CN101693739A - Chlorogenic acid synthesis associated protein and encoding gene thereof and application - Google Patents
Chlorogenic acid synthesis associated protein and encoding gene thereof and application Download PDFInfo
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Abstract
The invention discloses a chlorogenic acid synthesis associated protein and an encoding gene and application thereof. The protein provided by the invention can be a protein (a) or a protein (b), wherein (a)consists of amino acid sequences shown in sequence 1 in a sequence table; and (b) is an associated protein derived from the sequence 1, which is obtained by substituting and/or lacking and/or adding one or a plurality of amino acid residues of the amino acid sequences of the sequence 1 and being synthesized with chlorogenic acid. The invention also discloses the encoding gene of the protein. The encoding gene is guided into a target plant to obtain a transgenic plant with higher content of chlorogenic acid than the target plant. The chlorogenic acid synthesis associated protein and the encoding gene thereof have important value for solving the problem of the shortage of wild snow lotus resources.
Description
Technical field
The present invention relates to a kind of chlorogenic acid synthesis associated protein and encoding gene thereof and application.
Background technology
Snow Lotus Herb has another name called saussurea involucrata, and the composite family hieracioides belongs to.Be the famous and precious medicinal plant of a class commonly used among the people, belong to per nnial herb.Generally be distributed on the high mountain flowstone beach of height above sea level more than 4,000 meters.In " territory, northwest note " and " the little knowledge in garden mutually ", just relevant for the record of saussurea involucrata.Has dispelling cold and removing dampness, functions such as promoting blood circulation to restore menstrual flow, strong muscle is supporing yang, anti-inflammatory, analgesia, contraction uterus, the rheumatic arthritis that is used for the treatment of among the people delays senility, arteriosclerosis, termination of pregnancy, diseases such as women's cold and pain in the lower abdomen, amenorrhoea, retention of placenta, measles without adequate eruption, lung cold cough, impotence.Snow Lotus Herb thallophyta kind is more, though all can be used as medicine on an equal basis according to the record of Chinese medicine document, its quality has the branch of quality.According to relevant, the sales volume maximum is Xinjiang Tianshan saussurea involucrata (snow lotus) (S.involucrata Kar.et Kir.) and Saussurea medusa (S.medusa Maxim) in above several saussurea involucrata.And Saussurea medusa (Saussureamedusa Maxim.) is the former plant of Tibetan medicine " saussurea involucrata " medicinal material, is distributed in ground such as China Qinghai, Gansu, Tibet.Saussurea medusa is the rare medicinal herbs in the Tibetan medicine, has effects such as dispelling cold and removing dampness, promoting blood circulation and removing obstruction in channels, anticancer, anti-inflammatory and antifatigue, can treat multiple diseases such as rheumatic arthritis, menoxenia, carbuncle pyogenic infections from tumour or sore and high mountain incompatibility.
The main effective constituent of Saussurea medusa is the chlorogenic acid compound, as the promote blood circulation saussurea involucrata TONGMAI KOUFUYE of ball, Herba Saussureae Involueratae injection and treatment cerebral arteriosclerosis and ishemic stroke of the saussurea involucrata that is used for treating hemiplegia, be quality standard all with chlorogenic acid, total flavonoids content.
Chlorogenic acid another name: chlorogenic acid, caffeotannic acid; English name Chlorogenic acid, 3-caffeoyl quinic acid; Chemical name: the 3-caffetannic acid, 3-[[3-(3,4-dihydroxyphenyl)-1-oxy-2-propenyl] oxy]-1,4,5-trihydroxy-1, [1S-(1 α, 3 β, 4 α, 5 α)]; Systematic name: 1,3,4,5-tetrahydroxy hexahydrobenzoic acid-(3,4-dihydroxycinnamic acid ester); Name of product: chlorogenic acid; (1S, 3R, 4R, 5R)-and 3-[[3-(3, the 4-dihydroxy phenyl)-1-oxo-2-propenyl] oxygen]-1,4,5-trihydroxy-naphthenic acid; CAS NO:327-97-9; EINECS accession number: 206-325-6; Molecular formula and molecular weight: C
16H
18O
9, MW=354.30.
The biosynthesizing of chlorogenic acid, flavonoid compound realizes by phenylpropyl alcohol alkanes biosynthetic pathway.The plant transcription factor of having cloned comprises several big classes such as Myb, Myc (bHLH), ERF (Ethylene Response Factor), HD (Homeodomain), LIM zinc finger, WD40 and WRKY, mainly is the Myb class.The Myb transcription factor extensively is present in the higher plant, and its principal character is to contain 1-3 the incomplete multiple Myb-DNA binding domains of being made up of 51 or 52 amino-acid residues (R1, R2, R3).Each structural domain is made up of 3 alpha-helixs, second and the 3rd spiralization helix turn helix (HTH) structure wherein, and this structure is similar to λ supressor DNA identification binding domains.Number according to contained Myb-DNA binding domains can be divided into R1, R2R3,3 families of R1R2R3, and wherein R2R3 family has crucial regulating and controlling effect to Secondary Metabolism of Plant.
