 μ L in 37 ℃ of reactions after 1 hour 20 minutes, add again restriction enzyme BstEII1.5 μ L 60 ℃ of reactions 1 hour, reclaim the enzyme that purifying obtains containing Common Snapdragon Rosea1 gene and cut product; Restriction enzyme BglII and restriction enzyme BstEII, 10 * H Buffer, redistilled water, the volume ratio that contains the pMD19-T Simple carrier of Common Snapdragon Rosea1 gene are 3:3:5:19:20.BglII and BstEII double digestion pCAMBIA1302
+The system of-DEL recombinant plasmid and method are: get pCAMBIA1302
+-DEL recombinant plasmid 10 μ L, 10 * HBuffer2.5 μ L, restriction enzyme BglII 1.5 μ L, redistilled water 9.5 μ L are after reacting 1 hour 20 minutes under 37 ℃, add again restriction enzyme BstEII1.5 μ L 60 ℃ of reactions 1 hour, reclaim purifying and obtain pCAMBIA1302
+The enzyme of-DEL recombinant plasmid is cut product; Restriction enzyme BglII and restriction enzyme BstEII, 10 * H Buffer, redistilled water, pCAMBIA1302
+The volume ratio of-DEL recombinant plasmid is 3:3:5:19:20.The enzyme that reclaims is cut product utilization T4DNA ligase enzyme and is connected, and linked system and condition are: get the enzyme that contains Common Snapdragon Rosea1 gene and cut purified product 2 μ L, pCAMBIA1302
+The enzyme of-DEL recombinant plasmid is cut purified product 6 μ L, T4DNA ligase enzyme 1 μ L, behind 10 * T4 ligase enzyme Buffer, the 1 μ L mixing in 4 ℃ of connections spend the night (12～16 hours).Connecting product adopts heat shock conversion method product to transform bacillus coli DH 5 alpha, the picking mono-clonal, carry out the bacterium colony polymerase chain reaction and detect and extract the plasmid enzyme restriction checking, obtain containing the plant expression vector of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene, this expression vector sequence such as nucleotides sequence tabulation＜210〉3.
The above-mentioned plant expression vector that contains Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene, this expression vector can be used for improving simultaneously by gene engineering strategy the content of rosmarinic acid and salvianolic acid B in the red sage root.
3, preparation contains the agrobacterium tumefaciens bacterial strain of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene plant expression vector
The plant expression vector that will contain Common Snapdragon Rosea1 gene transforms agrobacterium tumefaciens EHA105 competent cell, adopt frozen-thawed to transform, agrobacterium tumefaciens EHA105 competent cell is the biomaterial that sell in market, produced by Australian CAMBIA company, 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, the plant expression vector 30ng that will contain Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene joins in the competent cell after dissolving, ice bath 40 minutes, liquid nitrogen flash freezer 1 minute, 37 ℃ of water-bath heat shocks 3 minutes, in centrifuge tube, add 400 μ L LB liquid nutrient mediums, 100 rev/mins of renewal cultivations of 28 ℃ of constant temperature 3～4 hours, to transform after product evenly coats on the 10mL LB solid medium (containing rifomycin and the 50 μ L concentration that 40 μ L concentration are 20mg/mL is the kantlex of 20mg/mL), in 28 ℃ of constant incubators, be inverted and cultivated 18～36 hours, the single bacterium colony of picking resistance, be inoculated in (containing rifomycin and the 25 μ L concentration that 20 μ L concentration are 20mg/mL is the kantlex of 20mg/mL) in the 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, acquisition contains the agrobacterium tumefaciens bacterial strain of the plant expression vector of Common Snapdragon Rosea1 gene and Common Snapdragon Delila 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 namely contains the plant expression vector of Common Snapdragon Delila gene, the Delila gene bacterium colony polymerase chain reaction result of the second swimming lane 1, the 3rd swimming lane 2, the 4th swimming lane 3 expression different strains, the 5th swimming lane-expression negative control; The 6th swimming lane+expression positive control namely contains the plant expression vector of Common Snapdragon Rosea1 gene, the 7th swimming lane 4, the 8th swimming lane 5, the 9th swimming lane " expression different strains Rosea1 gene bacterium colony polymerase chain reaction result, the tenth swimming lane-expression negative control.As seen from Figure 1, use the polymerase chain reaction special primer, can amplify simultaneously the specific DNA fragment of 663bp and 1939bp, illustrate that the plant expression vector that contains Common Snapdragon Rosea1 gene Common Snapdragon Delila gene successfully is transformed in the agrobacterium tumefaciens bacterial strain.
