CN106148355A - The application in regulation and control Lignanoids compounds synthesis of the Isatis indigotica Fort. IiAP2/ERF049 gene - Google Patents
The application in regulation and control Lignanoids compounds synthesis of the Isatis indigotica Fort. IiAP2/ERF049 gene Download PDFInfo
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- CN106148355A CN106148355A CN201610502516.5A CN201610502516A CN106148355A CN 106148355 A CN106148355 A CN 106148355A CN 201610502516 A CN201610502516 A CN 201610502516A CN 106148355 A CN106148355 A CN 106148355A
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- isatis indigotica
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
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- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to genetic engineering field, AP2/ERF family IiAP2/ERF049 transcription factor that in a kind of Isatis indigotica Fort., Lignanoids compounds content is relevant, encode the nucleotide sequence of this transcription factor, and the application in lariciresinol content in improving Isatis indigotica Fort..The present invention is by building the Overexpression vector of IiAP2/ERF049, by crucial transcription factor IiAP2/ERF049 genetic transformation Isatis indigotica Fort., break the bottleneck of lariciresinol synthesis, obtain the Isatis indigotica Fort. hairy root strain of high yield, provide high efficiency regulatory target spot for improving the yield of lariciresinol, improve larch turpentine cellulose content, alleviate lariciresinol supply shortage, reduce the cost in Chinese medicine extraction, reduce and pollute, cultivate the aspects such as excellent Isatis indigotica Fort. strain and there is huge application prospect.
Description
Technical field
The present invention relates to gene engineering technology field, specifically, be that Isatis indigotica Fort. IiAP2/ERF049 gene is at regulation and control wood fat
Application in chlorins compound synthesis.
Background technology
Isatis indigotica Fort. (Isatis indigotica Fort.), Cruciferae 1 year or 2 years SHENGCAO bases, begin to be loaded in " legendary god of farming's basis
Grass warp ", its root is used as medicine and is conventional Chinese medicine Radix Isatidis, commonly uses antiviral Chinese herbal medicine the successive dynasties for China.Modern clinic is used for flow
Row sexuality emits, epidemic encephalitis type B, mumps, acute, chronic hepatitis, herpes zoster etc., in severe acute respiratory syndrome
And the preventing and treating of influenza A H1N1 influenza virus serves important function (SARS).This seminar early stage passes through antiviral study in vitro
Specify that the Lignanoids compounds with lariciresinol as representative is the material base that Isatis indigotica Fort. plays antiviral activity effect.Closely
Over Nian, Zhong Nanshan teaches seminar it has also been found that the derivant of lariciresinol has significant anti-A type and Influenza B virus
Effect.But the content that lariciresinol is in Isatis indigotica Fort. root is seldom, only 47.14 μ g/g, have a strong impact on Isatis indigotica Fort. as one
The application of high-quality Chinese medicine.Therefore, start with from the key gene of regulation and control secondary metabolism, improve the kind matter of Isatis indigotica Fort., improve larch
The content of fat element becomes problem demanding prompt solution.
Plant secondary metabolic engineering is to utilize genetic engineering and molecular biology method to illustrate Secondary metabolites life
The mechanism of thing synthesis, checks on the information of metabolic pathway and relevant enzyme, regulates and controls the gene controlling Biosynthetic pathway,
Thus reach to transform on a molecular scale metabolic pathway, the controlled syntheses of goal of regulation and control product.Find by literature search, AP2/
ERF (APETALA2/ethylene-responsive factor) family's transcription factor can significantly regulate and control arabidopsis, Chinese lute and
The degree of lignification of Radix Dauci Sativae, can regulate and control alkaloids composition in Herba Catharanthi Rosei simultaneously, the synthesis of ter penoids in Herba Artemisiae Annuae, for
The synthesis of secondary metabolite has important regulating and controlling effect, is the important regulating and controlling target spot of lignanoid's synthesis.Experiment is simultaneously with hair
Shape root is bioreactor, has that inherited character is relatively stable, fast growth, metabolite content advantages of higher, is conducive to
The metabolic pathway of research plant, can also utilize some genetic engineering means to explore simultaneously and create new synthetic route, obtaining
It is worth higher product, the planting of medicinal materials soil can be saved, reduce and farmland is taken, be the effective of production Chinese medicine medicine resource
Means.
Chinese patent literature CN104805097A discloses Isatis indigotica Fort. (+)-Pinoresinol coded sequence of reductase enzyme protein and application, coding
Having the nucleotide sequence of the albumen of Isatis indigotica Fort. lariciresinol reductase activity, in plant, high expressed Isatis indigotica Fort. lariciresinol is also
Protoenzyme thus improve the yield of lariciresinol.Chinese patent literature CN104805096A discloses Isatis indigotica Fort. 4-coumaroyl A
Ligase protein family coded sequence and application, fallen to change by the expression of regulation and control Isatis indigotica Fort. 4-coumaroyl A family protein
The biosynthetic method of leaf pinoresinol, has important using value in terms of breeding and bioenergy.But about Isatis indigotica Fort.
IiAP2/ERF049 gene and the application in regulation and control Lignanoids compounds synthesis thereof yet there are no report.
Summary of the invention
It is an object of the invention to provide the AP2/ERF family that in a kind of Isatis indigotica Fort., Lignanoids compounds content is relevant
IiAP2/ERF049 transcription factor, encode the nucleotide sequence of this transcription factor, and lariciresinol contains in improving Isatis indigotica Fort.
