CN106148355B - Application of the woaded blue IiAP2/ERF049 gene in regulation Lignanoids compounds synthesis - Google Patents
Application of the woaded blue IiAP2/ERF049 gene in regulation Lignanoids compounds synthesis Download PDFInfo
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- CN106148355B CN106148355B CN201610502516.5A CN201610502516A CN106148355B CN 106148355 B CN106148355 B CN 106148355B CN 201610502516 A CN201610502516 A CN 201610502516A CN 106148355 B CN106148355 B CN 106148355B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—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
- 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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Abstract
The present invention relates to genetic engineering fields, the relevant AP2/ERF family IiAP2/ERF049 transcription factor of Lignanoids compounds content in specifically a kind of woaded blue, the nucleotide sequence for encoding the transcription factor, and its application in improving woaded blue in larch turpentine cellulose content.The Overexpression vector that the present invention passes through building IiAP2/ERF049, by crucial transcription factor IiAP2/ERF049 genetic transformation woaded blue, break the bottleneck of lariciresinol synthesis, the hairy stock system of woaded blue of high yield is obtained, high efficiency regulatory target spot is provided to improve the yield of lariciresinol, is improving larch turpentine cellulose content, alleviate lariciresinol supply shortage, the cost in traditional Chinese medicine extraction is reduced, pollution is reduced, cultivates excellent woaded blue strain etc. with huge application prospect.
Description
Technical field
The present invention relates to gene engineering technology fields, specifically, being that woaded blue IiAP2/ERF049 gene is regulating and controlling the wooden rouge
Application in chlorins compound synthesis.
Background technique
Woaded blue (Isatis indigotica Fort.), Cruciferae 1 year or biennial draft, first recorded in " legendary god of farming's sheet
Grass warp ", root is used as medicine as conventional Chinese medicine Radix Isatidis, commonly uses antiviral Chinese herbal medicine the successive dynasties for China.For flowing in modern clinic
Row sexuality emits, Japanese Type-B encephalitis, mumps, acute, chronic hepatitis, shingles zoster etc., in atypical pneumonia
(SARS) and in the prevention and treatment of H1N1virus important function is played.This seminar passes through antiviral study in vitro early period
Specify using lariciresinol as the Lignanoids compounds of representative be woaded blue play antiviral activity effect material base.Closely
Zhong Nanshan teaches seminar it has also been found that the derivative of lariciresinol has significant anti-A type and influenza B virus over year
Effect.However content of the lariciresinol in woaded blue root is few, only 47.14 μ g/g have seriously affected woaded blue as one
The application of high-quality Chinese medicine.Therefore, start with from the key gene of regulation secondary metabolism, improve the germplasm of woaded blue, improve larch
The content of rouge element becomes urgent problem to be solved.
Plant secondary metabolic engineering is that Secondary metabolites life is illustrated using genetic engineering and molecular biology method
The mechanism of object synthesis, checks on the information of metabolic pathway and relevant enzyme, regulates and controls to the gene of control Biosynthetic pathway,
Metabolic pathway is transformed on a molecular scale to reach, the controlled syntheses of goal of regulation and control product.It finds 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 carrot, while composition of alkaloids in catharanthus roseus can be regulated and controled, the synthesis of ter penoids in sweet wormwood, 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 many advantages, such as that hereditary capacity is relatively stable, the speed of growth is fast, metabolite content is high, is conducive to
The metabolic pathway of plant is studied, while can also be explored using certain genetic engineering means and create new synthetic route, is obtained
It is worth higher product, the planting of medicinal materials soil can be saved, reduces the occupancy to farmland, is the effective of production Chinese medicine medicine resource
Means.
Chinese patent literature CN104805097A discloses woaded blue rosin spirit coded sequence of reductase enzyme protein and application, coding
The nucleotide sequence of albumen with woaded blue lariciresinol reductase activity is high in plant to express woaded blue lariciresinol also
Protoenzyme is to improve the yield of lariciresinol.Chinese patent literature CN104805096A discloses woaded blue 4- coumaroyl A
Ligase protein family coded sequence and application, the expression by regulating and controlling woaded blue 4- coumaroyl A family protein are fallen with changing
The method of the biosynthesis of leaf rosin element has important application value in terms of breeding and bioenergy.But about woaded blue
IiAP2/ERF049 gene and its application in regulation Lignanoids compounds synthesis yet there are no report.
Summary of the invention
The purpose of the present invention is to provide a kind of relevant AP2/ERF families of Lignanoids compounds content in woaded blue
IiAP2/ERF049 transcription factor, the nucleotide sequence for encoding the transcription factor, and its lariciresinol contains in improving woaded blue
Application in amount.
In order to which the synthesis for excavating which AP2/ERF family transcription factor and lignanoid actually in woaded blue is closely related, this
Invention obtains 112 woaded blue AP2/ERF family transcription factors by the analysis to woaded blue transcript profile data, annotation.Detect MeJA
Different time points after induction (0,3,6,12, for 24 hours) AP2/ERF family transcription factor, lariciresinol route of synthesis gene and
The variation of Lignanoids compounds content is thresholding (lrl > 0.5) with Pearson correlation coefficients 0.5, constructs " transcription factor-base
Cause-compound " relevance network, screening obtain and Lignanoids compounds content and lignanoid's route of synthesis gene in woaded blue
Related AP2/ERF family transcription factor IiAP2/ERF049.Referring specifically to embodiment 1.