4, preparation transgenosis red sage root plant
(1) Agrobacterium tumefaciens mediated Common Snapdragon Rosea1 gene and the Common Snapdragon Delila gene transformation red sage root
The acquisition of aseptic red sage root test-tube plantlet can be adopted conventional organization culture technique method, and concrete grammar is as follows:
Salvia seeds is washed with flowing water, be 75% aqueous ethanolic solution surface sterilization 20 seconds with volume fraction, redistilled water flushing 2 times, each 4 minutes, be 0.1% mercuric chloride solution surface sterilization 10 minutes with massfraction, redistilled water flushing 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 cuts off from internode, will be with the stem segment cuttage of 2 axillalry buds on the 1/2MS substratum, every around successive propagation once.Transform the red sage root test-tube plantlet that used material is subculture 2～4 times, wherein 15 days seedling of succeeding transfer culture is used for gene transformation, and 30 days seedling of succeeding transfer culture is used for nucleic acid extraction.
Agrobacterium tumefaciens mediated red sage root genetic conversion system adopts Ye Panfa, and concrete grammar is as follows:
Choose 15 days red sage root aseptic seedling of succeeding transfer culture, its blade is cut into the fritter of 0.5 centimetre of 0.5 cm x, change that (pre-culture medium is that every 1LMS substratum contains the 6-benzyl aminoadenine that 10mL concentration is 1.0mg/mL in the preculture substratum over to, 1mL concentration is the substratum of the a-naphthylacetic acid of 1.0mg/mL), preculture is 1 day under normal culture condition, a part of blade that preculture is crossed changes in the good plant expression vector agrobacterium tumefaciens bacterium liquid that contains Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene of dilution, 100 rev/mins of dip-dyes of 28 ℃ of constant temperature 25～30 minutes, take out blade, blot with aseptic filter paper, transfer on the preculture substratum dark cultivate 2～3 days (having Agrobacterium to grow to blade edge); The pre-incubated blade of another part does not carry out the dip-dye of Agrobacterium, directly after the cultivation of preculture substratum is sprouted, bud is peeled off, and places on the 1/2MS substratum and takes root, as the unconverted blank plant of experiment; Cultivate after 2～3 days, blade is taken out from former substratum, be transferred to and select on the substratum (select substratum be contain Totomycin and the 10mL concentration that 10mL concentration is 3mg/mL in the 1L MS substratum be the substratum of the cephamycin of 200mg/mL), changed in per 10 ~ 15 days and once to select substratum; Treat that resistant buds is long during to about 0.5 ~ 1 centimetre of left and right sides, resistant buds is downcut, take root on the 1/2MS substratum that changes over to.In every 1L substratum, contain the cephamycin that 10mL concentration is 200mg/mL, 10mL concentration is screening and culturing 3～4 times in the 1/2MS substratum of Totomycin of 3mg/mL (per 10～15 days change a subculture), the plant that can normally take root moves 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, change a subculture about 4～6 weeks, the positive transformation plant of screening and contrast strain are transplanted to the scientific research greenhouse equal conditions and are cultivated, and transplant the mensuration that the transgenosis red sage root plant of cultivating 45 days is used for rosmarinic acid and content of danshinolic acid B.