Application in amount.
Closely related with the synthesis of lignanoid in order to excavate in the middle of Isatis indigotica Fort. which AP2/ERF family transcription factor actually, this
Inventing by the analysis to Isatis indigotica Fort. transcript profile data, annotation obtains 112 Isatis indigotica Fort. AP2/ERF family transcription factor.Detection MeJA
Different time points after induction (0,3,6,12,24h) AP2/ERF family transcription factor, lariciresinol route of synthesis gene and
The change of Lignanoids compounds content, with Pearson correlation coefficients 0.5 as thresholding (lrl > 0.5), builds " transcription factor-base
Cause-compound " relatedness network, screening obtains and Lignanoids compounds content and lignanoid's route of synthesis gene in Isatis indigotica Fort.
Related AP2/ERF family transcription factor IiAP2/ERF049.Referring specifically to embodiment 1.
A first aspect of the present invention, it is provided that a kind of Isatis indigotica Fort. IiAP2/ERF049 gene, its nucleotide sequence such as SEQ ID
Shown in NO:1.Described nucleotides sequence is classified as 684bp, and wherein, start codon is ATG, and termination codon is TGA.
The described sequence such as SEQ ID NO:1 is the open reading frame of Isatis indigotica Fort. IiAP2/ERF049 full-length genome DNA
(ORF), described Isatis indigotica Fort. IiAP2/ERF049 full-length genome DNA sequence is 1352bp, its nucleotide sequence such as SEQ ID
Shown in NO:2, containing six exons, five introns.
A second aspect of the present invention, it is provided that a kind of by the albumen of above-mentioned Isatis indigotica Fort. IiAP2/ERF049 gene code, i.e.
IiAP2/ERF049 transcription factor, its aminoacid sequence is as shown in SEQ ID NO:3, containing 227 aminoacid.
A third aspect of the present invention, it is provided that a kind of recombinant expression carrier containing above-mentioned Isatis indigotica Fort. IiAP2/ERF049 gene,
Recombinant bacterium or transgenic plant.
Preferably, when the recombinant expression carrier described in preparation or recombinant bacterium, it is used for expanding Isatis indigotica Fort. IiAP2/ERF049 base
The primer of cause to for:
Forward (F): 5'-ATGGTGAGCTTAAGAAGG-3'(SEQ ID NO:4)
Reversely (R): 5'-TCAGGTAGAAAGTGTACTGA-3'(SEQ ID NO:5).
Preferably, described recombinant expression carrier is plant expression vector.Preferred plasmid pIiAP2/ERF049-OVX.
Preferably, described recombinant bacterium, i.e. host cell, for escherichia coli, Agrobacterium etc..It is preferably Agrobacterium.More excellent
Select Agrobacterium rhizogenes C58C1.
A fourth aspect of the present invention, it is provided that above-mentioned Isatis indigotica Fort. IiAP2/ERF049 gene is lignanoids in improving Isatis indigotica Fort.
Application in compound content.Preferably, described Isatis indigotica Fort. IiAP2/ERF049 gene improves lariciresinol, Colophonium in Isatis indigotica Fort.
Alcohol, the content of Secoisolariciresinol.
A fifth aspect of the present invention, it is provided that above-mentioned IiAP2/ERF049 transcription factor is lignanoids in improving Isatis indigotica Fort.
Application in compound content.Preferably, above-mentioned IiAP2/ERF049 transcription factor improves lariciresinol, Colophonium in Isatis indigotica Fort.
Alcohol, the content of Secoisolariciresinol.
A sixth aspect of the present invention, it is provided that a kind of improve the method for lariciresinol content in Isatis indigotica Fort., comprises the following steps:
A, the structure recombinant expression carrier containing above-mentioned Isatis indigotica Fort. IiAP2/ERF049 gene；
B, the recombinant expression carrier conversion Agrobacterium built by step A, obtain recombinant bacterium；
The recombinant bacterium that C, step B obtain is transformed in Isatis indigotica Fort. tissue or cell, and screening obtains larch turpentine cellulose content and increases
Isatis indigotica Fort. plant.
Preferably, described improves the method for lariciresinol content in Isatis indigotica Fort., comprises the following steps:
(1) method using gene clone obtains the cDNA total length of Isatis indigotica Fort. IiAP2/ERF049；
(2) being connected with green fluorescent protein tag GFP by IiAP2/ERF049, IiAP2/ERF049 is subcellular fraction in detection
The location of level；
(3) IiAP2/ERF049 is connected to expression regulation sequence, forms the plant expression vector containing described gene, will
This plant expression vector transforming agrobacterium rhizogenes C58C1, it is thus achieved that the agrobacterium rhizogene strain of IiAP2/ERF049；
(4) agrobacterium rhizogene strain constructed by (3) is converted Isatis indigotica Fort. leaf dish, it is thus achieved that aggregated polymerase chain reaction
The transgenic hairy root of (Polymerase Chain Reaction, PCR) test positive；
(5) positive strain changes a subculture for every 9 days, measures hairy root mass change, arrives the growth platform phase to 45 days,
Draw growth rate curve；
(6) in real-time fluorescence quantitative PCR (Quantitative Real-time PCR, Q-PCR) detection Isatis indigotica Fort. hairy root
The expression of IiAP2/ERF049；
(7) lariciresinol and precursor chemical combination thereof during liquid chromatograph-second order ms is used in conjunction detection Isatis indigotica Fort. transgenic hairy root
Thing (+)-Pinoresinol, the content of its open-loop products Secoisolariciresinol, the hairy root that screening larch turpentine cellulose content significantly improves
Strain.