Summary of the invention
The purpose of this invention is to provide a kind of chlorogenic acid synthesis associated protein and encoding gene thereof and application.
Chlorogenic acid synthesis associated protein provided by the invention (MYB1) derives from Saussurea medusa (Saussurea.medusa), is following (a) or protein (b):
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and synthetic relevant with chlorogenic acid by sequence 1 deutero-protein.
MYB1 albumen is made up of 256 amino-acid residues.
In order to make the MYB1 in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table connect label as shown in table 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| ??Poly-Arg | 5-6 (being generally 5) | ??RRRRR |
| ??Poly-His | 2-10 (being generally 6) | ??HHHHHH |
| ??FLAG | ??8 | ??DYKDDDDK |
| ??Strep-tagII | ??8 | ??WSHPQFEK |
| ??c-myc | ??10 | ??EQKLISEEDL |
Above-mentioned (b) but in the MYB1 synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of MYB1 in above-mentioned (b) can be by the codon with sequence in the sequence table 2 one or several amino-acid residue of disappearance in the dna sequence dna shown in the 53rd to 823 Nucleotide of 5 ' end, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The gene (MYB1) of above-mentioned chlorogenic acid synthesis associated protein of encoding also belongs to protection scope of the present invention.
Described gene can be following 1) or 2) or 3) or 4) dna molecular:
1) encoding sequence be in the sequence table sequence 2 from the dna molecular shown in 5 ' the 53rd to 823 Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;
4) with 1) or 2) or 3) dna sequence dna that limits has 90% above homology, and the dna molecular of the chlorogenic acid synthesis associated protein of encoding.
Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Form by 969 Nucleotide shown in the sequence 1 in the sequence table, from 5 ' the 1st to 52 Nucleotide is 5 ' non-coding region (5 '-UTR) (52bp), the the 53rd to 823 Nucleotide is coding region (771bp), and the 824th to 969 Nucleotide is 3 ' non-coding region (3 '-UTR) (146bp).
The recombinant vectors that contains above arbitrary described gene also belongs to protection scope of the present invention.
The carrier that sets out can be selected various carrier known in the art for use, as commercially available carrier, comprises plasmid, clay etc.The encoding sequence of MYB1 operationally can be connected in expression regulation sequence, thereby form recombinant vectors.
Available existing plant expression vector construction contains the recombinant expression vector of described gene.
Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of inducing (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean storage protein gene) 3 ' end to transcribe as the Agrobacterium crown-gall nodule all has similar functions.
When using described gene constructed recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn, they can use separately or be used in combination with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.
For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase genes etc.) as adding the coding that in plant, to express, have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (as anti-weedkiller gene) etc.
The multiple clone site that described recombinant expression vector can insert described gene the pBI121 plasmid obtains.Specifically, described recombinant expression vector can be pBI121-SmMYB1; Described pBI121-SmMYB1 is substituted by described gene (MYB1) with the small segment between pBI121 plasmid BamHI and EcoRI site to obtain.
The expression cassette, transgenic cell line and the reorganization bacterium that contain above arbitrary described gene (MYB1) all belong to protection scope of the present invention.
Described gene (MYB1) total length that increases or arbitrary segmental primer are to also belonging to protection scope of the present invention.
Described gene (MYB1) and fragment thereof can obtain with the method for pcr amplification method, recombination method or synthetic entirely.For the pcr amplification method, can be according to the dna sequence dna of described gene, especially open reading frame sequence designs primer, with cDNA as template, the amplification and obtain.When sequence is longer, usually need to carry out twice or pcr amplification repeatedly, and then the fragment that each time amplification is come out is stitched together by proper order.In addition, can also synthesize aim sequence, can directly synthesize aim sequence, also can synthesize a plurality of polynucleotide small segments earlier, and then connect, thereby obtain aim sequence with the method for artificial chemosynthesis.After obtaining aim sequence, available recombination method comes large batch of acquisition aim sequence, normally it is cloned into carrier, changes cell again over to, separates obtaining aim sequence then from the host cell after the propagation by ordinary method.
Another object of the present invention provides a kind of method of cultivating the transgenic plant of chlorogenic acid content raising.
