CN105949289A - Flavanol regulatory protein MsMYB12L from functional apples, coding gene and application thereof - Google Patents
Flavanol regulatory protein MsMYB12L from functional apples, coding gene and application thereof Download PDFInfo
- Publication number
- CN105949289A CN105949289A CN201610300919.1A CN201610300919A CN105949289A CN 105949289 A CN105949289 A CN 105949289A CN 201610300919 A CN201610300919 A CN 201610300919A CN 105949289 A CN105949289 A CN 105949289A
- Authority
- CN
- China
- Prior art keywords
- plant
- sequence
- protein
- gene
- msmyb12l
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Nutrition Science (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a flavanol regulatory protein MsMYB12L from functional apples, a coding gene and application thereof. The protein provided by the invention is acquired from apples and named as MsMYB12L protein, and is (a1) or (a2) as the following: (a1) protein composed of the amino acid sequence shown as sequence 1 in the sequence table; (a2) a sequence 1 derived protein that is obtained by subjecting the amino acid sequence of the sequence 1 to substitution and/or deletion and/or adding by one or several amino acid residues and is related to the content of plant flavanol compound content. The gene coding the MsMYB12L protein (MsMYB12L gene) also belongs to the protection range of the invention. The invention discovers the MsMYB12L protein and its function, the MsMYB12L protein can be used for cultivating transgenic plants with changed flavanol compound content, and has significant application prospects in plant breeding.
Description
Technical field
The present invention relates to a kind of flavonol modulin MsMYB12L available from functional type Fructus Mali pumilae and encoding gene thereof and application.
Background technology
" doctor's food homology " is developing direction.Fructus Mali pumilae storage property is good, and supply cycle is long, is worldwide fruit, especially fruit
Containing higher proportion, human body be easier absorb free polyphenol, there is good antioxidation, antitumor, prevention
Cardiovascular and cerebrovascular disease and protecting the liver etc. acts on, and healthy nutritive value is high, has " one day Fructus Mali pumilae, doctor is away from me " (An apple
a day keeps the doctor away！) good reputation, the most considerable country is all classified as major consumers
Fruit and recommend energetically.But finding in recent years shows, on the one hand, in 1000 be bred as the most both at home and abroad
Multiple apple varieties 80% are the hybridization of kinds such as ' Jin Shuai ', grow directly from seeds or bud selection offspring, and this " inbreeding " is past
Toward bringing the problems such as the hereditary basis of kind is narrow and resistance goes down；On the other hand, characteristic, multi-resistance and multiformity fruit
Product become the important directions of industry development.
Malus sieversii and red meat modification (Malus sieversii f.neidzwetzkyana) thereof are that Herba Marsileae Quadrifoliae is cultivated in the world
Ancestors' kind of fruit, not only genetic diversity is the abundantest, and rich in the functions such as flavonoid, health-care components, is by
The degeneration-resistant precious gene bank with quality breeding.But because of reasons such as the farmland reclamation of wastelands, the genetic diversity of Malus sieversii just meets with
To heavy damage, endangered；China is apple production maximum in the world and country of consumption, wherein within 2012, produces Herba Marsileae Quadrifoliae
Really 39,500,000 tons, it is mainly used in eating raw, and nearly 70% is Fuji's kind that Flavonoid Content is relatively low.