The first aspect of the present invention provides a kind of woaded blue IiAP2/ERF049 gene, nucleotide sequence such as SEQ ID
Shown in NO:1.The nucleotides sequence is classified as 684bp, wherein initiation codon ATG, terminator codon TGA.
It is described if the sequence of SEQ ID NO:1 is the open reading frame of woaded blue IiAP2/ERF049 full-length genome DNA
(ORF), the woaded blue IiAP2/ERF049 full-length genome DNA sequence dna is 1352bp, nucleotide sequence such as SEQ ID
Shown in NO:2, containing there are six exon, five intrones.
The second aspect of the present invention provides a kind of albumen encoded by above-mentioned woaded blue IiAP2/ERF049 gene, i.e.,
IiAP2/ERF049 transcription factor, amino acid sequence contain 227 amino acid as shown in SEQ ID NO:3.
The third aspect of the present invention, provide a kind of recombinant expression carrier containing above-mentioned woaded blue IiAP2/ERF049 gene,
Recombinant bacterium or genetically modified plants.
Preferably, in the preparation recombinant expression carrier or recombinant bacterium, for expanding woaded blue IiAP2/ERF049 base
The primer pair of cause are as follows:
Positive (F): 5'-ATGGTGAGCTTAAGAAGG-3'(SEQ ID NO:4)
Reversely (R): 5'-TCAGGTAGAAAGTGTACTGA-3'(SEQ ID NO:5).
Preferably, the recombinant expression carrier is plant expression vector.Preferred plasmid pIiAP2/ERF049-OVX.
Preferably, the recombinant bacterium, i.e. host cell are Escherichia coli, Agrobacterium etc..Preferably Agrobacterium.It is more excellent
Select agrobacterium rhizogenes C58C1.
The fourth aspect of the present invention provides above-mentioned woaded blue IiAP2/ERF049 gene lignanoids in improving woaded blue
Close the application in object content.Preferably, the woaded blue IiAP2/ERF049 gene improves lariciresinol, rosin in woaded blue
The content of alcohol, Secoisolariciresinol.
The fifth aspect of the present invention provides above-mentioned IiAP2/ERF049 transcription factor lignanoids in improving woaded blue
Close the application in object content.Preferably, above-mentioned IiAP2/ERF049 transcription factor improves lariciresinol, rosin in woaded blue
The content of alcohol, Secoisolariciresinol.
The sixth aspect of the present invention provides a kind of method for improving larch turpentine cellulose content in woaded blue, comprising the following steps:
A, building contains the recombinant expression carrier of above-mentioned woaded blue IiAP2/ERF049 gene；
B, Agrobacterium is converted with the recombinant expression carrier of step A building, obtains recombinant bacterium；
C, the recombinant bacterium that step B is obtained is transformed into woaded blue tissue or cell, and screening obtains larch turpentine cellulose content and increases
Woaded blue plant.
Preferably, the method for improving larch turpentine cellulose content in woaded blue, comprising the following steps:
(1) the cDNA overall length of woaded blue IiAP2/ERF049 is obtained using the method for gene cloning；
(2) IiAP2/ERF049 is connected with green fluorescent protein tag GFP, detects IiAP2/ERF049 in subcellular
Horizontal positioning；
(3) IiAP2/ERF049 is connected to expression regulation sequence, forms the plant expression vector for containing the gene, it will
Plant expression vector transforming agrobacterium rhizogenes C58C1, obtains the agrobacterium rhizogene strain of IiAP2/ERF049；
(4) agrobacterium rhizogene strain constructed by (3) is converted into woaded blue leaf dish, obtains aggregated enzyme 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 mass change, until 45 days arrival growth platform phases,
Draw growth rate curve；
(6) real-time fluorescence quantitative PCR (Quantitative Real-time PCR, Q-PCR) detects in woaded blue hairy
The expression of IiAP2/ERF049；
(7) lariciresinol and its precursor chemical combination in detection woaded blue transgenic hairy root is used in conjunction in liquid chromatogram-second order ms
Object rosin spirit, the content of open-loop products Secoisolariciresinol screen hairy that larch turpentine cellulose content significantly improves
Strain.
Gene clone method described in step (1) refers to reverse transcription PCR (Reverse Transcription
Polymerase Chain Reaction, RT-PCR), the upstream primer used are as follows: 5'-ATGGTGAGCTTAAGAAGG-3'
(SEQ ID NO:4), downstream primer are as follows: 5'-TCAGGTAGAAAGTGTACTGA-3'(SEQ ID NO:5).
Detection IiAP2/ERF049 method used in the positioning of subcellsular level described in step (2) is polyethylene glycol Jie
The protoplast transformation led, protoplast used are rice protoplast.
PCR detection method described in step (4) are as follows: the rolb gene on Agrobacterium Ti plasmid is separately designed, on carrier
Specific primer (JDIiAP2/ERF049-F and the JDIiAP2/ of hygromycin gene hpt and Insert Fragment upstream and downstream
ERF049-R PCR amplification) is carried out, observes purpose band after agarose gel electrophoresis in the UV lamp, screening obtains the positive and turns base
Because of the hairy stock system of woaded blue.