Gene clone method described in step (1) refers to reverse transcriptional PCR (Reverse Transcription
Polymerase Chain Reaction, RT-PCR), the forward primer of use is: 5'-ATGGTGAGCTTAAGAAGG-3'
(SEQ ID NO:4), downstream primer is: 5'-TCAGGTAGAAAGTGTACTGA-3'(SEQ ID NO:5).
Detection IiAP2/ERF049 described in step (2) is that Polyethylene Glycol is situated between in the method used by the location of subcellsular level
The protoplast transformation led, protoplast used is rice protoplast.
PCR detection method described in step (4) is: the rolb gene separately designing on Agrobacterium Ti plasmid, on carrier
Specific primer (JDIiAP2/ERF049-F and JDIiAP2/ of hygromycin gene hpt and Insert Fragment upstream and downstream
ERF049-R) carrying out PCR amplification, observe purpose band after agarose gel electrophoresis under uviol lamp, screening obtains the positive and turns base
Because of Isatis indigotica Fort. hairy root strain.
In Q-PCR detection transgenic Isatis indigotica Fort. hairy root described in step (6), the expression of IiAP2/ERF049 gene is:
With Isatis indigotica Fort. house-keeping gene Actin and IiAP2/ERF049 gene, carrying out quantitative PCR detection, screening IiAP2/ERF049 gene is high
The transgenic hairy root strain expressed.
Detection method of content described in step (7) is: use Agilent 1200 liquid chromatograph-G6410A triple level Four bar
GC-MS detection hairy root in the volume of compounds, chromatographic column be Agilent ZORBAX SB-C18 (3.5 μm, 2.1
× 100mm), use acetonitrile-5mM ammonium acetate solution gradient elution, program is: within 0-4 minute, acetonitrile volume is increased to by 14%
50%, 5mM ammonium acetate solution volume is down to 50% by 86%；Within 4-4.5 minute, acetonitrile volume is increased to 85% by 50%, 5mM
Ammonium acetate solution volume is down to 15% by 50%；Within 4.5-8.5 minute, acetonitrile volume keeps 85%, 5mM ammonium acetate solution body
Long-pending holding 15%.Wherein parent ion peak and the daughter ion peak of lariciresinol is respectively 359.2 and 329.0；(+)-Pinoresinol is 357.1
With 151.1；Open loop lariciresinol is 361.1 and 164.6.Column temperature is 30 DEG C, flow velocity 0.3mL/ minute, sample size 5 μ L, uses
Two-point method calculates three kinds of compounds contents in hairy root, and computing formula is A0/C0=Ax/Cx, wherein A0And C0It is respectively standard substance
The peak area of solution and concentration, AxAnd CxIt is respectively peak area and the concentration of sample solution.
Isatis indigotica Fort. IiAP2/ERF049 transcription factor is proceeded to Isatis indigotica Fort. by the present invention, and screening obtains what lariciresinol significantly improved
Transgenic Isatis indigotica Fort. hairy root.When growing 45 days arrival growth platform phases, transgenic hairy root biological accumulation amount and wild type hairy
Root biological accumulation amount there was no significant difference, but in transgenic hairy root, lariciresinol content dramatically increases, wherein containing quantitative change
Changing lariciresinol content in the most obvious strain is 424.60 μ g/g, about improves compared with wild type control (51.13 μ g/g)
8.30 times.
The present invention is by building the Overexpression vector of IiAP2/ERF049, by crucial transcription factor IiAP2/ERF049
Genetic transformation Isatis indigotica Fort., breaks the bottleneck of lariciresinol synthesis, it is thus achieved that the Isatis indigotica Fort. hairy root strain of high yield, for improving larch turpentine
The yield of element provides high efficiency regulatory target spot, is improving larch turpentine cellulose content, is alleviating lariciresinol supply shortage, in reduction
Cost in medicine extraction, reduces and pollutes, cultivate the aspects such as excellent Isatis indigotica Fort. strain and have huge application prospect.
Accompanying drawing explanation
Fig. 1 screens and obtains the Isatis indigotica Fort. AP2/ERF transcription factor relevant to lariciresinol synthesis.
The domain schematic diagram of Fig. 2 IiAP2/ERF049 gene.
What Fig. 3 IiAP2/ERF049 albumen was special is positioned at the nucleus of cell, and wherein A-D is IiAP2/ERF049-
GFP albumen specifically expressing is at nucleus, and E-H is that blank GFP protein expression shows the green glow of GFP at Cytoplasm, A, E, and B, F are leaf
The HONGGUANG of green body, C, G are green glow and HONGGUANG combines, and D, H are that green glow, HONGGUANG, white light combine.
Fig. 4 IiAP2/ERF049 transcription factor is the plant expression vector pIiAP2/ERF049-OVX of process LAN in Isatis indigotica Fort.
Structure schematic diagram.
Fig. 5 transgenic hairy root Biomass Accumulation.
Fig. 6 positive hairy root strain PCR qualification result.
The transcriptional level of IiAP2/ERF049 in Fig. 7 transgenic hairy root.