Therefore, around " scientific conservation of Malus sieversii resource is opened up with Sustainable and highly-efficient use, cultivar hereditary basis
Exhibition, Apple Industry transition and upgrade promote to side structure reform and Practices Sustainable Increasing Income of Farmers and level of human health together ", enter
Row research cooperation and integration and demonstration, the present inventor cooperates with the professor of Cornell Univ USA, to open country, Xinjiang Herba Marsileae Quadrifoliae
Fruit and worldwide 97 parts of apple resources such as European Forest Fructus Mali pumilae have carried out genome and have resurveyed sequence and bioinformatics
Analyze, construct Xinjiang red meat Fructus Mali pumilae and the apple variety first generation of hybrid and backcross one, secondary segregating population, research is clearly
Technical parameter, flavonoid that Malus sieversii population genetic variations builds with genetic diversity feature, Core Germplasms contain
The hereditary variation feature of the character such as amount and development mechanism, it is proposed that the concept of " functional type Fructus Mali pumilae " and " wide row high level cadre,
Inter-row green covering, give grass fertilising, fertilize the soil support root " Modern orchard management philosophy, create conventional hybridization and biotechnology
The Fructus Mali pumilae high-efficient breeding technique system organically combined, has formulated a collection of new varieties and excellent germplasm, have developed Fructus Mali pumilae new product
Plant highly effective matched cultivation technical system.At present, authorized and declared patent of invention 10 remainder, field planting hybrid seedling 4
More than ten thousand strains, are bred as new varieties (being) 16；Deliver correlational study paper 120, wherein SCI paper more than 20 piece,
These achievements in research are totally in the top standard of international similar research.
Summary of the invention
It is an object of the invention to provide a kind of flavonol modulin MsMYB12L available from functional type Fructus Mali pumilae and coding thereof
Gene and application.
The protein that the present invention provides, available from Fructus Mali pumilae, named MsMYB12L albumen, is following (a1) or (a2):
(a1) protein being made up of the aminoacid sequence shown in sequence in sequence table 1；
(a2) by the aminoacid sequence of sequence 1 through the replacement of one or several amino acid residue and/or disappearance and/or
Add and the protein that by sequence 1 derives relevant to plant flavanols compounds content.
In order to make the MsMYB12L albumen in (a) be easy to purification and detection, can be in by sequence table shown in sequence 1
The amino terminal of protein of aminoacid sequence composition or carboxyl terminal connect upper label the most as shown in table 1.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
MsMYB12L albumen in above-mentioned (b) can synthetic, it is possible to first synthesizes its encoding gene, then carries out biology
Expression obtains.The encoding gene of the MsMYB12L albumen in above-mentioned (b) can be by by shown in sequence in sequence table 2
DNA sequence lacks the codon of one or several amino acid residue, and/or carries out the missense of one or several base pair
Sudden change, and/or hold the coded sequence connecting the label shown in table 1 to obtain at its 5 ' end and/or 3 '.
The gene (MsMYB12L gene) encoding described MsMYB12L albumen falls within protection scope of the present invention.
Described gene is following (1) or (2) or (3):
(1) DNA molecular shown in sequence 2 in coding region sequence table；
(2) the DNA sequence hybridization limited with (1) under strict conditions and coding contain with plant flavanols compounds
The protein DNA molecule that amount is relevant；
(3) DNA sequence limited with (1) has more than 90% homology and coding and plant flavanols compounds
The protein DNA molecule that content is relevant.
Above-mentioned stringent condition can be with 0.1 × SSPE (or 0.1 × SSC), and the solution of 0.1%SDS, at DNA or RNA
Hybrid experiment hybridizes at 65 DEG C and washes film.
Recombinant expression carrier containing described MsMYB12L gene, expression cassette, transgenic cell line, transgenic plant group
Knit or recombinant bacterium belongs to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression carrier of MsMYB12L gene.Described plant is expressed
Carrier includes double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Use MsMYB12L gene constructed heavy
During group expression vector, can be plus any enhancement mode, composing type, organizing specific type before its transcription initiation nucleotide
Or inducible promoter, they can be used alone or be used in combination with other plant promoter；Additionally, use
During the gene constructed recombinant expression carrier of MsMYB12L, it be also possible to use enhancer, including translational enhancer or transcriptional enhancer,
These enhancer regions can be ATG initiation codon or neighboring region start codon etc., but required and coded sequence
Reading frame identical, to ensure the correct translation of whole sequence.Described translation control signal and the source of start codon
It is widely, can be natural, it is also possible to be synthesis.Translation initiation region can come from transcription initiation region or
Structural gene.For the ease of transgenic plant cells or plant being identified and screening, plant used can be expressed and carry
Body is processed, as be added in plant express can produce color change enzyme or the gene of luminophor, have anti-
The antibiotic marker thing of property or anti-chemical reagent marker gene etc..From the security consideration of transgenic plant, can be not added with
Any selected marker, directly with phenotypic screen transformed plant.The carrier that sets out of described recombinant expression carrier can be
PRI101 carrier.Described recombinant expression carrier is concretely at Nde I and the BamH I restriction enzyme site of pRI101 carrier
Between the recombiant plasmid pRI101-MsMYB12L that obtains of the double chain DNA molecule shown in sequence 2 of insertion sequence table.