Q-PCR described in step (6) detects the expression of transgenosis woaded blue hairy middle IiAP2/ERF049 gene are as follows:
With woaded blue house-keeping gene Actin and IiAP2/ERF049 gene, quantitative PCR detection is carried out, screening IiAP2/ERF049 gene is high
The transgenic hairy root strain of expression.
Detection method of content described in step (7) are as follows: use the 1200 triple level four bars of liquid chromatogram-G6410A of Agilent
Mass spectrometer detects hairy middle 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 are as follows: acetonitrile volume is increased to by 14% within 0-4 minutes
50%, 5mM ammonium acetate solution volume are down to 50% by 86%；4-4.5 minutes acetonitrile volumes increase to 85%, 5mM by 50%
Ammonium acetate solution volume is down to 15% by 50%；Acetonitrile volume keeps 85%, 5mM ammonium acetate solution body within 4.5-8.5 minutes
Product keeps 15%.Wherein the parent ion peak of lariciresinol and daughter ion peak are respectively 359.2 and 329.0；Rosin spirit 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/ minutes, 5 μ L of sample volume, is used
Two-point method calculates three kinds of compounds contents in hairy, calculation formula A0/C0=Ax/Cx, wherein A0And C0Respectively standard items
The peak area and concentration of solution, AxAnd CxThe respectively peak area and concentration of sample solution.
Woaded blue IiAP2/ERF049 transcription factor is transferred to woaded blue by the present invention, and screening obtains what lariciresinol significantly improved
Transgenosis woaded blue hairy.When growing 45 days arrival growth platform phases, transgenic hairy root biological accumulation amount and wild type are hairy
There was no significant difference for root biological accumulation amount, however larch turpentine cellulose content dramatically increases in transgenic hairy root, wherein containing quantitative change
Changing larch turpentine cellulose content in most apparent strain is 424.60 μ g/g, is about improved compared with wild type control (51.13 μ g/g)
8.30 times.
The present invention passes through the Overexpression vector of building IiAP2/ERF049, by crucial transcription factor IiAP2/ERF049
Genetic transformation woaded blue breaks the bottleneck of lariciresinol synthesis, obtains the hairy stock system of woaded blue of high yield, to improve 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 pollution, cultivates excellent woaded blue strain etc. with huge application prospect.
Detailed description of the invention
Fig. 1 screening obtains woaded blue AP2/ERF transcription factor relevant to lariciresinol synthesis.
The structural domain schematic diagram of Fig. 2 IiAP2/ERF049 gene.
The special nucleus for being located in cell of Fig. 3 IiAP2/ERF049 albumen, wherein A-D is IiAP2/ERF049-GFP
For albumen specifically expressing in nucleus, E-H is blank GFP protein expression in cytoplasm, and A, E show that the green light of GFP, B, F are that leaf is green
The feux rouges of body, C, G are that green light and feux rouges merge, and D, H are that green light, feux rouges, white light merge.
The plant expression vector pIiAP2/ERF049-OVX that Fig. 4 IiAP2/ERF049 transcription factor is overexpressed in woaded blue
Building schematic diagram.
Fig. 5 transgenic hairy root Biomass Accumulation.
The positive hairy stock system PCR qualification result of Fig. 6.
The transcriptional level of IiAP2/ERF049 in Fig. 7 transgenic hairy root.
IiAP2/ERF049 gene is overexpressed in Fig. 8 woaded blue can be such that lariciresinol content increases, and wherein A is rosin spirit
Content, B are larch turpentine cellulose content, and C is Secoisolariciresinol content.
Specific embodiment
It elaborates below with reference to embodiment to specific embodiment provided by the invention.
The reagents and materials used in the present invention are commercially available or can prepare by literature method.Primer in the following example and
Sequencing is completed by Suzhou Jin Weizhi Biotechnology Co., Ltd, test method without specific conditions in embodiment, usually
According to normal conditions such as Sambrook et al. " molecular cloning: lab guide " (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to normal conditions, or according to the normal condition proposed by manufacturer.
Unless otherwise stated, otherwise percentage and number are calculated by weight.
Embodiment 1: the screening of woaded blue AP2/ERF transcription factor
Respectively from arabidopsis database (DATF, http://datf.cbi.pku.edu.cn/) and Chinese cabbage database
(http://brassicadb.org/brad/) downloading 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), crucifer AP2/ERF database is established according to its amino acid sequence.With
The database is compared that (e-value value is set as 10 by TBLASTN algorithm with woaded blue transcript profile database-5), it identifies and extracts
The gene homologous with crucifer AP2/ERF is as woaded blue AP2/ERF candidate gene in woaded blue transcript profile database.
The woaded blue AP2/ERF of above-mentioned acquisition is analyzed using bioinformatics software Vector NTI 11.5 and MEGA 5.05
The nucleotide sequence of candidate gene extracts open reading frame (open reading frame, ORF), and translates into amino acid sequence
Column.Using Pfam database (http://pfam.janelia.org/) and SMART analysis tool (http://smart.embl-
Heidelberg.de/) the AP2/ERF functional domain of obtained amino acid is identified, it is final to determine that woaded blue AP2/ERF is compiled
Code gene (IiAP2/ERFs) totally 112.