The method of the transgenic plant that cultivation chlorogenic acid content provided by the present invention improves, the gene of the described chlorogenic acid synthesis associated protein of coding can be imported in the purpose plant (as vegetable cell or tissue), obtain the transgenic plant that chlorogenic acid content improves than purpose plant; Described purpose plant is the plant with synthetic chlorogenic acid ability.Specifically, described recombinant expression vector can be imported in the purpose plant, obtain the transgenic plant that chlorogenic acid content improves.
Utilize any carrier that can guide foreign gene in plant, to express,, can obtain transgenic cell line and transfer-gen plant that chlorogenic acid content improves the gene transfered plant cell of encoding said proteins.Carry that described expression carrier can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed tissue cultivating is become plant.By the plant transformed host both can be the monocotyledons with synthetic chlorogenic acid ability, also can be the dicotyledons with synthetic chlorogenic acid ability, as: tobacco, Caulis et Folium Brassicae capitatae etc.
The transgenic plant that the chlorogenic acid content that above method prepares improves can be applicable to produce chlorogenic acid.
The invention provides a kind of chlorogenic acid synthesis associated protein from Saussurea medusa, this albumen belongs to the R2R3-Myb family member, and coding chlorogenic acid key enzyme structure gene (as, genes such as 4CL2 and C3H) is had regulating and controlling effect.With the recombinant expression vector transformation of tobacco that contains described protein coding gene, the transgene tobacco of acquisition is compared with commentaries on classics empty carrier contrast tobacco with wild-type tobacco, and chlorogenic acid content is significantly improved.Chlorogenic acid synthesis associated protein of the present invention and encoding gene thereof have important value to solving saussurea involucrata wild resource problem of shortage.
Description of drawings
Fig. 1 is Saussurea medusa MYB1 gene cDNA and protein amino acid sequence.
Fig. 2 is the secondary structure prediction of Saussurea medusa MYB1 gene mRNA.
The comparison of proteic prediction secondary structure of Fig. 3 Saussurea medusa MYB1 and the known chicken of structure (Gallus gallus) Myb transcriptional regulator 1A5J secondary structure.
Fig. 4 is the homology of Saussurea medusa MYB1 and other R2R3-MYB family protein.
Fig. 5 is the sequence alignment of R2R3-MYB family protein in Saussurea medusa MYB1 and the tobacco (Nicotiana tabacum L).
Fig. 6 is a CGA content in the blade in the plant of each strain system among the embodiment 2.
Fig. 7 is transgene tobacco T
1For the chlorogenic acid content HPLC collection of illustrative plates of seedling, peak 1 and 2 is two kinds of isomerss of chlorogenic acid.
Fig. 8 is transgene tobacco T
1Differential expression analytical results for CGA biosynthesizing relative enzyme gene in the seedling.
Embodiment
Further set forth the present invention below in conjunction with specific embodiment, following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method." molecular cloning experiment guide " (Science Press 2002) such as J. Sa nurse Brooker for example, " fine works molecular biology experiment guide " (Science Press 1998) described conditions such as F. Ao Sibai, or the condition of being advised according to used product manufacturer.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.
The discovery of embodiment 1, chlorogenic acid synthesis associated protein MYB1 and encoding gene MYB1 thereof
One, the foundation in saussurea involucrata cDNA library
(former plant is picked up from 4500-5000 height above sea level mountain range, Qilian, Gansu Province to Saussurea medusa, and be accredited as Saussurea medusa through the Chen Yilin researcher of plant institute) (Saussurea.medusa) 1-15 days callus red colour systems, in the MS substratum of additional 2mg/LNAA and 0.5mg/L BA, cultivate under 25 ± 1 ℃, 16h illumination/8h dark, subculture expands numerous.
Take by weighing 1-14 days red colour system callus, every day, 50mg amounted to the 700mg biased sample in mortar, extracted total RNA with TRIZOL Reagent (available from GIBCO company).Get 1-3 μ 1 total RNA sample, with SMARTcDNA Library Construction Kit (CLONTEC; The PolyT primer of the CDSIII restriction enzyme site that Catlog#:K1051-1) carries synthesizes the first chain cDNA.Adopt LD PCR (long distance PCR) the amplification first chain cDNA to obtain double-stranded cDNA.
Add the Taq enzyme among the protease K digesting removal cDNA, cut cDNA with Sfi I enzyme again, with CHROMASPIN-400 cDNA is carried out size separation then.Collection is connected the cDNA that collects greater than the cDNA of 500bp with λ TripIEx2Vector (Promega).After the DNA that connects was packed by packaging protein Packagene Lambda DNAPackaging System (available from Promega company), it was 0.01% that the adding gelatin makes final concentration, and the DMSO final concentration is 7%, can be constant in 1 year titre of-70 ℃ of following preservations.Obtained saussurea involucrata cDNA library, the library is divided into 20 Ya Wenku, avoided freezing repeatedly molten, each Ya Wenku comes from when original amplified library the regenerant of different culture dish.