In Fig. 8 Isatis indigotica Fort., process LAN IiAP2/ERF049 gene can make lariciresinol content increase, and wherein A is (+)-Pinoresinol
Content, B is larch turpentine cellulose content, and C is Secoisolariciresinol content.
Detailed description of the invention
The detailed description of the invention provided the present invention below in conjunction with embodiment elaborates.
Agents useful for same of the present invention and raw material the most maybe can be prepared by literature method.Primer in the following example and
Order-checking is completed by Jin Weizhi bio tech ltd, Suzhou, the experimental technique of unreceipted actual conditions in embodiment, generally
According to normal condition such as Sambrook et al. " molecular cloning: lab guide " (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to normal condition, or according to the condition proposed by manufacturer.
Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.
Embodiment 1: the screening of Isatis indigotica Fort. AP2/ERF transcription factor
Respectively from arabidopsis data base (DATF, http://datf.cbi.pku.edu.cn/) and Chinese cabbage data base
(http://brassicadb.org/brad/) downloads and obtains 159 (Arabidopsis of arabidopsis AP2/ERF transcription factor
Thaliana AP2/ERFs, AtAP2/ERFs) and 321 (Chinese cabbage of Chinese cabbage AP2/ERF transcription factor
AP2/ERFs, BraAP2/ERFs), set up crucifer AP2/ERF data base according to its aminoacid sequence.Use
This data base and Isatis indigotica Fort. transcript profile data base are compared by TBLASTN algorithm, and (e-value value is set to 10-5), identify and extract
With the gene of crucifer AP2/ERF homology as Isatis indigotica Fort. AP2/ERF candidate gene in Isatis indigotica Fort. transcript profile data base.
Application bioinformatics software Vector NTI 11.5 and MEGA 5.05 analyzes the Isatis indigotica Fort. AP2/ERF of above-mentioned acquisition
The nucleotide sequence of candidate gene, extracts open reading frame (open reading frame, ORF), and translates into aminoacid sequence
Row.Application Pfam data base (http://pfam.janelia.org/) and SMART analytical tool (http://smart.embl-
Heidelberg.de/) obtained amino acid whose AP2/ERF functional domain is identified, finally determines that Isatis indigotica Fort. AP2/ERF compiles
Code gene (IiAP2/ERFs) totally 112.
According to the annotation of Isatis indigotica Fort. AP2/ERF encoding gene, different after MeJA (0.5 μM) process that seminar's early stage is set up
In the gene expression profile data storehouse of time point (0,1,3,6,12 and 24h) Isatis indigotica Fort. hairy root, under extraction MeJA (0.5 μM) process not
With AP2/ERF encoding gene expressing information in time point Isatis indigotica Fort. hairy root and Lignanoids compounds route of synthesis gene expression amount
Information (sets the expression of 0h as 1 as reference).After using the method in embodiment 7 to measure MeJA (0.5 μM) process simultaneously
(+)-Pinoresinol, laricin, larch quality, the different larch of open loop in different time points (0,1,3,6,12 and 24h) Isatis indigotica Fort. hairy root
Four kinds of Lignanoids compounds changes of contents of quality.
Use canonical correlation analysis (canonical correlation analysis) method in SPSS software, by MeJA
Different time points under inducing action (0,3,6,12,24h) change of 112 AP2/ERF family transcription factor expression levels and LC-
MS/MS measures different time points lignanoid under the MeJA inducing action that obtains, and ((+)-Pinoresinol, laricin, larch quality, open loop are different
Larch quality) changes of contents integrates, and builds " transcription factor-compound " relatedness network；Identical method is used to incite somebody to action
Different time points under MeJA inducing action (0,3,6,12,24h) change of 112 AP2/ERF family transcription factor expression levels
Integrate with different time points Lignanoids compounds route of synthesis gene expression amount change under MeJA inducing action, build
" transcription factor-gene " relatedness network.With Pearson correlation coefficients 0.5 as thresholding (lrl > 0.5), screening obtains and falls respectively
Leaf spongiosa element synthesizes closely-related AP2/ERF family transcription factor, AP2/ERF family closely-related with approach gene expression
Two groups of transcription factor of obtaining of screening are compared by transcription factor, coincidence person be with lignanoid's route of synthesis gene expression and
Lignanoid synthesizes the AP2/ERF family transcription factor of equal significant correlation, and the selection result is shown in Fig. 1.As shown in Figure 1, No. 049 AP2/
ERF family transcription factor IiAP2/ERF049 is the closeest with the expression of Lignanoids compounds synthesis and lignanoid's route of synthesis gene
(AP2/ERF family transcription factor can be further divided into five subfamilies of AP2, DREB, ERF, RAV, Soloist) is closed in cut.
Embodiment 2: the clone of Isatis indigotica Fort. IiAP2/ERF049 gene
1. Isatis indigotica Fort. DNA and the extraction of total serum IgE
Take Isatis indigotica Fort. aseptic seedling about 100mg, rapid grind into powder in liquid nitrogen, use CTAB method to extract Isatis indigotica Fort. DNA；Separately take ancient name for Chinese cabbage
Blue aseptic seedling about 100mg, rapid grind into powder in liquid nitrogen, add the 1.5ml centrifuge tube being pre-loaded with plant tissue lysate
In, mixing of fully vibrating, then according to TIANGEN TRNzol-A+It is total that the description of total RNA extraction reagent box extracts Isatis indigotica Fort.