It is different after (0.5 μM) of the MeJA processing established from seminar's early period according to the annotation of woaded blue AP2/ERF encoding gene
In the gene expression profile database at time point (0,1,3,6,12 and for 24 hours) woaded blue hairy, extract under (0.5 μM) of MeJA processing not
With time point woaded blue hairy middle AP2/ERF encoding gene expressing information and Lignanoids compounds route of synthesis gene expression amount
Information (sets the expression of 0h as 1 as reference).After measuring (0.5 μM) of MeJA processing using the method in embodiment 7 simultaneously
Different time points (0,1,3,6,12 and for 24 hours) hairy middle rosin spirit of woaded blue, laricin, larch quality, the different larch of open loop
Four kinds of Lignanoids compounds changes of contents of quality.
With canonical correlation analysis in SPSS software (canonical correlation analysis) method, by MeJA
Different time points under inducing action (0,3,6,12, for 24 hours) variations of 112 AP2/ERF family transcription factor expression levels and LC-
(rosin spirit, laricin, larch quality, open loop are different for different time points lignanoid under the MeJA inducing action that MS/MS is measured
Larch quality) changes of contents integrated, construct " transcription factor-compound " relevance network；It will using identical method
Different time points under MeJA inducing action (0,3,6,12, for 24 hours) variations of 112 AP2/ERF family transcription factor expression levels
It is integrated, is constructed with different time points Lignanoids compounds route of synthesis gene expression amount variation under MeJA inducing action
" transcription factor-gene " relevance network.It is thresholding (lrl > 0.5) with Pearson correlation coefficients 0.5, screening is obtained and fallen respectively
Leaf spongiosa element synthesizes closely related AP2/ERF family transcription factor, the AP2/ERF family closely related with approach gene expression
Transcription factor compares the transcription factor that two groups are screened, coincidence person be with lignanoid's route of synthesis gene expression and
Lignanoid synthesizes significant relevant AP2/ERF family transcription factor, and the selection result is shown in Fig. 1.As shown in Figure 1, No. 049 AP2/
ERF family transcription factor IiAP2/ERF049 is synthesized with Lignanoids compounds and the expression of lignanoid's route of synthesis gene is close
(AP2/ERF family transcription factor can be further divided into five subfamilies of AP2, DREB, ERF, RAV, Soloist) is closed in cut phase.
Embodiment 2: the clone of woaded blue IiAP2/ERF049 gene
1. the extraction of woaded blue DNA and total serum IgE
Woaded blue aseptic seedling about 100mg is taken, rapid grind into powder in liquid nitrogen extracts woaded blue DNA using CTAB method；Separately take ancient name for Chinese cabbage
The 1.5ml centrifuge tube for being pre-loaded with plant tissue lysate is added in blue aseptic seedling about 100mg, rapid grind into powder in liquid nitrogen
In, sufficiently oscillation mixes, then according to TIANGEN TRNzol-A+It is total that the specification of total RNA extraction reagent box extracts woaded blue
RNA.Using 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 woaded blue IiAP2/ERF049 gene
Using extracted 1 μ g of total serum IgE as template, full formula gold TansScript First-Strand cDNA is used
Synthesis Supermix kit synthesizes woaded blue cDNA.
Gene-specific primer is designed according to the open reading frame of IiAP2/ERF049 gene:
Positive (F): 5'-ATGGTGAGCTTAAGAAGG-3'(SEQ ID NO:4)
Reversely (R): 5'-TCAGGTAGAAAGTGTACTGA-3'(SEQ ID NO:5).
Use Trans Start Fast@Pfu DNA Polymerase kit carries out PCR amplification, reaction system and item
Part is as follows: template (cDNA) 1 μ L, positive each 1 μ L, EasyPfu DNA Polymerase1 μ L, 10 × EasyPfu of anti-primer
5 μ L, 2.5mM dNTPs of Buffer, 2.5 μ L, 5 × PCR buffer 5 μ L, 13.5 μ L of deionized water constitute 25 μ L reactants
System.PCR reaction condition are as follows: 94 DEG C initial denaturation 1 minute, (94 DEG C are denaturalized 20 seconds, and 45 DEG C are annealed 20 seconds, 72 DEG C for 35 amplification cycles
Extend 1 minute), 72 DEG C are continued to extend 5 minutes.
The PCR product of acquisition connects after the recycling of target fragment glue with pGEMT-easy carrier through 1% agarose gel electrophoresis
It connects, converts bacillus coli DH 5 alpha, after being incubated overnight, choose monoclonal, send sequencing (limited by Suzhou Jin Weizhi biotechnology after bacterial examination
Company completes), it is that successful clone obtains that sequencing result is consistent with IiAP2/ERF049 sequence in woaded blue transcript profile database
Woaded blue IiAP2/ERF049 open reading frame (ORF) (SEQ ID NO:1).Wherein, initiation codon ATG is terminated close
Numeral is TGA.
In addition using woaded blue DNA as template, the genomic DNA of woaded blue AP2/ERF class transcription factor IiAP2/ERF049 is expanded
(SEQ ID NO:2), reaction system and reaction condition are same as above, and only extension of time is set as 3 minutes in reaction.