Described plant tissue concretely plant callus, can be more specifically plant embryo callus.Described plant
Can be monocotyledon or dicotyledon.Described dicotyledon concretely rosaceous plant.Described Rosaceae is planted
Thing concretely Malus.Described Malus concretely Fructus Mali pumilae, can be more specifically ' Wang Lin ' Fructus Mali pumilae.
The present invention also protects the application of described MsMYB12L albumen, for as follows (b1) or (b2):
(b1) the flavanols compounds content of plant is regulated and controled；
(b2) the flavanols compounds content of plant is increased.
Described plant can be monocotyledon or dicotyledon.Described dicotyledon concretely rosaceous plant.
Described rosaceous plant concretely Malus.Described Malus concretely Fructus Mali pumilae, can be more specifically ' king
Woods ' Fructus Mali pumilae.
The present invention also protects described MsMYB12L gene cultivating the transgenic plant that flavanols compounds content increases
In application.Described plant can be monocotyledon or dicotyledon.Described dicotyledon concretely Rosaceae
Plant.Described rosaceous plant concretely Malus.Described Malus concretely Fructus Mali pumilae, more specifically
It can be ' Wang Lin ' Fructus Mali pumilae.
The present invention also protects a kind of method cultivating transgenic plant, comprises the steps: described MsMYB12L gene
Importing is set out plant, obtain flavanols compounds content be higher than described in set out the transgenic plant of plant.Described set out
Plant can be monocotyledon or dicotyledon.Described dicotyledon concretely rosaceous plant.Described Flos Rosae Multiflorae
Section plant concretely Malus.Described Malus concretely Fructus Mali pumilae, can be more specifically ' Wang Lin ' Herba Marsileae Quadrifoliae
Really.Described MsMYB12L gene specifically can be by the plant that sets out described in recombinant expression carrier importing described in any of the above.Institute
State in method, the callus of the plant that specifically described MsMYB12L channel genes can be set out, then callus is trained
Educate for plant.Described callus concretely embryo callus.Carry the restructuring table of described MsMYB12L gene
Reach carrier can pass through Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance,
Agriculture bacillus mediated conventional biology methods such as grade is transformed in plant cell or tissue.
The present invention also protects a kind of method obtaining Transgenic plant tissue, comprises the steps: described MsMYB12L
Channel genes sets out plant tissue, obtain flavanols compounds content be higher than described in the set out transgenic of plant tissue plant
Fabric texture.The described plant that sets out can be monocotyledon or dicotyledon.Described dicotyledon concretely Flos Rosae Multiflorae
Section plant.Described rosaceous plant concretely Malus.Described Malus concretely Fructus Mali pumilae, more
Body can be ' Wang Lin ' Fructus Mali pumilae.Described MsMYB12L gene specifically can be imported by recombinant expression carrier described in any of the above
The described plant tissue that sets out.Described plant tissue concretely plant callus.Described callus concretely embryo
Property callus.
The present invention also protects method described in described MsMYB12L albumen or described MsMYB12L gene or any of the above
Application in plant breeding.The purpose of described plant breeding is to cultivate the plant that flavanols compounds content increases.
Flavanols compounds described in any of the above is for having the compound of female ring shown in formula (I)；
Flavanols compounds described in any of the above is compound shown in formula (II)；
R1=H, OH or OG；
R2=OH or H；
R3=H or OH.