(2) polymerase chain reaction of transgenosis red sage root plant is detected
Adopting Bioer company DNA of plants to extract test kit extracts contrast red sage root plant, transforms described gene red sage root strain and transforms the DNA that does not contain described gene red sage root strain, utilize respectively the upstream and downstream primer pair goal gene of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene complete encoder block to carry out the polymerase chain reaction detection, electrophorogram is seen 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 namely contains the combinations of plant expression vector of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene, the second swimming lane B represents not contain the dna profiling contrast, it is non-transformed red sage root strain that the 3rd swimming lane CK-represents to contrast red sage root strain, the 4th swimming lane CK+ represents to transform and does not contain the contrast strain that described gene red sage root strain namely transforms the pCAMBIA1302+ intermediate carrier, and all the other swimming lane 1-30 represent different transgenic lines.As seen from Figure 2, to transform red sage root genomic dna as template amplification, it is the specific DNA fragment that to observe simultaneously 663bp and 1939bp under the ultraviolet ray of 302nm at wavelength, and take the non-transformed red sage root with transform when not containing described gene red sage root genomic dna as template, do not amplify any fragment, it is correct described vector construction being described and transforming strategy, and goal gene has inserted in the red sage root genome.The acquisition of transgenosis red sage root plant provides basic material for the red sage root strain of screening high level rosmarinic acid and rosmarinic acid.
5, the expression of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene in real time fluorescent quantitative reverse transcription-polymerase chain reaction detection transgenosis red sage root plant
(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, the primer of Common Snapdragon Delila gene and red sage root house-keeping gene Actin muscle-β gene of being fit to of design.The Rosea1 gene, Delila 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; QDel-F, qDel-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’；
qDel-F：5’-GAGTTCCCATCAGTTGGTTCTTG-3’，
qDel-R：5’-CTTTGTGAGGTTCCGCTTCG-3’；
SmACT-F：5’-AGGAACCACCGATCCAGACA-3’，
SmACT-R：5’-GGTGCCCTGAGGTCCTGTT-3’。
(2) extraction of the total RNA of the red sage root
Adopt the OMEGA E.Z.N.A. of company
TMPlant RNA Kit extracts test kit to carry out according to the test kit specification sheets, and method is consistent with the extracting method of the total BNA of Common Snapdragon.
(3) cDNA the first chain is synthetic
The synthetic Takara of the utilization PrimeScript RT reagent Kit of the company reverse transcription test kit of cDNA the first chain carries out, and reaction system is: 5 * PrimeScrip
TMBufe 2 μ L, PrimeScrip
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 with the reaction mixture mixing, were hatched 30 minutes for 37 ℃, and 85 ℃ of reactions got cDNA in 5 seconds, and cDNA is diluted 50 times with redistilled water, and-20 ℃ save backup
(4) expression of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene in real time fluorescent quantitative reverse transcription-polymerase chain reaction detection transgenosis red sage root plant
Take non-transformed red sage root strain as blank, to transform the negative contrast of plant expression vector red sage root strain that does not contain described gene, measure the expression amount of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene with real time fluorescent quantitative reverse transcription-polymerase chain reaction.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 annealing are also collected fluorescent signal, 25 seconds); 95 ℃, 1 minute; 60 ℃, 1 minute; 60 ℃～95 ℃, raise 0.5 ℃ in per 30 seconds, collect first order fluorescence.The fluorescent signal value adopted " the relatively relative quantification method of Ct value " to carry out the analysis of genetic expression after reaction finished.Concrete treatment process is according to " iQ
TM5 multiple real time fluorescence quantifying PCR specification sheetss " carry out.Detected result is seen Fig. 3.In Fig. 3,1-30 represents different transgenic lines; CK-represents non-transformed red sage root strain, and the CK+ representative transforms the strain of the plant expression vector that does not contain described gene, and *, * *, * * * represent that respectively experimental group and control group relatively have the significance difference opposite sex (P＜0.05,0.01 and 0.001).As seen from Figure 3, compare with adjoining tree, external source goal gene Common Snapdragon Delila gene and Rosea1 gene all have expression in various degree simultaneously in transformation plant 1,3,5,7,8,9,14,18,19,21,22,25,26,27,28,29, the relative expression quantity of Delila and Rosea1 gene has significant difference (P＜0.5) compared with the control, shows overexpression in the red sage root of described goal gene.