The genomic DNA of IiAP2/ERF049 is 1352bp, open reading frame 684bp, by the base of IiAP2/ERF049
Because group DNA is compared with open reading frame, it is found that the genomic DNA of IiAP2/ERF049 contains there are six exon, five include
Son.The ORF finder function of NCBI derives its albumen coded sequence (SEQ ID NO:3), using SMART (http: //
Smart.embl-heidelberg.de/) software, predicting IiAP2/ERF049 albumen includes an AP2 structural domain (121-
178 amino acid), it is the transcription factor of an AP2/ERF class, altogether includes 227 amino acid (as shown in Figure 2).
Embodiment 3: the Subcellular Localization of woaded blue IiAP2/ERF049 gene
According to the content of 2 bioinformatic analysis of example it is found that IiAP2/ERF049 gene is that have an AP2 structural domain
AP2/ERF class transcription factor.Further to verify property of the IiAP2/ERF049 gene as transcription factor, we are constructed
The subcellular localization carrier of IiAP2/ERF049 gene, passes through rice transformation protoplast, it was demonstrated that IiAP2/ERF049 is positioned at
Nucleus meets the characteristic of transcription factor.
1. the building of subcellular localization carrier
In the present embodiment, according to pCAMBIA1301-GFP carrier sequence and IiAP2/ERF049 gene ORF region sequence,
Design has the primer of restriction enzyme site, and 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, which is obtained, contains Nco I and Spe I restriction enzyme site
The code area IiAP2/ERF049, respectively use the code area digestion IiAP2/ERF049 Nco I and Spe I and pCAMBIA1301-
GFP carrier, recycles IiAP2/ERF049 gene and pCAMBIA1301-GFP large fragment, connection conversion, and picking single colonie send survey
Correct IiAP2/ERF049 gene subcellular localization carrier is sequenced in sequence, final obtain.
2. protoplast transformation and observation
According to the operating instruction of the big extraction reagent kit of QENGEN plasmid, extracts and obtain purity and the higher IiAP2/ of concentration
ERF049 gene subcellular localization vector plasmid, then rice transformation protoplast.The plasm that specific method is mediated referring to PEG
Body method for transformation.The protoplast of pCAMBIA1301-GFP empty carrier is converted using under the same terms as negative control.
Convert complete rice protoplast, 25 DEG C of room temperature culture 18 hours after use confocal laser scanning microscope, hair
Existing IiAP2/ERF049 is specifically located in nucleus, consistent with the function of transcription factor (as shown in Figure 3).
Embodiment 4: the plant of woaded blue IiAP2/ERF049 gene is overexpressed the building of binary vector
In the present embodiment, using the bacterium solution containing IiAP2/ERF049 as template, the forward primer with restriction enzyme site is designed:
5'-AAGGATCCATGGTGAGCTTAAGAAGG-3'(SEQ ID NO:8, HI containing Bam restriction enzyme site) and reverse primer: 5'-
CCACTAGTGGTAGAAAGTGTACTGA-3'(SEQ ID NO:9, I containing Spe restriction enzyme site).Use NEB Phusion@It is super
Fidelity PCR MasterMix carries out PCR amplification, obtains the target gene for having restriction enzyme site, is inserted into PHB- after digestion
Flag expression vector, picking single colonie send sequencing after conversion, and correct IiAP2/ERF049 overexpression double base is sequenced in final obtain
Carrier (as shown in Figure 4).
Embodiment 5: the IiAP2/ERF049 over-express vector genetic transformation woaded blue blade and turn base that agrobacterium rhizogenes mediates
Because of hairy acquisition
1.IiAP2/ERF049 over-express vector transforming agrobacterium rhizogenes
In this embodiment, the IiAP2/ERF049 plant binary over-express vector that will be obtained in embodiment 4, using freeze thawing
Method is transferred in agrobacterium rhizogenes C58C1, and the positive bacterial strain of PCR identification is as successfully transferred to the agrobacterium rhizogenes of over-express vector
(C58C1-pIiAP2/ERF049-OVX)。
2. agrobacterium rhizogenes mediates the conversion of IiAP2/ERF049 gene leaf disk method
2.1 leaf disk method step of converting are as follows:
A. it with the positive bacterium colony on sterile toothpick picking YEB selection plate, is inoculated in 2ml YEB fluid nutrient medium, 28
DEG C, 200 revs/min of shaken cultivations are stayed overnight；
B. by C58C1 empty bacterium and the bacterium liquid activation of C58C1-PHB-flag, C58C1-pIiAP2/ERF049-OVX two
It is secondary, take 20 μ L to be added in 2mL YEB fluid nutrient medium every time, 28 DEG C, 200 revs/min of overnight incubations.Wherein C58C1 empty bacterium makes
With the culture medium of YEB (40mg/L rifampin), other bacterial strains use the training of YEB (40mg/L rifampin+75mg/L kanamycins)
Support base；
C. it takes 30mL YEB culture medium, is added the activated Agrobacterium of 1mL, 28 DEG C, 200 revs/min of overnight incubations；
D.5, it is centrifuged 10 minutes for 000 rev/min, abandons supernatant, the MS fluid nutrient medium of 100 μM of acetosyringones is contained with 20mL
Thallus is resuspended；
E. it takes woaded blue aseptic seedling, blade is cut into about 1cm in super-clean bench2Size immerses in the thallus being resuspended, 28 DEG C,
200 revs/min shaken cultivation 10 minutes；
F. leaf dish is taken out, the bacterium solution above leaf dish is blotted using aseptic filter paper, faces up and is put on MS culture medium,
(25 soil 1) DEG C dark culture 2 days in constant incubator.