R1=H, R2=OH, R3It it is catechin during=H
R1=OH, R2=H, R3It it is epicatechin during=H
R1=H, R2=OH, R3It it is nutgall catechin during=OH
R1=OH, R2=H, R3It it is epigallo catechin during=OH
R1=OG, R2=H, R3It it is L-Epicatechin gallate during=H
R1=OG, R2=H, R3It it is epigallocatechin gallate (EGCG) during=OH
Present invention finds MsMYB12L albumen and function thereof, can be used for cultivating turning of flavanols compounds content change
Gene plant, has major application prospect in plant breeding.
Accompanying drawing explanation
Fig. 1 is photo before DMACA dyeing.
Fig. 2 is photo after DMACA dyeing.
Detailed description of the invention
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiment
Method, if no special instructions, is conventional method.Test material used in following embodiment, if no special instructions,
It is and is commercially available from routine biochemistry reagent shop.Quantitative test in following example, is respectively provided with three times and repeats in fact
Test, results averaged.
PRI101 carrier (also known as " pRI101-AN DNA "): Takala company, Code No.3262.Agrobacterium LBA4404:
Tiangen company, catalog number: CC2901.' Wang Lin ' Fructus Mali pumilae: list of references: " The bHLH transcription
factor MdbHLH3 promotes anthocyanin accumulation and fruit colouration in
response to low temperature in apples》。
Embodiment 1, MsMYB12L albumen and the discovery of encoding gene thereof
It is found that a new albumen from " purplish red No. 1 " Fructus Mali pumilae, as shown in the sequence 1 of sequence table, it is named
MsMYB12L albumen.Being MsMYB12L gene by the unnamed gene of coding MsMYB12L albumen, its open reading frame is such as
Shown in the sequence 2 of sequence table.
In GENBANK, the albumen the highest with sequence 1 homology is as shown in the sequence 3 of sequence table.By the sequence of sequence table
The named reference protein of protein shown in row 3, the named crt gene of its encoding gene, such as the sequence 4 of sequence table
Shown in.
Embodiment 2, the Function Identification of MsMYB12L albumen
One, construction recombination plasmid
By shown in the sequence 2 of the sequence table of synthetic double chain DNA molecule insert pRI101 carrier Nde I and
Between BamH I restriction enzyme site, obtain recombiant plasmid pRI101-MsMYB12L.
By shown in the sequence 4 of the sequence table of synthetic double chain DNA molecule insert pRI101 carrier Nde I and
Between BamH I restriction enzyme site, obtain control plasmid.
Two, the acquisition of genetically modified organism
1, recombiant plasmid pRI101-MsMYB12L is imported Agrobacterium LBA4404, obtain recombinational agrobacterium.
2, take the recombinational agrobacterium thalline that step 1 obtains, suspend with 30ml MS fluid medium, obtain OD600nm=0.8
Bacteria suspension, add 30 μ L acetosyringones, obtain infecting liquid.
3, take the embryo callus of ' Wang Lin ' Fructus Mali pumilae, be seeded on MS solid medium, cultivate 15 days for 25 DEG C.
4, after completing step 3, taking callus, be immersed in that step 2 obtains infects in liquid, and 160rpm room temperature is shaken
Swing 15-20min, then blot surface with sterilizing filter paper.
5, after completing step 4, take callus, be placed in and co-culture culture medium (containing 1mg/L 2,4-D and 0.5mg/L
The MS solid medium of 6-BA) on, 25 DEG C of dark culturing 36 hours.
6, after completing step 5, take callus, be placed in screening culture medium (containing 50mg/L kanamycin, 250mg/L carboxylic
Bian penicillin, 1mg/L 2, the MS solid medium of 4-D and 0.5mg/L 6-BA) on, 25 DEG C of dark culturing 20-30 days.
7, after completing step 6, take the resistant calli that can grow in screening culture medium, carry out PCR qualification.
Primer used by PCR qualification is to as follows:
35s-F:5 '-GACGCACAATCCCACTATCC-3 '；
MYB12L-R:5 '-GACTACTCTCTCCAACTC-3 '；
35s-F is corresponding to the section in the 35S promoter on carrier framework, and MYB12L-R is corresponding in MsMYB12L gene
Section, target sequence length is about 1000bp.
PCR identifies that positive callus is and turns MsMYB12L gene callus.