6, the content of rosmarinic acid and salvianolic acid B in the usefulness 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), take aqueous acetic acid, acetonitrile and the methyl alcohol of massfraction as 0.4% as the eluent gradient wash-out, the eluent gradient elution program is 0.01～5 minute, massfraction is that 0.4% aqueous acetic acid volume reduces to 90% by 95%, and the acetonitrile volume rises to 10% by 5%; 5～25 minutes, massfraction was that 0.4% aqueous acetic acid volume reduces 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, massfraction was that 0.4% aqueous acetic acid volume reduces 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 by weighing respectively rosmarinic acid and each 10.0mg of salvianolic acid B standard substance, join to such an extent that concentration is the standard substance mother liquor of 10.0mg/mL take the methanol solution of volume fraction as 75% as solvent, place 4 ℃ of refrigerators to save backup.During experiment, get respectively the standard solution that the standard substance mother liquor is diluted to concentration gradient, sample introduction analysis behind the 0.22 μ m filtering with microporous membrane.
The eluent gradient elution program that adopts among the present invention, rosmarinic acid retention time are 24.82 ± 0.08 minutes, and the salvianolic acid B retention time is 28.17 ± 0.42 minutes.Peak shape is good, can guarantee separating of other liposoluble ingredient in rosmarinic acid and salvianolic acid B and the red sage root.
(2) drawing standard curve
Draw respectively rosmarinic acid and salvianolic acid B 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, 0.22 sample introduction analysis behind the μ m filtering with microporous membrane, the drawing standard curve.According to above-mentioned corresponding chromatographic condition sample introduction, record collection of illustrative plates and chromatographic parameter carry out regression analysis with the rosmarinic acid peak area to standard substance concentration respectively, and the equation of linear regression that obtains rosmarinic acid is:
y
1=6×10
-8x
1-0.015(r
1 2=0.9990)
X in the formula
1The peak area of rosmarinic acid in the expression sample, y
1The concentration of expression rosmarinic acid, the mg/mL of unit, r
1The expression relation conefficient.With the salvianolic acid B peak area standard substance concentration is carried out regression analysis, the equation of linear regression that obtains salvianolic acid B is:
y
2=4×10
-8x
2-0.006(r
2 2=0.9990)
X in the formula
2The peak area of salvianolic acid B in the expression sample, y
2The concentration of expression salvianolic acid B, the mg/mL of unit, r
2The expression relation conefficient.
(3) preparation sample solution
30 ℃ of red sage root dry root are dried to constant weight, place mortar to grind, take by weighing respectively transgenosis and each 25mg of contrast red sage root plant dried powder, place the 1.5mL centrifuge tube, add 500 μ L volume fractions and be 75% methanol aqueous solution, 30 ℃ with the ultrasonic extraction of frequency 30KHz 20 minutes, and 12000 rev/mins centrifugal 6 minutes.Shift supernatant stand-by to new centrifuge tube; Residue extracts twice again with same procedure, merges three times supernatant liquor, through 0.22 μ m filtering with microporous membrane, analyze with high performance liquid chromatograph, record rosmarinic acid peak and salvianolic acid B area in each sample, the substitution equation of linear regression calculates the content of rosmarinic acid and salvianolic acid B.