Two, screening saussurea involucrata MYB1 transcription factor cDNA
Carry out the cDNA library screening with degenerated primer (MYB1df and MYB1dr) and obtain positive monoclonal, and obtain the specific probe fragment; According to probe fragment design Auele Specific Primer (MYB1pf and MYB1pr), be that template is carried out the NO excalation part of PCR acquisition library screening with cDNA; Right with the primer that MYB1gf and MYB1gr form, be that template is carried out the full-length cDNA that PCR finally obtains the MYB1 gene with cDNA.
MYB1df：5’-AAR?WSN?TGY?MGN?YTN?MGN?TGG-3’；
MYB1dr：5’-CCA?RTA?RTT?YTT?NAY?NTS?RTT?RTC-3’。
R=A/G, W=A/T, M=A/C, H=A/T/C, Y=C/T, S=G/C, any base of N=.
MYB1pf：5’-GTT?GCA?GGT?TAC?GAT?GGG?TG-3’；
MYB1pr：5’-CAT?TGT?CAC?TTC?TTC?CAG?GCA-3’。
MYB1gf：5’-GAATTAAGATGGTAAGAGCACC-3’；
MYB1gr：5’-TAGAAGAGACAAATTCGAGGG-3’。
The sequence of MYB1cDNA and proteins encoded thereof is seen Fig. 1.
MYB1cDNA is shown in the sequence 2 of sequence table.Total length 969bp is made up of the ORF (Open Reading Frame) of 3 of 5 of 52bp (1-52bp) '-UTR (5 '-un-translated region), 146bp (824-969bp) '-UTR (3-un-translatedregion) and 771bp (53-823bp).3 '-UTR comprises Poly (A) tail of a 26bp.Whole cDNA sequence all is rich in A+T, accounts for 63% of whole bases.5 '-UTR in A+T content be 63%, 3 '-UTR in A+T content up to 77%, even Poly (A) is not counted, also up to 71%.The ORF zone is then low slightly, also reaches 61%.The ATG and the contiguous sequence (AAGATGG) that are positioned at 53-55bp meet eukaryotic initiation password ATG prediction conserved sequence (A/GXXATGG), and the TAA of 821-823bp is a termination codon, confirms that further this cDNA is the full-length gene that comprises complete open reading frame.The Blast of MYB1 gene ORF district in NbGI (
Http:// tigrblast.tigr.org/tgi/) analytical results shows, the myb gene LBM1 of known function in the nucleotide sequence of MYB1 gene and the tobacco (
AB028649), LBM3 (
AB028651) and NtMYBAS1 (
AF198499) consensus nucleic acid sequence (Identities) be respectively 66%, 60% and 61%.Wherein, LBM1 and LBM3 are derived from N.tomentosiformis, can be induced by the mosaic virus of tobacco, SA, wounding signal, and then (for example: the expression of pathogenesis-related proteins gene (PR) the PAL gene) and in the tobacco, generation defensive raction regulate genes involved in the phenylpropionic acid approach of downstream.MYBAS1 is from N.tabacum cv samsun, can be in pollen sac specifically expressing, and then activate downstream PAL expression of gene, promote the growth of pollen tube.The secondary structure prediction of MYB1 gene mRNA is seen Fig. 2.
MYB1 albumen is shown in the sequence 1 of sequence table.Form by 256 amino-acid residues.Among the MYB1, polare Aminosaeren accounts for 32.42% of total amino acid content; Charge residue accounts for 31.64%; Hydrophobic amino acid accounts for 29.69%; Basic aminoacids accounts for 12.89% of total amino acid; N-holds (1-115aa), and basic aminoacids accounts for 20.87%, and acidic amino acid only accounts for 7.83%, thereby has formed the N-end of an alkalescence; Acidic amino acid accounts for 11.33% of total amino acid content; C-holds (116-256aa), and acidic amino acid accounts for 14.18%, and basic aminoacids only accounts for 6.38%, thereby has formed a tart C-end.This structure with an alkaline N-end and an acid C-end of MYB1 meets the general feature of Myb transcriptional regulator.MYB1 infers that molecular weight is 28.6kD, and iso-electric point pI=8.91 is basic protein, forms relevant with its amino acid.Proteic C-end (121aa-256aa) is rich in hydrophilic amino acid, and especially Serine (S) accounts for 18.4% of this zone total amino acid, and exists with the form of oligomer, and this is the common feature in transcriptional regulator activation structure territory.Structural domain is searched software PPSEARCH on-line analysis result and is shown that MYB1 protein sequence N-end contains conservative MYB primitive (R2R3DNA is in conjunction with the territory), and the C-end has acid α-Helix, the feature that is had jointly for the R2R3-MYB family protein.The proteic secondary structure prediction of MYB1 is seen Fig. 3.The amino acid sequence analysis result of the MYB primitive (R2R3DNA is in conjunction with the territory) that the N-end is conservative shows, MYB1 albumen and tobacco LBM1, LBM3 and NtMYBAS1 have 70.9%, 68% and 53.4% consistence respectively, then are difficult to find conforming zone in C-end active region.The PSORT on-line analysis of nuclear localization signal forecasting software is the result show, this MYB1 protein sequence contains nuclear localization signal, has the localized potential ability of nuclear.The MYB1 aminoacid sequence is derived from different plants and the known MYB protein sequence of function carries out multisequencing comparison (DNAMAN) with selected, the results are shown in Figure 4 and Fig. 5, show that MYB1 is the albumen with R2R3Myb-DNA-binding-domain.MYB1 albumen is 48-71% at the aminoacid sequence of MYB elementary area and the sequence identity of other plant MYB.Wherein, be about 59.2% with the consensus amino acid sequence of corn P gene protein and Arabidopis thaliana AtMYB12, with the consistence of Arabidopis thaliana AtMYB75 and AtMYB90 be 57.3%, and with the consistence of petunia Ph2 be 69.9%.