8, the MsMYB12L gene callus that turns step 7 obtained is seeded to screening culture medium (that is mould containing 50mg/L card
Element, 250mg/L carboxylic Bian penicillin, the MS solid medium of 1mg/L 2,4-D and 0.5mg/L 6-BA) on carry out subculture
Cultivate.
Three, the acquisition of control tissue
Control plasmid replaces recombiant plasmid pRI101-MsMYB12L carry out step 2, obtains turning crt gene callus.
PRI101 carrier replacement recombiant plasmid pRI101-MsMYB12L is carried out step 2, obtains turning empty carrier callus.
Four, the qualification of tissue
1, Qualitative Identification (DMACA staining)
DMACA staining principle: in acid condition, can be with flavanol compound to dimethylaminocinnamaldehyde (DMACA)
(flavanols compounds includes three classes to compound: the first kind is natural flavan-3-alcohol class；Equations of The Second Kind is flavane-3,4-two
Alcohols, i.e. leucoanthocyanidin；3rd class is procyanidin) colour developing generation blueness, its color and concentration are positive relationship.
List of references: " Radix Camelliae sinensis procyanidin extraction process and the optimization of detection method ".
Callus to be measured is respectively as follows: and turns MsMYB12L gene callus, turns empty carrier callus and Colombia
Arabidopsis thaliana ecotype callus.
Operate in accordance with the following steps:
(1) 1 parts by volume methanol and 1 parts by volume 6M HCl/water solution are mixed, obtain methanol-hydrochloric acid mixed solution.
(2) DMACA is dissolved in methanol-hydrochloric acid mixed solution, obtains the DMACA solution of 0.3g/100ml.
(3) taking callus to be measured, dye in DMACA solution 15min.
Before DMACA dyeing, Fig. 1 is shown in by photo.Turning MsMYB12L gene callus is buff.Columbia ecotype
Arabidopsis callus and turn empty carrier callus and be light yellow.
Fig. 2 is shown in by photo after DMACA dyeing.Turn MsMYB12L gene callus and dyed navy blue by DMACA, meaning
Taste the accumulation of a large amount of flavanols compounds.Columbia ecotype Arabidopsis callus and turn empty carrier wound healing
Tissue is not all dyed to navy blue, is shown as lightpink.Result shows, MsMYB12L albumen can regulate and control flavanol compound
The synthesis of compound.
Carrying out five times repeating test, result is consistent.
2, Quantitative measurement
Callus to be measured be respectively as follows: turn MsMYB12L gene callus, turn crt gene callus, turn zero load
Body callus and Columbia ecotype Arabidopsis callus.
Operate in accordance with the following steps:
(1) take 0.2g callus to be measured, transfer in centrifuge tube after liquid nitrogen grinding.
(2) taking the centrifuge tube that step (1) obtains, (volume basis contains to add 1ml 70% containing 1mg/ml ascorbic acid
Amount) aqueous acetone solution, 4 DEG C, stand under dark condition and extract 30min, collect supernatant, residue precipitation in centrifuge tube.
(3) taking the centrifuge tube that step (2) obtains, (volume basis contains to add 1ml 70% containing 1mg/ml ascorbic acid
Amount) aqueous acetone solution, 4 DEG C, stand under dark condition and extract 30min, collect supernatant, residue precipitation in centrifuge tube.
(4) taking the centrifuge tube that step (5) obtains, (volume basis contains to add 1ml 70% containing 1mg/ml ascorbic acid
Amount) aqueous acetone solution, 4 DEG C, stand under dark condition and extract 30min, collect supernatant, residue precipitation in centrifuge tube.
(5) take the centrifuge tube that step (4) obtains, add the 1ml 70% (volume basis containing 1mg/ml ascorbic acid
Content) aqueous acetone solution, 4 DEG C, stand under dark condition and extract 10 hours, collect supernatant.
(6) it is upper that supernatant that the supernatant that step (2) obtained, step (3) obtain, step (4) obtain
The supernatant mixing that clear liquid and step (5) obtain, obtains mixed liquor.