With the content of rosmarinic acid and salvianolic acid B in the high-performance liquid chromatogram determination transgenosis red sage root strain, the results are shown in Figure 4, Fig. 5, in Fig. 4, Fig. 5,1 is rosmarinic acid, 2 is salvianolic acid B.By Fig. 4, Fig. 5 as seen, in 45 days genetically modified red sage root dry root of growth, the content of rosmarinic acid is 26.96 ± 0.90mg/g, the content of salvianolic acid B reaches 63.64 ± 1.94mg/g, is respectively rosmarinic acid in the non-transformed common red sage root dry root of contemporaneously (16.90 ± 0.48mg/g) and (30.04 ± 0.89mg/g) 1.60 times and 2.12 times of salvianolic acid Bs.
The present embodiment has obtained the transgenosis red sage root plant of rosmarinic acid and the common high yield of salvianolic acid B with the gene engineering strategy of cotransformation Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene; with high effective liquid chromatography for measuring the content of rosmarinic acid and salvianolic acid B in the transgenosis red sage root, for large-scale production rosmarinic acid and salvianolic acid B and the final red sage root resource scarcity problem that solves provide a kind of Perfected process.
Claims (4)
1. a transgenosis improves the method for rosmarinic acid and content of danshinolic acid B in the red sage root simultaneously, it is characterized in that the method comprises the steps:
(1) Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene cloning
Extracting the total RNA of Common Snapdragon and reverse transcription becomes cDNA for subsequent use; adopt Primer Premier 5.0 softwares; upstream primer, the downstream primer of design Common Snapdragon Delila and Rosea1 genes encoding frame, and before the upstream primer of Common Snapdragon Delila gene, introduce BamH I restriction enzyme site and protection bases G A
GGATCC, before downstream primer, introduce Sac I restriction enzyme site and protection base CAG
GAGCTC, Common Snapdragon Delila upstream region of gene primer sequence is Delila-F:5 '-GA
GGATCCATGGCTACTGGTATCCAAAACCAAAAG-3 ', the downstream primer sequence is Delila-R:5 '-CAG
GAGCTCAACTTCAAGACTTCATAGTAACTTTCTG-3 '; Before the upstream primer of Common Snapdragon Rosea1 gene, introduce BglII restriction enzyme site and protection bases G A
AGATCT, before downstream primer, introduce BstEII restriction enzyme site and protection base CAG
GGTAACC, the upstream primer of Common Snapdragon Rosea1 gene is Rosea1-F:5 '-GA
AGATCTATGGAAAAGAATTGTCGTGGAGT-3 ', downstream primer are Rosea1-R:5 '-CAG
GGTAACCTTAATTTCCAATTTGTTGGGCCT-3 '; Take Common Snapdragon cDNA as template, Common Snapdragon Delila gene and Rosea1 gene increase respectively through the polymerase chain reaction, the amplified production that obtains 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 change escherichia coli DH5a over to, the volume ratio that connects product and escherichia coli DH5a is 1:10, connecting product changes in the 100 μ L escherichia coli DH5a competent cells, random choose gained positive colony, detect through the bacterium colony polymerase chain reaction, sequence verification obtains the encoding sequence of described gene, and sequence is as follows:
Common Snapdragon Rosea1 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
Common Snapdragon Delila gene:
atggctactg gtatccaaaa ccaaaagata gtgcctgaga atttgaggaa 50
gcaacttgct attgctgtga gaagtatcca atggagttat gcaattttct 100
ggtccaattc agttgcacaa ccaggggtct tggagtgggg tgatgggttc 150
tacaatggag atattaaaac tcgaaaaact gtacaatctg tcgaattgaa 200
tcaagatcag ctgggattgc agagaagtga tcaattgaga gaactttatg 250
agtctctttc acttggtgaa accaacacac aagctaaaag gcctactgct 300