G. leaf dish dark culture is taken out two days later, uses aseptic water washing five times in super-clean bench, aseptic filter paper blots, and is placed in 1/
On 2MS (500mg/L cephalosporin) culture medium, in (25 soil 1) DEG C dark culture；
H. about after two weeks, leaf dish edge grows hairy, carefully cuts when root long pending is to 3-5cm, every 15d subculture one
It is secondary, and constantly reduce cephalosporin concentration (300mg/L → 100mg/L → 0mg/L), in addition to C58C1 empty bacterium induce it is hairy
In addition root, other groups add 10mg/L hygromycin on the basis of 1/2MS+ cephalosporin, screening positive clone continues (25 soil 1)
DEG C dark culture；
I. when cephalosporin concentration is reduced to 0mg/L, continue culture about two weeks, select that growth conditions are good, and branch is more
Strain be numbered, it is wild type hairy that C58C1 empty bacterium, which induces, number are as follows: WT1, WT2, WT3 ...；C58C1-
It is blank hairy that PHB-flag, which is induced, number are as follows: CK-1, CK-2, CK-3 ...；C58C1-pIiAP2/ERF049-
It is transgenic hairy root that OVX, which is induced, is numbered respectively are as follows: OVX049-1, OVX049-2, OVX049-3 ...
J. the root of hair strain positive through PCR confirmation, takes 100mg to be transferred in 200mL1/2MS fluid nutrient medium, is placed in shaking flask
In cabinet, 120 revs/min, 25 DEG C of dark cultures changed a culture solution every 9 days and record the variation of hairy root growth amount, until 45 days receive
It obtains.As shown in figure 5, mapping using weight in wet base variation as ordinate by abscissa of growth time.Hairy grows rapidly after 18d,
Enter the growth platform phase when 36d, though biomass slightly increases when 45d, variation is unobvious, with hairy of wild type and blank hair
Shape root is compared, and the transgenic hairy root biomass accumulation of overexpression IiAP2/ERF049 changes without conspicuousness.
The YEB culture medium are as follows: take yeast extract 1g/L, peptone 5g/L, beef extract 5g/L, sucrose respectively
7g agar is added if preparing solid medium in 5g/L, magnesium sulfate seven water 0.24g/L, pH=7.2.121 DEG C of high temperature and pressure
Sterilizing 20 minutes, it is spare.
The 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, it is spare.Wherein MS culture medium is purchased from Phyto Technology Laboratories (U.S.).
The fluid nutrient medium are as follows: 1/2MS culture medium+30g/L sucrose, pH5.8-6.0.
2.2 positive hairy identification methods are as follows: containing Ri plasmid, rolb and rolc thereon in agrobacterium rhizogenes cell
Gene can induce and maintain root of hair form, choose rolb gene to detect gained be hairy, rather than adventitious root.PHB-flag
Carrier has hygromycin gene (hpt), using hpt gene and vector-specific primers (JDPDK) to IiAP2/
ERF049 transgenosis root of hair carries out Molecular Identification, and C58C1-PHB-flag, C58C1-pIiAP2/ERF049-OVX bacterium solution are as sun
Property control.
Total DNA is extracted according to CTAB method used in embodiment 2, carries out PCR identification, the primer by template of total DNA
Sequence is as follows:
Rolb gene magnification primer are as follows:
rolb-F:5'-CGAGGGGATCCGATTTGCTT-3'(SEQ ID NO:10)
rolb-R:5'-GACGCCCTCCTCGCCTTCCT-3'(SEQ ID NO:11)
Hpt gene magnification primer are as follows:
hpt-F:5'-CGATTTGTGTACGCCCGACAGTC-3'(SEQ ID NO:12)
hpt-R:5'-CGATGTAGGAGGGCGTGGATATG-3'(SEQ ID NO:13)
JDIiAP2/ERF049 gene magnification primer are as follows:
JDIiAP2/ERF049-F:5'-CTCAAGCAAGGATCCATGGTGA-3'(SEQ ID NO:14)
JDIiAP2/ERF049-R:5'-CGATACCGTCACTAGTGGTAGAA-3'(SEQ ID NO:15)
PCR amplification is carried out using Takara rTaq enzyme, reaction system and condition are as follows: 1 μ L of template (DNA), positive anti-primer
Each 1 μ L, 2 × rTaq PCR SuperMix, 10 μ L, 7 μ L of deionized water, totally 20 μ L reaction system.PCR reaction condition are as follows: 94 DEG C
Initial denaturation 5 minutes, 35 amplification cycles (94 DEG C are denaturalized 30 seconds, and 55 DEG C are annealed 30 seconds, and 72 DEG C extend 1 minute), 72 DEG C after reneing
It stretches 5 minutes.