(7) take the mixed liquor that step (6) obtains, add 3ml ether ,-20 DEG C, stand under dark condition (split-phase,
Upper strata is ether phase, and lower floor is acetone phase, and flavanols compounds is contained mutually in lower floor), collect lower floor's phase.
(8) take lower floor's phase that 2ml step (7) obtains, prepared by (2) that add 1ml methanol and 0.5ml step 1
DMACA solution, room temperature stand 20min, then 643nm measure absorbance.
With (+)-Catechin (sigma product) makes standard curve, standard curve equation is as follows: y=2.816x
-0.012, R2=0.996 (y: light absorption value；X: flavanols compounds concentration, unit are mg/g).
Absorbance is brought into standard curve equation, thus calculates the flavanols compounds content in seed to be measured.
Carry out five times repeating test, repeat test takes the meansigma methods of 10 parts of calluss to be measured every time.
The flavanols compounds content turning MsMYB12L gene callus is 124.562mg/kg.Turn crt gene
The flavanols compounds content of callus is 63.254mg/kg.Turn the flavanol compound chemical combination of empty carrier callus
Thing content is 21.758mg/kg.The flavanols compounds content of Columbia ecotype Arabidopsis callus is
21.604mg/kg.What " kg " in this section referred to is callus fresh weight.
Claims (10)
1. a protein, is following (a1) or (a2):
(a1) protein being made up of the aminoacid sequence shown in sequence in sequence table 1；
(a2) by the aminoacid sequence of sequence 1 through the replacement of one or several amino acid residue and/or disappearance and/or
Add and the protein that by sequence 1 derives relevant to plant flavanols compounds content.
2. the gene of protein described in coding claim 1.
3. gene as claimed in claim 2, it is characterised in that: described gene is following (1) or (2) or (3):
(1) coding region DNA molecular as shown in sequence 2 in sequence table；
(2) the DNA sequence hybridization limited with (1) under strict conditions and coding contain with plant flavanols compounds
The protein DNA molecule that amount is relevant；
(3) DNA sequence limited with (1) has more than 90% homology and coding and plant flavanols compounds
The protein DNA molecule that content is relevant.
4. contain the recombinant expression carrier of gene described in Claims 2 or 3, expression cassette, transgenic cell line, turn base
Because of plant tissue or recombinant bacterium.
5. the application of protein described in claim 1, for as follows (b1) or (b2):
(b1) the flavanols compounds content of plant is regulated and controled；
(b2) the flavanols compounds content of plant is increased.
6. the answering in cultivating the transgenic plant that flavanols compounds content increases of gene described in Claims 2 or 3
With.
7. the method cultivating transgenic plant, comprises the steps: channel genes described in Claims 2 or 3
Set out plant, obtain flavanols compounds content be higher than described in set out the transgenic plant of plant.
8. the method cultivating Transgenic plant tissue, comprises the steps: gene described in Claims 2 or 3
Importing is set out plant tissue, obtain flavanols compounds content be higher than described in set out the transgenic plant group of plant tissue
Knit.
9. method as claimed in claim 8, it is characterised in that: described plant tissue is plant callus.