gcattatcac cagaagacct cactgatgct gagtggtttt tcttggtttg 350
catgtctttc atattcaata ttggccaagg gttgcctgga agaacattag 400
cacgaaatca agcagtatgg ctatgcaacg ctcatcgtgc ggacaccaaa 450
gttttctcgc gttctttgct tgcaaagagt gcgtcaattc agacagttgt 500
gtgctttcca tattcagaag gtgtagttga gctgggagca acagagctag 550
taccggagga tttgaatcta atccagcata taaaaacttc attcttggac 600
agtcctgcca ccgttcccaa gattcccaac tatgtctcca acagtattac 650
aaacaacaat gacctcattt gtgaagcgct tgaacatgct aatataccag 700
aaaacgatct tgatcagctt ttgaattgtc cagacacgaa catatgttct 750
cctgataaca gtttggatga ctttgcagac aatttactca tagacgaatc 800
gaatttggca gaaggcatca atggggaggt tcctcaaaca caaagctggc 850
ctttcatgga tgatgcaatc agcaattgtc tcaatagttc tatgaattct 900
agtgactgta tatctcaaac tcatgaaaat ctagagtctt ttgctccact 950
ttctgatgga aaagggccac cggagacgaa taattgtatg cacagcactc 1000
aaaaatgcaa tcagcagata gaaaacacgg gtgtccaagg cgatgaggtc 1050
cattatcaag gggtactttc caatcttttg aagagttccc atcagttggt 1100
tcttggtccc tacttcagaa atgggaatag agaatcaagc ttcgttagtt 1150
ggaacaagga tggatcgtcg ggtactcatg ttccccgaag cggaacctca 1200
caaagatttc tgaagaaagt actttttgaa gtagctagaa tgcatgaaaa 1250
ctccaggctt gatgctggta aacaaaaggg caacagtgac tgccttgcaa 1300
agccaacggc tgatgaaatt gatagaaacc acgtcttgtc agagagaaaa 1350
cgcagagaga aaataaacga acggtttatg attcttgcat ccctagtccc 1400
atccggtggc aaggttgaca aagtatcaat actagaccat acaatagatt 1450
acttgagagg gcttgagagg aaagtcgacg agctggaatc taacaaaatg 1500
gtaaagggcc gggggcggga atcaactaca aaaactaaac tacacgatgc 1550
cattgagagg acctctgata attatggcgc aacaaggaca agtaacgtca 1600
agaaaccgtt gacaaacaag agaaaggctt ctgatacgga caagattgga 1650
gccgtaaata gcagaggtcg attgaaagat tccttaacag ataatataac 1700
tgtgaacatt acaaacaagg atgtgttgat tgtcgtgact tgttcttcca 1750
aggagtttgt attgcttgaa gtgatggaag ccgtaagacg actaagtttg 1800
gattccgaaa ctgttcaatc ttccaacaga gatggaatga tatctattac 1850
cataaaagcc aagtgcaagg gattgaaggt tgcatcagca agtgtgatca 1900
aacaagctct tcagaaagtt actatgaagt cttgaagtt 1939
(2) make up the plant expression vector that contains Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene
With EcoR I and HindIII double digestion plant expression vector pBI121 and pCAMBIA-1302, the enzyme that reclaims is cut product utilization T4DNA ligase enzyme and is connected, connecting product adopts the heat shock conversion method to transform bacillus coli DH 5 alpha, the picking mono-clonal, detect and extract plasmid through the bacterium colony polymerase chain reaction and do enzyme and cut the detection screening positive clone, obtain pCAMBIA-1302
+Intermediate carrier; The pMD19-T Simple carrier and the pCAMBIA1302 that contain Common Snapdragon Delila gene with BamH I and Sac I double digestion
+Intermediate carrier, the enzyme of recovery are cut product and are connected with the T4DNA ligase enzyme, connect product employing heat shock conversion method and transform bacillus coli DH 5 alpha, and the picking mono-clonal carries out the bacterium colony polymerase chain reaction and detects and extract plasmid enzyme restriction checking acquisition pCAMBIA1302
+-DEL recombinant plasmid; The pMD 19-T Simple carrier and the pCAMBIA1302 that contain Common Snapdragon Rosea1 gene with BglII and BstEII double digestion
+-DEL recombinant plasmid, the enzyme that reclaims is cut product utilization T4DNA ligase enzyme and is connected, connecting product adopts the heat shock conversion method to transform bacillus coli DH 5 alpha, the picking mono-clonal, detect and extract the plasmid enzyme restriction checking through the bacterium colony polymerase chain reaction, obtain containing the plant expression vector of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene, this expression vector sequence is shown in Nucleotide tabulation＜210〉3;