Acquisition of embodiment 2, transgenic plant and evaluation
First-generation transfer-gen plant is T
0For plant, T
1T is shown in representative
0The seed that produces for selfing and by plant that it grew up to.
One, the acquisition of transgenic plant
1, the structure of recombinant vectors
Artificial preparation must arrive the upstream and have the BamHI restriction enzyme site, and the downstream has the MYB1 gene of EcoRI restriction enzyme site.
Dna fragmentation is imported between the BamHI and EcoRI restriction enzyme site of pBI121 plasmid (clontech), (skeleton carrier is pBI121, has inserted sequence 2 from the 45th to 898 Nucleotide of 5 ' end between BamHI and EcoRI restriction enzyme site to obtain recombinant vectors pBI121-SmMYB1.
Two, the acquisition of transgene tobacco
1, the preparation of reorganization Agrobacterium
PBI121-SmMYB1 (or pBI121) is imported agrobacterium tumefaciens lba4404 (Invitrogen with the frozen-thawed method; Cat.No.18313-015) in, make up LBA4404-SmMYB1 engineering bacteria (or contrast bacterium), concrete steps are as follows:
(1) take out pBI121-SmMYB1 (or pBI121) from-70 ℃ of cryogenic refrigerators, line the YEB flat board of the attached 50mg/L of containing kantlex (Kanacymin), 100mg/L Rifampin (Rifampicin) respectively, 28 ℃ of inversions have been cultured to single bacterium colony and have grown;
(2) picking list colony inoculation contains in the YEB liquid nutrient medium of 50mg/L kantlex (Kanacymin), 50mg/L Rifampin (Rifampicin) in 10mL, and 28 ℃, 180rpm shaking culture spend the night;
(3) get 100 μ L bacterium liquid and be inoculated into 25mL and do not contain in the antibiotic YEB liquid nutrient medium, continue to be cultured to logarithmic phase (about about 2h) under the same terms, under the room temperature, 4000rpm is centrifugal, collect thalline, and use MS
0Liquid nutrient medium is diluted to OD
600=0.3-0.6 is standby.
2, the acquisition of transgene tobacco
The tobacco Transformation Program is with reference to " plant genetic engineering " (Wang Guanlin and the Fang Hong skin of bamboo, 2002), and concrete steps are as follows:
(1) chooses eugonic tobacco Wisconsin 38 (W38) (available from Dohanykutato Intezet of the Chinese Academy of Agricultural Sciences Chinese tobacco genetic breeding research (north) center) aseptic seedling (switching back 10-15d) blade, be cut into 1cm with sharp knife blade
2Small pieces discard middle arteries and veins;
(2) tobacco leaf is soaked in the bacterium liquid of step 1 preparation, guarantees that the paddle cutout edge contacts with bacterium liquid fully, soak 5min;
(3) the bacterium liquid that inclines will infect blade and transfer to inhale on the sterilization thieving paper and remove unnecessary bacterium liquid;
(4) blade is transferred to common culture medium (MS
0+ 6-BA 1mg/L+NAA 0.1mg/L, pH5.8) in, vacuum side of blade is cultivated 48h up altogether in the dark;
(5) blade after will cultivating altogether is transferred to division culture medium (MS
0+ 6-BA 1mg/L+NAA 0.1mg/L+Kan150mg/L+Cef 400mg/L, pH5.8) on, illumination cultivation;
(6) behind the 10d blade is transferred to subculture medium (MS
0+ Kan 150mg/L+Cef 400mg/L, pH5.8) on;
(7) budlet to be differentiated grows to 2-3cm, and its cutting-out is transferred to root media (MS
0+ Kan150mg/L, pH5.8).The seedling that grows is candidate's transfer-gen plant (or changeing the empty carrier adjoining tree)
3, PCR-Southern identifies
(1) preparation of probe
The MYB1 gene was that Myb DNA is in conjunction with the territory at 425bp in the past, has higher conservative property, in non-DNA binding domains homology not highly then, therefore be chosen in non-DNA binding domains design detection primer and carry out PCR-Southern probe mark fragment analysis, to avoid detecting false positive at conservative region.