RNA.Use 1% agarose gel electrophoresis identification of dna and total serum IgE quality, with Nano drop 2000C spectrophotometer
(Thermo Scientific, San Jose, California, USA) detects DNA and total serum IgE purity and concentration；
2. the clone of Isatis indigotica Fort. IiAP2/ERF049 gene
With the total serum IgE 1 μ g that extracted as template, use full formula gold TansScript First-Strand cDNA
Synthesis Supermix test kit synthesis Isatis indigotica Fort. cDNA.
Open reading frame design gene-specific primer according to IiAP2/ERF049 gene:
Forward (F): 5'-ATGGTGAGCTTAAGAAGG-3'(SEQ ID NO:4)
Reversely (R): 5'-TCAGGTAGAAAGTGTACTGA-3'(SEQ ID NO:5).
Use Trans Start Fast@Pfu DNA Polymerase test kit carries out PCR amplification, reaction system and bar
Part is as follows: template (cDNA) 1 μ L, each 1 μ L of positive anti-primer, EasyPfu DNA Polymerase1 μ L, 10 × EasyPfu
Buffer 5 μ L, 2.5mM dNTPs 2.5 μ L, 5 × PCR buffer 5 μ L, deionized water 13.5 μ L, constitute 25 μ L reactants
System.PCR reaction condition is: 94 DEG C of denaturations 1 minute, and (94 DEG C of degeneration 20 seconds, anneal 20 seconds for 45 DEG C, 72 DEG C for 35 amplification cycles
Extend 1 minute), 72 DEG C are continued to extend 5 minutes.
The PCR primer obtained is through 1% agarose gel electrophoresis, and target fragment glue connects with pGEMT-easy carrier after reclaiming
Connect, convert bacillus coli DH 5 alpha, after incubated overnight, choose monoclonal, after bacterium inspection, send order-checking (limited by Suzhou gold only intelligence biotechnology
Company completes), sequencing result the be successful clone consistent with IiAP2/ERF049 sequence in Isatis indigotica Fort. transcript profile data base obtains
The open reading frame (ORF) (SEQ ID NO:1) of Isatis indigotica Fort. IiAP2/ERF049.Wherein, start codon is ATG, terminates close
Numeral is TGA.
Additionally with Isatis indigotica Fort. DNA as template, the genomic DNA of amplification Isatis indigotica Fort. AP2/ERF class transcription factor IiAP2/ERF049
(SEQ ID NO:2), ibid, simply in reaction, extension of time is set to 3 minutes for reaction system and reaction condition.
The genomic DNA of IiAP2/ERF049 is 1352bp, and open reading frame is 684bp, by the base of IiAP2/ERF049
Because group DNA is compared with open reading frame, finding that the genomic DNA of IiAP2/ERF049 contains six exons, five include
Son.The ORF finder function of NCBI derives its albumen coded sequence (SEQ ID NO:3), use SMART (http: //
Smart.embl-heidelberg.de/) software, it was predicted that go out IiAP2/ERF049 albumen and comprise an AP2 domain (121-
178 aminoacid), it is the transcription factor of an AP2/ERF class, comprises 227 aminoacid (as shown in Figure 2) altogether.
Embodiment 3: the Subcellular Localization of Isatis indigotica Fort. IiAP2/ERF049 gene
According to the content of example 2 bioinformatic analysis, IiAP2/ERF049 gene is to have an AP2 domain
AP2/ERF class transcription factor.For checking IiAP2/ERF049 gene is as the character of transcription factor further, we construct
The Subcellular Localization carrier of IiAP2/ERF049 gene, by rice transformation protoplast, it was demonstrated that IiAP2/ERF049 is positioned
Nucleus, meets the characteristic of transcription factor.
1. the structure of Subcellular Localization carrier
In the present embodiment, according to pCAMBIA1301-GFP carrier sequence and IiAP2/ERF049 gene ORF region sequence,
Designing the primer with restriction enzyme site, wherein forward primer contains Nco I restriction enzyme site (5'-
AACCATGGGAATGGTGAGCTTAAGAAGG-3', SEQ ID NO:6), reverse primer contains Spe I restriction enzyme site (5'-
GGACTAGTGGTAGAAAGTGTACTGATCT-3', SEQ ID NO:7).PCR obtains containing Nco I and Spe I restriction enzyme site
IiAP2/ERF049 coding region, use Nco I and Spe I enzyme action IiAP2/ERF049 coding region and pCAMBIA1301-respectively
GFP carrier, reclaims IiAP2/ERF049 gene and pCAMBIA1301-GFP large fragment, connects and converts, and picking list bacterium colony send survey
Sequence, the IiAP2/ERF049 gene Subcellular Localization carrier that final acquisition order-checking is correct.
2. protoplast transformation and observation
According to the IiAP2/ that the operating instruction of the big extraction reagent kit of QENGEN plasmid, extraction acquisition purity and concentration are higher
ERF049 gene Subcellular Localization vector plasmid, then rice transformation protoplast.Concrete grammar is with reference to the protoplasm of PEG mediation
Body method for transformation.The protoplast of pCAMBIA1301-GFP empty carrier is converted as negative control under the same terms.
The rice protoplast converted, room temperature 25 DEG C is used confocal laser scanning microscope after cultivating 18 hours, is sent out
Existing IiAP2/ERF049 is positioned in nucleus specifically, consistent with the function of transcription factor (as shown in Figure 3).