(3) preparation contains the agrobacterium tumefaciens bacterial strain of the plant expression vector of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene
The plant expression vector that will contain Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene transforms agrobacterium tumefaciens EHA105 competent cell, adopt the frozen-thawed method to transform, product after the conversion is inverted in 28 ℃ of constant incubators and was cultivated 18～36 hours, the single bacterium colony of picking resistance carries out the screening of bacterium colony polymerase chain reaction, obtains to contain the agrobacterium tumefaciens bacterial strain of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene plant expression vector;
(4) preparation transgenosis red sage root plant
Adopt conventional organization culture technique 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 15 days red sage root aseptic seedling blade of succeeding transfer culture is cut into small pieces, change over to and contain the 6-benzyl aminoadenine that 10mL concentration is 1.0mg/mL in every 1L substratum, 1mL concentration is preculture 1 day on the MS substratum of a-naphthylacetic acid of 1.0mg/mL, contaminate the red sage root blade that preculture is crossed with the agrobacterium tumefaciens bacterial strain that contains Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene plant expression vector, the dip-dye method is that 100 rev/mins of 28 ℃ of constant temperature were contaminated 25～30 minutes, place every 1L substratum to contain the 6-benzyl aminoadenine that 10mL concentration is 1.0mg/mL in the blade of contaminating, 1mL concentration is that dark the cultivation had Agrobacterium to grow to blade edge in 2～3 days on the MS substratum of a-naphthylacetic acid of 1.0mg/mL, blade is taken out from substratum, be transferred in the MS substratum that contains the cephamycin that Totomycin that 10mL concentration is 3mg/mL and 10mL concentration is 200mg/mL in every 1L substratum and cultivate, grow behind the resistant buds it to be transferred on the 1/2MS substratum that contains the cephamycin that Totomycin that 10mL concentration is 3mg/mL and 10mL concentration is 200mg/mL in every 1L substratum and take root, obtain the regeneration red sage root plant of hygromycin resistance, with the DNA of the specific detection primer polymerase chain reaction (PCR) amplification of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene regeneration red sage root plant, wavelength is that the positive strain that can observe simultaneously 663bp and 1939bp purpose band under the ultraviolet ray of 302nm is transgenosis red sage root plant.
2. transgenosis according to claim 1 improves the method for rosmarinic acid and content of danshinolic acid B in the red sage root simultaneously, it is characterized in that containing in the step (2) of plant expression vector of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene at structure, describedly with EcoR I and HindII double digestion be: get plant expression vector pBI121,10 * M Buffer, restriction enzyme EcoR I, restriction enzyme HindII, redistilled water is in 37 ℃ of reactions hours 10 minutes, restriction enzyme EcoRI and restriction enzyme HindII, 10 * M Buffe, redistilled water, the volume ratio of plant expression vector pBI121 is 3:3:5:19:20;
Get plant expression vector pCAMBIA-1302,10 * MBufer, restriction enzyme EcoRI, restriction enzyme HindII, redistilled water in 37 ℃ of reactions 2 hours 10 minutes, the volume ratio of restriction enzyme EcoR I and restriction enzyme HindII, 10 * MBuffer, redistilled water, plant expression vector pCAMBIA-1302 is 3:3:5:19:20.