It is as follows to prepare the used primer of PCR-Southern probe:
MYB1snf：5’-AGATGGAAATGCTGAAACC-3’；
MYB1snr：5’-GAGCCCAAAGGTGAGTACA-3’。
The used template of preparation probe is the cDNA of the red callus of Saussurea medusa.The probe length for preparing is 325bp, uses digoxigenin labeled.
(2) PCR-Southern identifies
Transgene tobacco is carried out PCR-Southern identify that template is the cDNA that the RNA reverse transcription of transgene tobacco obtains, probe is the probe that step (1) obtains, and the PCR primer is as follows:
MYB1sf：5’-GTAC?ggatcc?GAATTAAGATGGTAAGAGCACC-3’；
MYB1sr：5’-AGCA?gagctc?TAGAAGAGACAAATTCGAGGG-3’。
The result shows, has obtained the T of 7 adopted SmMYB1 that become a full member (SS-1, SS-6, SS-22, SS-30, SS-31, SS-56, SS-61)
0For the tobacco strain be.Must be with changes empty carrier contrast tobacco (S0).
Three, T
1Acquisition for transgene tobacco
With T
0Seed for the transgene tobacco results, with 70% (volumn concentration) ethanol surface sterilization 30 seconds, with the redistilled water flushing of sterilizing once, use 0.5% (quality percentage composition) clorox sterilization 10 minutes again, redistilled water with sterilization washes 4 times, and sowing is at the MS that contains kantlex 250mg/l
0On the solid medium, be placed on 25 ℃, cultivate under 16h illumination/8h dark.The genetically modified tobacco seed in five week backs is sprouted the back root can be normally profound, grows true leaf, is T
1For transgene tobacco.Root can not extend behind the not genetically modified seed germination, does not also have true leaf to grow, slowly albefaction death.
Equally, preparation T
1In generation, changeed empty carrier contrast tobacco.
Four, T
1Chlorogenic acid (CGA) assay for the transgene tobacco regrowth
T
1For transgene tobacco, T
1In generation, changeed empty carrier contrast tobacco, wild-type tobacco, and each strain is 4; Get and sprout back 40 days seedling leaves, grind into powder in the liquid nitrogen; By per 0.1 gram fresh weight proportioning 1ml 80% (volumn concentration) methyl alcohol, extracted 24 hours under the room temperature; 13000rpm got supernatant liquor in centrifugal 10 minutes.
Detect the content of CGA in the supernatant liquor with RP-HPLC.RP-HPLC chromatographic condition: AGILENT 1100series; C18 reverse-phase chromatographic column (250x4.6mm); Sample size 10 μ l; Detect wavelength 345nm; Moving phase solution A (1% formic acid), solution B (methyl alcohol); Elution program: 0-2min:A 90%B 10%; 2-12min:A 90% → 80%B 10% → 20%; 12-22min:A 80% → 50%, and B 20% → 50%; 22-27min:A 50% → 0, and B 50% → 100%.
CGA content is as shown in Figure 6 in the blade in the plant of each strain system.CGA content is respectively 1.8 times and 1.6 times of wild-type and empty carrier contrast apparently higher than wild-type and empty carrier contrast (P=0.01) in the SS-22 blade.SS-1, SS-6, SS-31 strain are that CGA content also is slightly higher than wild-type and empty carrier contrast in the blade.Wherein, strain is that SS-22 and wild-type tobacco HPLC figure see Fig. 7.
Five, transgene tobacco T
1For in the seedling with CGA biosynthesizing involved enzyme expression of gene difference
Identify the T of wild-type tobacco and transgene tobacco SS-22 respectively
1In seedling, several and CGA biosynthesizing involved enzyme expression of gene difference.
The result of RT-PCR as shown in Figure 8.
Can show tentatively that from Fig. 8 compare with wild-type that the expression amount of 4CL2 and C3H obviously raises among the SS-22, other gene does not then have noticeable change.