Embodiment 4: the structure of the plant process LAN binary vector of Isatis indigotica Fort. IiAP2/ERF049 gene
In the present embodiment, with the bacterium solution containing IiAP2/ERF049 as template, the forward primer of design band restriction enzyme site:
5'-AAGGATCCATGGTGAGCTTAAGAAGG-3'(SEQ ID NO:8, containing Bam HI restriction enzyme site) and reverse primer: 5'-
CCACTAGTGGTAGAAAGTGTACTGA-3'(SEQ ID NO:9, containing Spe I restriction enzyme site).Use NEB Phusion@Super
Fidelity PCR MasterMix carries out PCR amplification, it is thus achieved that with the genes of interest of restriction enzyme site, is inserted into PHB-after enzyme action
Flag expression vector, after conversion, picking list bacterium colony send order-checking, the IiAP2/ERF049 process LAN double base that final acquisition order-checking is correct
Carrier (as shown in Figure 4).
Embodiment 5: Agrobacterium rhizogenes mediation IiAP2/ERF049 over-express vector genetic transformation Isatis indigotica Fort. blade and turn base
Acquisition because of hairy root
1.IiAP2/ERF049 over-express vector transforming agrobacterium rhizogenes
In this embodiment, the IiAP2/ERF049 plant binary over-express vector that will obtain in embodiment 4, use freeze thawing
Method proceeds in Agrobacterium rhizogenes C58C1, and PCR identifies that positive bacterial strain is the Agrobacterium rhizogenes successfully proceeding to over-express vector
(C58C1-pIiAP2/ERF049-OVX)。
2. Agrobacterium rhizogenes mediation IiAP2/ERF049 gene leaf disk method converts
2.1 leaf disk method step of converting are as follows:
A. select the positive bacterium colony on flat board with sterile toothpick picking YEB, be inoculated in 2ml YEB fluid medium, 28
DEG C, 200 revs/min of shaken cultivation are overnight；
B. by C58C1 sky bacterium and the bacterium liquid activation two of C58C1-PHB-flag, C58C1-pIiAP2/ERF049-OVX
Secondary, take 20 μ L every time and add in 2mL YEB fluid medium, 28 DEG C, 200 revs/min of overnight incubation.Wherein C58C1 sky bacterium makes
By the culture medium of YEB (40mg/L rifampicin), other bacterial strains use the training of YEB (40mg/L rifampicin+75mg/L kanamycin)
Support base；
C. take 30mL YEB culture medium, add the Agrobacterium that 1mL activates, 28 DEG C, 200 revs/min of overnight incubation；
D.5,000 rev/min is centrifuged 10 minutes, abandons supernatant, with the 20mL MS fluid medium containing 100 μMs of acetosyringones
Resuspended thalline；
E. take Isatis indigotica Fort. aseptic seedling, blade is cut into about 1cm by super-clean bench2Size, immerses in resuspended good thalline, 28 DEG C,
200 revs/min of shaken cultivation 10 minutes；
F. leaf dish is taken out, uses aseptic filter paper the bacterium solution above leaf dish to be blotted, face up and be put in MS culture medium,
(25 soil 1) DEG C light culture 2 days in constant incubator.
G. leaf dish light culture takes out two days later, uses aseptic water washing five times in super-clean bench, and aseptic filter paper blots, and is placed in 1/
In 2MS (500mg/L cephamycin) culture medium, in (25 soil 1) DEG C light culture；
After the most about two weeks, leaf plate edge grows hairy root, carefully cuts during root length pending to 3-5cm, every 15d subculture one
Secondary, and constantly reduce cephamycin concentration (300mg/L → 100mg/L → 0mg/L), the hairy induced except C58C1 sky bacterium
Root, other groups are additionally added 10mg/L hygromycin, screening positive clone on the basis of 1/2MS+ cephamycin, are continued (25 soil 1)
DEG C light culture；
I. when cephamycin concentration reduces to 0mg/L, continuing to cultivate about two weeks, select growth conditions good, branch is more
Strain be numbered, C58C1 sky bacterium induce for wild type hairy root, numbered: WT1, WT2, WT3 ...；C58C1-
PHB-flag induce for blank hairy root, numbered: CK-1, CK-2, CK-3 ...；C58C1-pIiAP2/ERF049-
OVX induce for transgenic hairy root, the most numbered: OVX049-1, OVX049-2, OVX049-3 ...
J. confirm positive root of hair strain through PCR, take 100mg and proceed to, in 200mL1/2MS fluid medium, be placed in shaking flask
In cabinet, 120 revs/min, 25 DEG C of light culture, changed a culture fluid and record the change of hairy root growth amount, receiving to 45 days every 9 days
Obtain.As it is shown in figure 5, be changed to vertical coordinate with weight in wet base, map with growth time for abscissa.Hairy root grows rapidly after 18d,
Enter the growth platform phase during 36d, though Biomass slightly raises during 45d, but change inconspicuous, with wild type hairy root and blank hair
Shape root is compared, and the transgenic hairy root biomass accumulation of overexpression IiAP2/ERF049 changes without significance.
Described YEB culture medium is: take yeast extract 1g/L, peptone 5g/L, beef extract 5g/L, sucrose respectively
5g/L, magnesium sulfate seven water 0.24g/L, pH=7.2, if preparation solid medium, add 7g agar.121 DEG C of High Temperature High Pressure
Sterilizing 20 minutes, standby.