After reaction, Ago-Gel detects PCR as a result, wherein rolb and hpt gene can amplify respectively to PCR
The specific fragment of 423bp and 812bp size uses primer JDIiAP2/ERF049-F and JDIiAP2/ on PHB-flag carrier
It is equal with IiAP2/ERF049 length scale that ERF049-R amplifies the sequence come, therefore successfully imports pIiAP2/ERF049-OVX
Hairy stock system can detecte the segment of about 897bp.
Observed under ultraviolet lamp with rolB, hpt gene and JDIiAP2/ERF049 band of the same size, as a result such as Fig. 6
Shown, that show screening acquisition is hairy of the IiAP2/ERF049 containing target gene induced by C58C1 agrobacterium rhizogenes.
Embodiment 6: IiAP2/ERF049 transcriptional level detects in transgenic hairy root
In the present embodiment, the method for extracting total RNA according to used in embodiment 2 extracts difference IiAP2/ in embodiment 5
The RNA that ERF049 gene overexpression is transgenosis woaded blue hairy, and further Overturn ratio is cDNA.It is designed by primer 3
The real-time quantitative PCR primer (table 1) of IiAP2/ERF049 gene and Actin reference gene across introne, uses TAKARA
SYBR-Green PCR Mastermix kit carries out real-time fluorescence quantitative PCR analysis.Instrument is Thermal Cycler
Dice, two-step method carry out PCR amplification, 95 DEG C initial denaturation 10 seconds, 40 amplification cycles (95 DEG C 5 seconds；60 DEG C 30 seconds), separation reaction
(95 DEG C 15 seconds；60 DEG C 30 seconds；95 DEG C 15 seconds).Use 2-ΔΔCtMethod carries out later data processing, as a result as shown in fig. 7, calculating public
Formula is as follows:
Destination gene expression amount=2-ΔΔCt
Δ Δ Ct=(CT target gene-CT reference gene)Experimental group-(CT target gene-CT reference gene)Control
1 real-time quantitative PCR primer sequence of table
Embodiment 7: lariciresinol, rosin spirit, the different larch of open loop in LC-MS/MS measurement transgenic hairy root are utilized
Rouge cellulose content
1. the preparation of standard solution
Precision weighs lariciresinol, rosin spirit, Secoisolariciresinol standard items 2mg respectively, and it is molten that 2mL methanol is added
Solution, is configured to the standard solution of 1mg/mL, spare；5 samples are taken a sample test at random, are averaged according to three kinds of ingredients in 5 samples
Concentration determines each component concentration in hybrid standard product.It is final to determine that each component concentration is respectively as follows: larch in hybrid standard product
Rouge element 300ng/mL, rosin spirit 100ng/mL, Secoisolariciresinol 100ng/mL.
2. the preparation of sample solution
Hairy sample of harvest is dried overnight in 45 DEG C of drying boxes, is fully ground in mortar.Weigh 100mg powder-like
Product are put into 10mL centrifuge tube, and 5mL methanol is added, during which oscillation was shaken ultrasonic (40W) extraction 30 minutes after mixing every 5 minutes
It is primary to swing mixing；4,000 revs/min are centrifuged 5 minutes, pour out supernatant in a clean pipe, repeat above operation primary.Merge two
Secondary extracting solution uses volumetric flask constant volume to 10mL.It takes 1mL extracting solution vacuum to volatilize, 500 μ L initial flows is added in each sample cell
Dynamic phase, be vortexed 1 minute sample dissolution, and 14,000 revs/min are centrifuged 10 minutes, and 0.22 μm of organic membrane filtration of supernatant takes
Subsequent filtrate sample introduction.
3. Mass Spectrometer Method condition
Mass spectrum use electric spray ion source, ion mode, atomizer gas pressure 40psi, 350 DEG C of gas temperature, gas
10 liters/min of body flow velocity, capillary voltage 4000V, mass spectrometry parameters are 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, and 35 DEG C of column temperature, 0.3 ml/min of flow velocity, 5 μ L of sample volume, single needle operation 8.5
Minute.
3 gradient elution program of table
5. the measurement and calculating of sample
Hairy middle rosin spirit, lariciresinol, Secoisolariciresinol content, calculation formula are calculated using two-point method
For A0/C0=Ax/Cx.Wherein A0And C0The respectively peak area and concentration of standard solution, AxAnd CxThe respectively peak of sample solution
Area and concentration.
In the present invention, hairy middle rosin spirit of IiAP2/ERF049 gene overexpression, lariciresinol, the different fallen leaves of open loop
The content of rosin element improves (as shown in Figure 8).Compared with wild type, the transgenosis for being overexpressed IiAP2/ERF049 gene is hairy
Larch turpentine cellulose content is 4.39-8.30 times of wild type in root, and rosin alcohol content is 1.50-3.42 times of wild type, open loop
Isolariciresinol content is 5.75-10.57 times of wild type.It as a result is three duplicate average values of biology, error line table
Show standard deviation, statistical analysis is examined using t-test.
It is fallen in short, the present invention by the overexpression IiAP2/ERF049 transcription factor in woaded blue, obtains a large amount of generate
The hairy stock system of leaf rosin element is alleviating lariciresinol supply shortage, is reducing the cost in traditional Chinese medicine extraction, reducing pollution,
Excellent woaded blue strain etc. is cultivated with huge application prospect.