Sequence table
＜110〉Institute of Botany, Chinese Academy of Sciences
＜120〉chlorogenic acid synthesis associated protein and encoding gene thereof and application
<130>CGGNARY92589
<160>2
<210>1
<211>256
<212>PRT
＜213〉Saussurea medusa (Saussurea.medusa)
<400>1
Met?Val?Arg?Ala?Pro?Cys?Phe?Asp?Lys?His?Gly?Ile?Lys?Arg?Gly?Ala
1???????????????5???????????????????10??????????????????15
Trp?Ser?Gln?Glu?Glu?Asp?Asn?Lys?Leu?Arg?Ala?His?Ile?Gln?Arg?Ser
20??????????????????25??????????????????30
Gly?His?Ser?Asn?Trp?Arg?Gln?Leu?Pro?Lys?Leu?Ala?Gly?Leu?Ser?Arg
35??????????????????40??????????????????45
Cys?Gly?Lys?Ser?Cys?Arg?Leu?Arg?Trp?Val?Asn?Tyr?Leu?Arg?Pro?Thr
50??????????????????55??????????????????60
Ile?Lys?His?Gly?Asn?Phe?Thr?Lys?Asp?Glu?Lys?Asp?Val?Val?Val?Ala
65??????????????????70??????????????????75??????????????????80
Leu?His?Asn?Lys?Leu?Gly?Asn?Lys?Trp?Ser?Ala?Ile?Ala?Ala?Arg?Leu
85??????????????????90??????????????????95
Pro?Gly?Arg?Ser?Asp?Asn?Glu?Ile?Lys?Asn?Tyr?Trp?His?Thr?His?Leu
100?????????????????105?????????????????110
Lys?Asn?Arg?Ala?Gln?Thr?Asp?Gln?Thr?Val?Leu?Gln?Thr?Lys?Gln?Asp
115?????????????????120?????????????????125
Gly?Asn?Ala?Glu?Thr?Ser?Lys?Gly?Lys?Gly?Thr?Pro?Lys?Ala?Gly?Cys
130?????????????????135?????????????????140
Val?Val?Arg?Lys?Pro?Asn?Val?Lys?Ser?Gln?Gln?Glu?Val?Glu?Ile?Leu
145?????????????????150?????????????????155?????????????????160
Leu?Ala?Val?Leu?Ser?Ser?Ala?Ser?Ser?Ser?Ser?Phe?Ser?Ser?Ser?Thr
165?????????????????170?????????????????175
Thr?Ser?Asp?Gln?Asn?Glu?Ser?Ser?Pro?Pro?Ser?Leu?Ser?Val?Ser?Asp
180?????????????????185?????????????????190
Ala?Asn?Val?Thr?Pro?Gln?Cys?Ser?Glu?Glu?Ala?Ala?Arg?Ser?Leu?Cys
195?????????????????200?????????????????205
Ile?Asp?Gln?Phe?Leu?Val?Glu?Glu?Asp?Ser?Ile?Met?Leu?Ser?Ser?Asp
210?????????????????215?????????????????220
Lys?Met?Tyr?Ser?Pro?Leu?Gly?Ser?Val?Asp?Tyr?Phe?Ile?Ser?Gln?Asp
225?????????????????230?????????????????235?????????????????240
Asn?Val?Met?Asp?Asp?Val?Leu?Leu?Trp?Pro?Asn?Leu?Asp?Phe?Tyr?Tyr
245?????????????????250?????????????????255
<210>2
<211>969
<212>DNA
＜213〉Saussurea medusa (Saussurea.medusa)
<400>2
gatccccagc?aaaagatctg?cagagaaaaa?aagtgtacaa?acaagaatta?agatggtaag?????60
agcaccatgt?tttgacaaac?acggaatcaa?gagaggtgca?tggagtcaag?aagaagacaa????120
caaactaagg?gcccatatac?agagatccgg?ccattctaac?tggcgtcaac?ttcccaagtt????180
agctggtctg?tctagatgcg?gaaaaagttg?caggttacga?tgggtgaatt?atttacgtcc????240
aaccataaag?catgggaatt?ttacaaaaga?tgaaaaagat?gtcgttgttg?ctttacataa????300
caagcttggg?aacaaatggt?cagcgattgc?tgcacgattg?cctggaagaa?gtgacaatga????360
aataaaaaac?tattggcata?cgcatttgaa?aaatcgggct?cagacagatc?aaactgtgtt????420
acaaactaag?caagatggaa?atgctgaaac?cagtaagggt?aagggtaccc?ctaaagcagg????480
atgtgtggta?agaaaaccaa?acgtaaaaag?ccaacaggaa?gttgaaattt?tattggcagt????540
attatcatct?gcatcatcat?cttctttctc?ttcttcaaca?acaagtgatc?aaaatgaatc????600
atcaccacct?tcgttgagtg?tctcagatgc?aaatgttaca?ccacaatgtt?ctgaagaagc????660
ggccagaagt?ttatgtattg?atcagttttt?agtggaagag?gatagcatca?tgttatcaag????720
tgataaaatg?tactcacctt?tgggctcagt?cgattatttc?atctcccagg?ataacgttat????780
ggatgatgtg?cttttatggc?caaacttgga?tttctattat?taatctaagt?tgattcgtaa????840
ttaattatgt?caaacatcaa?ttattttgtc?ccgttctccc?tcgaatttgt?ctcttctaaa????900
ttattgctta?actctgttaa?tcatgaataa?aatattcttg?tcaaaaaaaa?aaaaaaaaaa????960
aaaaaaaaa????????????????????????????????????????????????????????????969
Claims (10)
1. protein is following (a) or protein (b):
(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and synthetic relevant with chlorogenic acid by sequence 1 deutero-protein.