Described MS culture medium is MS culture medium+sucrose (30g/L)+agar (6g/L), pH5.8-6.0,121 DEG C of high temperature height
Pressure sterilizing 20 minutes, standby.Wherein MS culture medium is purchased from Phyto Technology Laboratories (U.S.).
Described fluid medium is: 1/2MS culture medium+30g/L sucrose, pH5.8-6.0.
2.2 positive hairy root authentication methods are as follows: containing Ri plasmid in Agrobacterium rhizogenes cell, rolb and rolc thereon
Gene can induce and maintain root of hair form, chooses rolb gene to detect gained as hairy root, rather than adventitious root.PHB-flag
Carrier has hygromycin gene (hpt), utilizes hpt gene and vector-specific primers (JDPDK) to IiAP2/
ERF049 transgenic root of hair carries out Molecular Identification, and C58C1-PHB-flag, C58C1-pIiAP2/ERF049-OVX bacterium solution is as sun
Property comparison.
Extract STb gene according to the CTAB method that embodiment 2 is used, carry out PCR qualification, the primer with STb gene for template
Sequence is as follows:
Rolb gene amplification primer is:
rolb-F:5'-CGAGGGGATCCGATTTGCTT-3'(SEQ ID NO:10)
rolb-R:5'-GACGCCCTCCTCGCCTTCCT-3'(SEQ ID NO:11)
Hpt gene amplification primer is:
hpt-F:5'-CGATTTGTGTACGCCCGACAGTC-3'(SEQ ID NO:12)
hpt-R:5'-CGATGTAGGAGGGCGTGGATATG-3'(SEQ ID NO:13)
JDIiAP2/ERF049 gene amplification primer is:
JDIiAP2/ERF049-F:5'-CTCAAGCAAGGATCCATGGTGA-3'(SEQ ID NO:14)
JDIiAP2/ERF049-R:5'-CGATACCGTCACTAGTGGTAGAA-3'(SEQ ID NO:15)
Takara rTaq enzyme is used to carry out PCR amplification, reaction system and condition as follows: template (DNA) 1 μ L, positive anti-primer
Each 1 μ L, 2 × rTaq PCR SuperMix 10 μ L, deionized water 7 μ L, totally 20 μ L reaction system.PCR reaction condition is: 94 DEG C
Denaturation 5 minutes, 35 amplification cycles (94 DEG C of degeneration 30 seconds, anneal 30 seconds for 55 DEG C, and 72 DEG C extend 1 minute), 72 DEG C are continued to prolong
Stretch 5 minutes.
After PCR reaction terminates, agarose gel detection PCR result, wherein rolb and hpt gene can amplify respectively
The specific fragment of 423bp and 812bp size, uses primer JDIiAP2/ERF049-F and JDIiAP2/ on PHB-flag carrier
The sequence that ERF049-R expands out is equal with IiAP2/ERF049 length scale, therefore successfully imports pIiAP2/ERF049-OVX
Hairy root strain the fragment of about 897bp can be detected.
Observe under uviol lamp and rolB, hpt gene and JDIiAP2/ERF049 band of the same size, result such as Fig. 6
Shown in, show screening acquisition is the hairy root containing genes of interest IiAP2/ERF049 induced by C58C1 Agrobacterium rhizogenes.
Embodiment 6: IiAP2/ERF049 transcriptional level detection in transgenic hairy root
In the present embodiment, the method for extracting total RNA used according to embodiment 2, extracts different IiAP2/ in embodiment 5
The RNA of ERF049 gene overexpression transgenic Isatis indigotica Fort. hairy root, and Overturn ratio is cDNA further.Designed by primer 3
IiAP2/ERF049 gene and Actin reference gene, across the real-time quantitative PCR primer (table 1) of intron, use TAKARA
SYBR-Green PCR Mastermix test kit carries out real-time fluorescence quantitative PCR analysis.Instrument is Thermal Cycler
Dice, two-step method carries out PCR amplification, 95 DEG C of denaturations 10 seconds, 40 amplification cycles (95 DEG C 5 seconds；60 DEG C 30 seconds), separating reaction
(95 DEG C 15 seconds；60 DEG C 30 seconds；95 DEG C 15 seconds).Use 2-ΔΔCtMethod carries out later data process, and result is as it is shown in fig. 7, calculate public affairs
Formula is as follows:
Destination gene expression amount=2-ΔΔCt
Δ Δ Ct=(CT genes of interest-CT reference gene)Experimental group-(CT genes of interest-CT reference gene)Comparison
Table 1 real-time quantitative PCR primer sequence
Embodiment 7: utilize LC-MS/MS to measure lariciresinol, (+)-Pinoresinol, the different larch of open loop in transgenic hairy root
Fat cellulose content
1. the preparation of standard solution
Precision weighs lariciresinol, (+)-Pinoresinol, Secoisolariciresinol standard substance 2mg respectively, adds 2mL methanol molten
Solve, be configured to the standard solution of 1mg/mL, standby；Take a sample test 5 samples at random, according to three kinds of compositions average in 5 samples
Concentration determines each concentration of component in hybrid standard product.Finally determine that in hybrid standard product, each concentration of component is respectively as follows: larch
Fat element 300ng/mL, (+)-Pinoresinol 100ng/mL, Secoisolariciresinol 100ng/mL.