The preferred embodiment of the present invention has been described in detail above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent on the premise of not violating the inventive spirit of the present invention
Variation or replacement, these equivalent variation or replacement are all included in the scope defined by the claims of the present application.
Claims (9)
1. a kind of woaded blue IiAP2/ERF049 gene, nucleotide sequence is as shown in SEQ ID NO:1.
2. a kind of IiAP2/ERF049 transcription factor of woaded blue IiAP2/ERF049 gene coding as described in claim 1,
Amino acid sequence is as shown in SEQ ID NO:3.
3. a kind of recombinant expression carrier or recombinant bacterium containing woaded blue IiAP2/ERF049 gene as described in claim 1.
4. the recombinant expression carrier or recombinant bacterium of woaded blue IiAP2/ERF049 gene according to claim 3, feature exist
In, in the preparation recombinant expression carrier or recombinant bacterium, in the primer pair for expanding woaded blue IiAP2/ERF049 gene,
One primer sequence is as shown in SEQ ID NO:4, and another primer sequence is as shown in SEQ ID NO:5.
5. the recombinant expression carrier or recombinant bacterium of woaded blue IiAP2/ERF049 gene according to claim 3, feature exist
In the recombinant expression carrier is plant expression vector.
6. the recombinant expression carrier or recombinant bacterium of woaded blue IiAP2/ERF049 gene according to claim 3, feature exist
In the recombinant bacterium is Escherichia coli or Agrobacterium.
7. a kind of woaded blue IiAP2/ERF049 gene as described in claim 1 is in improving woaded blue in larch turpentine cellulose content
Using.
8. a kind of IiAP2/ERF049 transcription factor as claimed in claim 2 is in improving woaded blue in larch turpentine cellulose content
Using.
9. a kind of method for improving larch turpentine cellulose content in woaded blue, which comprises the following steps:
A, building contains the recombinant expression carrier of woaded blue IiAP2/ERF049 gene as described in claim 1；
B, Agrobacterium is converted with the recombinant expression carrier of step A building, obtains recombinant bacterium；
C, the recombinant bacterium that step B is obtained is transformed into woaded blue tissue or cell, and screening obtains the ancient name for Chinese cabbage that larch turpentine cellulose content increases
Blue strain.
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| CN110129334B (en) * | 2019-05-21 | 2021-08-24 | 上海中医药大学 | Application of IiWRKY34 in regulation and control of Isatis Indigotica lignan biosynthesis and stress-resistant reaction |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102226193A (en) * | 2011-06-17 | 2011-10-26 | 西南大学 | Brassica napus, parent species Chinese cabbage, cabbage MYBL2 (v-myb avian myeloblastosis viral oncogene homolog-like 2) gene family and application thereof |
| CN104278053A (en) * | 2013-07-05 | 2015-01-14 | 中国科学院上海生命科学研究院 | Plant drought resistance ability improving method |
| CN104805097A (en) * | 2014-01-23 | 2015-07-29 | 中国人民解放军第二军医大学 | Coding sequences and applications of isatis indigotica fortune pinoresinol reductase protein |
| CN104805096A (en) * | 2014-01-23 | 2015-07-29 | 中国人民解放军第二军医大学 | Coding sequences and applications of 4-coumarate coenzyme A ligase protein family of isatis indigotica fortune |
| CN105175522A (en) * | 2015-10-26 | 2015-12-23 | 深圳市铁汉生态环境股份有限公司 | Lotus corniculatus AP2/ERF transcription factor and encoding gene and application thereof |
-
2016
- 2016-06-30 CN CN201610502516.5A patent/CN106148355B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102226193A (en) * | 2011-06-17 | 2011-10-26 | 西南大学 | Brassica napus, parent species Chinese cabbage, cabbage MYBL2 (v-myb avian myeloblastosis viral oncogene homolog-like 2) gene family and application thereof |
| CN104278053A (en) * | 2013-07-05 | 2015-01-14 | 中国科学院上海生命科学研究院 | Plant drought resistance ability improving method |
| CN104805097A (en) * | 2014-01-23 | 2015-07-29 | 中国人民解放军第二军医大学 | Coding sequences and applications of isatis indigotica fortune pinoresinol reductase protein |
| CN104805096A (en) * | 2014-01-23 | 2015-07-29 | 中国人民解放军第二军医大学 | Coding sequences and applications of 4-coumarate coenzyme A ligase protein family of isatis indigotica fortune |
| CN105175522A (en) * | 2015-10-26 | 2015-12-23 | 深圳市铁汉生态环境股份有限公司 | Lotus corniculatus AP2/ERF transcription factor and encoding gene and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| PREDICTED: Brassica oleracea var. oleracea ethylene-responsive transcription factor-like protein At4g13040 (LOC106309599), transcript variant X3, mRNA;CBI Reference Sequence: XM_013746656.1;《GenBank》;20151025;全文 |
| PREDICTED: ethylene-responsive transcription factor-like protein At4g13040 [Brassica rapa];NCBI Reference Sequence: XP_009107675.1;《GenBank》;20140916;全文 |
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