10. protein described in claim 1, or, gene described in Claims 2 or 3, or, claim 7 or
Method described in 8 or 9, the application in plant breeding.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610300919.1A CN105949289B (en) | 2016-05-06 | 2016-05-06 | Obtained from the flavanols modulin MsMYB12L and its encoding gene of functional form apple and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610300919.1A CN105949289B (en) | 2016-05-06 | 2016-05-06 | Obtained from the flavanols modulin MsMYB12L and its encoding gene of functional form apple and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105949289A true CN105949289A (en) | 2016-09-21 |
| CN105949289B CN105949289B (en) | 2019-03-05 |
Family
ID=56914473
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610300919.1A Expired - Fee Related CN105949289B (en) | 2016-05-06 | 2016-05-06 | Obtained from the flavanols modulin MsMYB12L and its encoding gene of functional form apple and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105949289B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109295069A (en) * | 2018-09-19 | 2019-02-01 | 昆明理工大学 | The application of panax japonicus majoris transcription factor gene PjMYB1 |
| CN113461794A (en) * | 2021-08-19 | 2021-10-01 | 云南省烟草农业科学研究院 | Kit and method for regulating seed germination and application thereof |
-
2016
- 2016-05-06 CN CN201610300919.1A patent/CN105949289B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| NONE: "Accession NO.: XP_008337875.1，PREDICTED: transcription factor MYB12 isoform X2 [Malus domestica]", 《GENBANK DATABASE》 * |
| ROGER P HELLENS等: "Transient expression vectors for functional genomics,", 《PLANT METHODS》 * |
| YA-RU WANG等: "Different coloration patterns between the red- and white-fleshedfruits of malus crabapples", 《SCIENTIA HORTICULTURAE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109295069A (en) * | 2018-09-19 | 2019-02-01 | 昆明理工大学 | The application of panax japonicus majoris transcription factor gene PjMYB1 |
| CN113461794A (en) * | 2021-08-19 | 2021-10-01 | 云南省烟草农业科学研究院 | Kit and method for regulating seed germination and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105949289B (en) | 2019-03-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108164588A (en) | Application of the cotton transport protein GhBASS5 genes in plant salt tolerance | |
| CN105837671B (en) | Obtained from the flavonols modulin MsMYB22 and its encoding gene of functional form apple and application | |
| CN105820224A (en) | Anthocyanin-regulation protein MsMYB111 from functional apple and encoding gene and application thereof | |
| CN105820223A (en) | Anthocyanin-regulation protein MsMYB16 from functional apple and encoding gene and application thereof | |
| CN104928396B (en) | A kind of method using EST SSR molecular marker Rapid identification capsicum variety gold green pepper purity | |
| CN105566463B (en) | One kind albumen relevant to Chlorophyll synthesis and its encoding gene and application | |
| CN105837669B (en) | Obtained from the anthocyanin modulin MsARF3 and its encoding gene of functional form apple and application | |
| CN116144701A (en) | Application method of ginkgo bZIP transcription factor GbbZIP08 in promotion of plant flavonoid synthesis | |
| CN105949289A (en) | Flavanol regulatory protein MsMYB12L from functional apples, coding gene and application thereof | |
| CN106754999A (en) | A kind of related GST GFPs of Cotton Fiber of Natural Brown Cotton OPC transhipment and its application | |
| Ogata et al. | Virus-mediated transient expression techniques enable functional genomics studies and modulations of betalain biosynthesis and plant height in quinoa | |
| CN102964437A (en) | Soybean nuclear factor protein and encoding genes of protein and applications of protein and encoding genes | |
| CN113337521B (en) | Application of knockout OsNAC78 gene in reduction of antioxidant enzyme activity of rice | |
| CN107033228B (en) | Obtained from the flavanols and anthocyanin modulin MsMYBPA1 and its encoding gene of functional form apple and application | |
| CN117305498B (en) | Molecular marker combination for identifying pea varieties, primer combination and application of molecular marker combination | |
| CN110128520A (en) | Moso bamboo PheHDZ45 albumen and its encoding gene and application | |
| CN106883292B (en) | Obtained from the MsLBD13 albumen and its encoding gene of functional form apple and application | |
| CN106243207B (en) | Obtained from the MsSUT4 albumen and its encoding gene of functional form apple and application | |
| CN102690811B (en) | Molecular marker SIsv1363 closely linked with millet leaf color gene | |
| CN106749580A (en) | Plant salt tolerance GAP-associated protein GAP TaPUB15 D and its encoding gene and application | |
| CN105949288A (en) | Flavanol regulatory protein MsMYB6L acquired from functional apples, coding gene and application thereof | |
| CN107022014B (en) | Obtained from the MsTMT1L albumen and its encoding gene of functional form apple and application | |
| CN103849605B (en) | A kind of method cultivating resistance plant and special protein thereof and gene | |
| CN107022013B (en) | Obtained from the MsMYBL2 albumen and its encoding gene of functional form apple and application | |
| Song et al. | Expressing the maize anthocyanin regulatory gene Lc increased flavonoid content in the seed of white pericarp rice and purple pericarp rice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190305 |