2. coding claim 1 described proteic gene.
3. gene according to claim 2 is characterized in that: described gene is following 1) or 2) or 3) or 4) dna molecular:
1) its encoding sequence be in the sequence table sequence 2 from the dna molecular shown in 5 ' the 53rd to 823 Nucleotide;
2) dna molecular shown in the sequence 2 in the sequence table;
3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;
4) with 1) or 2) or 3) dna sequence dna that limits has 90% above homology, and the dna molecular of the chlorogenic acid synthesis associated protein of encoding.
4. the recombinant expression vector that contains claim 2 or 3 described genes.
5. recombinant expression vector as claimed in claim 4 is characterized in that: described recombinant expression vector is that the multiple clone site that claim 2 or 3 described genes insert the pBI121 plasmids is obtained.
6. contain claim 2 or 3 described expression of gene boxes, transgenic cell line or reorganization bacterium.
7. total length and any segmental primer thereof of amplification claim 2 or 3 described genes are right.
8. a method of cultivating the transgenic plant of chlorogenic acid content raising is that claim 2 or 3 described genes are imported in the purpose plants, obtains the transgenic plant that chlorogenic acid content improves than purpose plant; Described purpose plant is the plant with synthetic chlorogenic acid ability.
9. method as claimed in claim 8 is characterized in that: claim 2 or 3 described genes import in the described purpose plant by claim 4 or 5 described recombinant expression vectors; Described purpose plant is a tobacco.
10. the application of transgenic plant in producing chlorogenic acid that improve of the chlorogenic acid content for preparing of the described method of claim 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910178164A CN101693739A (en) | 2009-10-15 | 2009-10-15 | Chlorogenic acid synthesis associated protein and encoding gene thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910178164A CN101693739A (en) | 2009-10-15 | 2009-10-15 | Chlorogenic acid synthesis associated protein and encoding gene thereof and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101693739A true CN101693739A (en) | 2010-04-14 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910178164A Pending CN101693739A (en) | 2009-10-15 | 2009-10-15 | Chlorogenic acid synthesis associated protein and encoding gene thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101693739A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484544A (en) * | 2019-08-31 | 2019-11-22 | 贵州大学 | Tobacco gene LBM1, its screening technique and the application in regulation plant epidermal hair development |
| CN110904067A (en) * | 2019-12-20 | 2020-03-24 | 中国烟草总公司郑州烟草研究院 | Tobacco chlorogenic acid synthetic gene NtHQT and application thereof |
| CN114395566A (en) * | 2022-03-28 | 2022-04-26 | 江苏省农业科学院 | Application of sweet potato ERF transcription factor IbERF4 in promoting synthesis of plant chlorogenic acid substances |
-
2009
- 2009-10-15 CN CN200910178164A patent/CN101693739A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110484544A (en) * | 2019-08-31 | 2019-11-22 | 贵州大学 | Tobacco gene LBM1, its screening technique and the application in regulation plant epidermal hair development |
| CN110904067A (en) * | 2019-12-20 | 2020-03-24 | 中国烟草总公司郑州烟草研究院 | Tobacco chlorogenic acid synthetic gene NtHQT and application thereof |
| CN110904067B (en) * | 2019-12-20 | 2022-08-26 | 中国烟草总公司郑州烟草研究院 | Tobacco chlorogenic acid synthetic gene NtHQT and application thereof |
| CN114395566A (en) * | 2022-03-28 | 2022-04-26 | 江苏省农业科学院 | Application of sweet potato ERF transcription factor IbERF4 in promoting synthesis of plant chlorogenic acid substances |
| CN114395566B (en) * | 2022-03-28 | 2022-05-24 | 江苏省农业科学院 | Application of sweet potato ERF transcription factor IbERF4 in promoting synthesis of plant chlorogenic acid substances |
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Open date: 20100414 |