3. transgenosis according to claim 1 improves the method for rosmarinic acid and content of danshinolic acid B in the red sage root simultaneously, it is characterized in that containing in the step (2) of plant expression vector of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene at structure, describedly with BamHI and Sac I double digestion be: get the pMD19-T Simple carrier that contains Common Snapdragon Delila gene, 10 * K Buffer, restriction enzyme BamH I, restriction enzyme SacI, redistilled water is in 37 ℃ of reactions 2 hours 20 minutes, restriction enzyme BamH I and restriction enzyme Sac I, 10 * K Buffer, redistilled water, the volume ratio that contains the pMD19-T Simple carrier of Common Snapdragon Delila gene is 3:3:5:19:20;
Get pCAMBIA1302
+Intermediate carrier, 10 * K Buffer, restriction enzyme BamH I, restriction enzyme Sac I, redistilled water were in 37 ℃ of reactions hours 20 minutes, and the volume ratio of restriction enzyme BamH I and restriction enzyme Sac I, 10 * K Buffer, redistilled water, pCAMBIA1302+ intermediate carrier is 3:3:5:19:20.
4. transgenosis according to claim 1 improves the method for rosmarinic acid and content of danshinolic acid B in the red sage root simultaneously, it is characterized in that containing in the step (2) of plant expression vector of Common Snapdragon Rosea1 gene and Common Snapdragon Delila gene at structure, describedly with BglII and BstEII double digestion be: get the pMD19-TSimple carrier that contains Common Snapdragon Rosea1 gene, 10 * HBuffer, restriction enzyme BglII, redistilled water in 37 ℃ the reaction 1 hour 20 minutes after, add again restriction enzyme BstEII at 60 ℃ of reactions 1 hour, restriction enzyme BglII and restriction enzyme BstEII, 10 * H Buffer, redistilled water, the volume ratio that contains the pMD19-T Simple carrier of Common Snapdragon Rosea1 gene is 3:3:5:19:20;
Get pCAMBIA1302
+-DEL recombinant plasmid, 10 * H Buffer, restriction enzyme BglII, redistilled water are in 37 ℃ of reactions after 1 hour 20 minutes, add restriction enzyme BstEII 60 ℃ of lower reactions 1 hour, the volume ratio of restriction enzyme BglII and restriction enzyme BstEII, 10 * H Buffer, redistilled water, pCAMBIA1302+-DEL recombinant plasmid is 3:3:5:19:20 again.
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| CN103834685A (en) * | 2013-11-29 | 2014-06-04 | 陕西师范大学 | Carrier capable of simultaneously improving rosmarinic acid and salvianolic acid B content of red sage root and use thereof |
| CN104004747A (en) * | 2014-05-29 | 2014-08-27 | 陕西师范大学 | Method for improving insect resistance and active component of salvia miltiorrhiza by using tomato gene and endogenous gene of salvia miltiorrhiza |
| CN104472357A (en) * | 2014-11-25 | 2015-04-01 | 浙江理工大学 | Inducing method capable of promoting danshinolic acid accumulation in hairy salvia miltiorrhiza root |
| WO2020258896A1 (en) * | 2019-06-25 | 2020-12-30 | 陕西鸿道生物分析科学技术研究院有限公司 | Strain and method for producing rosmarinic acid |
| CN115725625A (en) * | 2022-08-03 | 2023-03-03 | 四川农业大学 | Application of SmTAT2 gene in preparation of transgenic plants with high phenolic acid compounds yield |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103834685A (en) * | 2013-11-29 | 2014-06-04 | 陕西师范大学 | Carrier capable of simultaneously improving rosmarinic acid and salvianolic acid B content of red sage root and use thereof |
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| CN104472357A (en) * | 2014-11-25 | 2015-04-01 | 浙江理工大学 | Inducing method capable of promoting danshinolic acid accumulation in hairy salvia miltiorrhiza root |
| WO2020258896A1 (en) * | 2019-06-25 | 2020-12-30 | 陕西鸿道生物分析科学技术研究院有限公司 | Strain and method for producing rosmarinic acid |
| CN115725625A (en) * | 2022-08-03 | 2023-03-03 | 四川农业大学 | Application of SmTAT2 gene in preparation of transgenic plants with high phenolic acid compounds yield |
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