2. the preparation of sample solution
The hairy root sample of results is overnight dried in 45 DEG C of drying baker, is fully ground in mortar.Weigh 100mg powder-like
Product are put in 10mL centrifuge tube, add 5mL methanol, and after vibration mixing, ultrasonic (40W) extracts 30 minutes, and period shook every 5 minutes
Swing mixing once；4,000 revs/min centrifugal 5 minutes, pours out supernatant in a clean pipe, repeats above operation once.Merge two
Secondary extracting solution, use volumetric flask constant volume to 10mL.Take 1mL extracting solution vacuum to volatilize, each sample cell adds 500 μ L initial flow
Dynamic phase, 1 minute sample dissolution of vortex, 14,000 revs/min are centrifuged 10 minutes, and supernatant, with the 0.22 organic membrane filtration of μm, takes
Subsequent filtrate sample introduction.
3. Mass Spectrometer Method condition
Mass spectrum uses electric spray ion source, ion mode, atomizer gas pressure 40psi, gas temperature 350 DEG C, gas
Rate of flow of fluid 10 liters/min, capillary voltage 4000V, mass spectrometry parameters is as shown in table 2.
The Mass Spectrometry Conditions that table 2 optimizes
4. chromatographic separation condition
Using Agilent ZORBAX SB-C18 (3.5 μm, 2.1 × 100mm) chromatographic column, acetonitrile-5mM ammonium acetate is water-soluble
Liquid gradient elution, elution program is as shown in table 3, column temperature 35 DEG C, flow velocity 0.3 ml/min, sample size 5 μ L, and single needle runs 8.5
Minute.
Table 3 gradient elution program
5. the mensuration of sample and calculating
Two-point method is used to calculate (+)-Pinoresinol, lariciresinol, Secoisolariciresinol content in hairy root, computing formula
For A0/C0=Ax/Cx.Wherein A0And C0It is respectively peak area and concentration, the A of standard solutionxAnd CxIt is respectively the peak of sample solution
Area and concentration.
In the present invention, (+)-Pinoresinol, lariciresinol, the different fallen leaves of open loop in IiAP2/ERF049 gene overexpression hairy root
The content of pinoresinol all improves (as shown in Figure 8).Compared with wild type, the transgenic hairy of process LAN IiAP2/ERF049 gene
In root, lariciresinol content is 4.39-8.30 times of wild type, and (+)-Pinoresinol content is 1.50-3.42 times of wild type, open loop
Isolariciresinol content is 5.75-10.57 times of wild type.Result is the meansigma methods repeated three biologys, error line table
Showing standard deviation, statistical analysis uses t-test inspection.
In a word, the present invention is by overexpression IiAP2/ERF049 transcription factor in Isatis indigotica Fort., it is thus achieved that a large amount of generations fall
The hairy root strain of leaf pinoresinol, is alleviating lariciresinol supply shortage, is reducing the cost in Chinese medicine extraction, reducing and pollute,
Cultivate the aspects such as excellent Isatis indigotica Fort. strain and there is huge application prospect.
Below preferred embodiment to the invention is illustrated, but the invention is not limited to described
Embodiment, those of ordinary skill in the art it may also be made that all equivalents on the premise of the invention spirit
Modification or replacement, modification or the replacement of these equivalents are all contained in the application claim limited range.
Claims (9)
1. an Isatis indigotica Fort. IiAP2/ERF049 gene, its nucleotide sequence is as shown in SEQ ID NO:1.
2. an IiAP2/ERF049 transcription factor for Isatis indigotica Fort. IiAP2/ERF049 gene code as claimed in claim 1, its
Aminoacid sequence is as shown in SEQ ID NO:3.
3. the recombinant expression carrier containing Isatis indigotica Fort. IiAP2/ERF049 gene as claimed in claim 1 or recombinant bacterium.
The recombinant expression carrier of Isatis indigotica Fort. IiAP2/ERF049 gene the most according to claim 3 or recombinant bacterium, its feature exists
In, when the recombinant expression carrier described in preparation or recombinant bacterium, for expanding the primer centering of Isatis indigotica Fort. IiAP2/ERF049 gene,
Article one, primer sequence is as shown in SEQ ID NO:4, and another primer sequence is as shown in SEQ ID NO:5.
The recombinant expression carrier of Isatis indigotica Fort. IiAP2/ERF049 gene the most according to claim 3 or recombinant bacterium, its feature exists
In, described recombinant expression carrier is plant expression vector.
The recombinant expression carrier of Isatis indigotica Fort. IiAP2/ERF049 gene the most according to claim 3 or recombinant bacterium, its feature exists
In, described recombinant bacterium is escherichia coli or Agrobacterium.
7. an Isatis indigotica Fort. IiAP2/ERF049 gene as claimed in claim 1 is in improving Isatis indigotica Fort. in lariciresinol content
Application.
8. an IiAP2/ERF049 transcription factor as claimed in claim 2 is in improving Isatis indigotica Fort. in lariciresinol content
Application.
9. one kind is improved the method for lariciresinol content in Isatis indigotica Fort., it is characterised in that comprise the following steps:
A, the structure recombinant expression carrier containing Isatis indigotica Fort. IiAP2/ERF049 gene as claimed in claim 1；
B, the recombinant expression carrier conversion Agrobacterium built by step A, obtain recombinant bacterium；
The recombinant bacterium that C, step B obtain is transformed in Isatis indigotica Fort. tissue or cell, and screening obtains the ancient name for Chinese cabbage that larch turpentine cellulose content increases
Blue strain.
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